Robert John Aitken

Melatonin alleviates heat stress-induced oxidative stress and apoptosis in human spermatozoa

Publisher: Elsevier BV

Date: 02-2021

DOI: 10.1016/J.FREERADBIOMED.2021.01.014

Polyclonal antibodies to a 32‐KDA deglycosylated polypeptide from porcine zonae pellucidae will prevent human gamete interaction in vitro

Publisher: Wiley

Date: 11-1987

DOI: 10.1002/MRD.1120180306

Abstract: The major deglycosylated polypeptides of the porcine zona pellucida (ZP), with molecular masses of 66, 52, 36, and 32 kDa, were purified to homogeneity with one-dimensional sodium dodecyl sulphate olyacrylamide gel electrophoresis (SDS/PAGE). Immunofluorescence studies demonstrated that antibodies to the DGZP fraction, and the 66- and 32-kDa polypeptides, bound predominantly to the outer ZP however, only the first two of these antisera formed an immunoprecipitate around the outer human ZP. In immunoblotting experiments using polyclonal antisera raised to these molecules all four polypeptides exhibited cross-reactivity with each other and their parental glycoprotein families (ZP 1-4). In addition, the antisera were tested in an in vitro human gamete bioassay to determine their contraceptive potential antibodies to the 32-kDa deglycosylated polypeptide inhibited human gamete interaction to the greatest extent, 5.3% (+/- 1.2%), relative to a control value of 100%.

Prospective analysis of sperm-oocyte fusion and reactive oxygen species generation as criteria for the diagnosis of infertility

Publisher: Elsevier BV

Date: 02-1991

DOI: 10.1016/S0002-9378(11)80017-7

Abstract: We have undertaken a prospective analysis of the diagnostic significance of three different criteria of human sperm function including the conventional semen profile, measurements of hamster-oocyte fusion, and determinations of reactive oxygen species generation in 139 couples. The latter, who were characterized by a lack of detectable abnormalities in the female partner, were followed up for a maximum of 4 years to determine the incidence of spontaneous pregnancy in the absence of therapeutic intervention. Assessments of monthly fecundity with life table analysis techniques revealed a highly significant, positive relationship between fertility and hamster-oocyte fusion rates that were measured in the presence of the ionophore, A23187. Conversely, reactive oxygen species generation was shown to be negatively associated with both the outcome of the sperm-oocyte fusion assay and fertility in vivo. The clinical significance of these diagnostic techniques was emphasized by the fact that within the same data set, the conventional semen profile was of no significant diagnostic value.

Heat Shock Protein member A2 forms a stable complex with angiotensin converting enzyme and protein disulfide isomerase A6 in human spermatozoa

Publisher: Oxford University Press (OUP)

Date: 16-12-2016

DOI: 10.1093/MOLEHR/GAV073

Abstract: Given the importance of the chaperone Heat Shock Protein A2 (HSPA2) in the regulation of male fertility, this study aimed to identify and characterize additional proteins that may rely on the activity of this chaperone in human spermatozoa. In view of the findings in this study we propose that angiotensin converting enzyme (ACE) and protein disulfide isomerase A6 (PDIA6) are novel interacting proteins of HSPA2 and that this multimeric complex may participate in key elements of the fertilization cascade. The molecular chaperone HSPA2 plays a pivotal role in the remodelling of the sperm surface during capacitation. Indeed, human spermatozoa that are deficient in HSPA2 protein expression lack the ability to recognize human oocytes, resulting in repeated IVF failure in a clinical setting. Moreover, our recent work has shown that defective HSPA2 function induced by oxidative stress leads to the aberrant surface expression of one of its interacting proteins, arylsulfatase A, and thus contributes to a loss of sperm-zona pellucida adhesion. Human spermatozoa were collected from fertile donors, capacitated and prepared for Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) analysis. Protein complexes resolved via BN-PAGE were excised and their constituents were identified using mass spectrometry. The interactions between ACE, PDIA6 and HSPA2 were then confirmed using immunoprecipitation and proximity ligation assays and the localization of these proteins was assessed in isolated spermatozoa and commercially available human testis tissue sections. Finally, pharmacological inhibition of ACE was performed to assess the role of ACE in human sperm capacitation. Herein we have identified ACE and PDIA6 as potential HSPA2-interacting proteins and shown that this assemblage resides in membrane raft microdomains located in the peri-acrosomal region of the sperm head. Additionally, the surface expression of PDIA6, but not ACE, was shown to be dynamically regulated during sperm capacitation and, like that of previously characterized HSPA2-interacting proteins, this surface expression proved vulnerable to oxidative stress. In terms of the functional significance of this protein complex, pharmacological inhibition of ACE significantly reduced the ability of human spermatozoa to undergo an agonist induced acrosome reaction (P < 0.01). While these results provide a descriptive analysis of the PDIA6/ACE/HSPA2 complex, this study provides the impetus for further investigation into the role of PDIA6 and ACE in human sperm function. As our research group, and others, have shown that HSPA2 is compromised in the spermatozoa of men with oocyte recognition defects, the characterization of these HSPA2-interacting proteins provides important insight into the complexity of the cellular pathways that may be affected in the spermatozoa of infertile in iduals. Large scale proteomics data can be accessed through the Proteomics Identifications Database (PRIDE). This work was supported by the National Health and Medical Research Council. Grant # APP1046346. The authors have no competing interests to declare.

Contraceptive potential of antibodies to the zona pellucida

Publisher: Bioscientifica

Date: 05-1988

DOI: 10.1530/JRF.0.0830325

Abstract: The notion of a contraceptive vaccine based on gamete-specific surface antigens was first proposed over a decade ago, as the result of in-vitro and in-vivo studies, and in recent years has been the subject of intensive research. In particular, the zona pellucida has attracted much attention as a potential target for immunological intervention in the fertilization process. Such is the rapidly-expanding nature of research into the biochemical and biological characterization of this structure, that a review of the implications for the development of a contraceptive vaccine seems timely.

A new method for evaluation of the acrosome reaction in viable human spermatozoa

Publisher: Hindawi Limited

Date: 24-04-2009

DOI: 10.1111/J.1439-0272.1991.TB02537.X

Abstract: The acrosome reaction of human spermatozoa was induced by changes of temperature. Spermatozoa were collected from fertile donors and a patient group, and selected by the "swim-up" method. The spermatozoa were treated in two different ways: Protocol I: 24 hours at room temperature followed by additional incubation at 37 degrees C for 3 hours (control), and protocol II: 24 hours at 4 degrees C followed by additional incubation at 37 degrees C for 3 hours. The acrosome reaction of the viable spermatozoa was evaluated by a new method utilizing indirect immunofluorescence with anti-outer acrosomal membrane antibodies and exposure to a hypo-osmotic medium. In fertile donors as well as in the patient group, significant induction of the acrosome reaction (20%) was evident after exposure to low temperature (4 degrees C). The spontaneous rate of acrosome reaction in the control group was below 7%.

The role of molecular chaperones in spermatogenesis and the post-testicular maturation of mammalian spermatozoa

Publisher: Oxford University Press (OUP)

Date: 20-04-2012

DOI: 10.1093/HUMUPD/DMS009

Abstract: Spermatogenesis culminates in production of one of the most highly differentiated cells in biology, the spermatozoon. The gametes that emerge from the testes are, however, functionally immature and only acquire full functionality once they have completed a process of post-testicular maturation in the epididymis and female reproductive tract. Remarkably, this acquisition of sperm function occurs while these cells are transcriptionally and translationally silent and is therefore highly dependent on post-translational modifications to their existing protein complement. In this review, we consider the emerging roles of several prominent molecular chaperone families in orchestrating both the morphological differentiation of male germ cells during spermatogenesis and their functional transformation during sperm maturation. Journal databases were searched using key words, including chaperone, heat shock protein, testes, spermatogenesis, spermatozoa, epididymal maturation, capacitation and fertilization. In the past two decades, molecular chaperones have been acknowledged to play key roles in controlling both the morphological transformation of germ cells during spermatogenesis and the post-testicular maturation of these cells as they transit the male and female reproductive tracts. Furthermore, there is mounting evidence that aberrant chaperone expression may be a major contributing factor to the defective sperm function seen in many cases of male infertility. Molecular chaperones are critically involved in all phases of sperm development. Targeted disruption of these proteins has the ability to arrest spermatogenesis, compromise sperm maturation and inhibit fertilization. These proteins therefore hold considerable promise as targets for novel contraceptive strategies and as diagnostic biomarkers for male infertility.

Fertility rates and future population trends: will Europe's birth rate recover or continue to decline?

Publisher: Wiley

Date: 02-2006

DOI: 10.1111/J.1365-2605.2005.00639.X

Abstract: Europe has long completed its demographic transition from high birth and death rates to low birth and death rates. But the demographic transition paradigm that has been very useful for explaining global demographic trends during the 20th century and that still has strong predictive power when it comes to projecting future trends in countries that still have high fertility, has nothing to say about the future of fertility in Europe. The currently popular notion of a 'second demographic transition' is a useful way to describe a bundle of behavioural and normative changes that recently happened in Europe, but it has no predictive power. The social sciences have not yet come up with a useful theory to predict the future fertility level of post-demographic transition societies. We even do not know whether the trend will be up or down. Given the lack of a predictive theory, this paper will try to do two things: (i) Summarize different substantive arguments that would either suggest the assumption of a recovery of fertility rates in Europe or alternatively, imply further declines. (ii) Convert this discussion of the uncertainty of future fertility trends into probabilistic population projections for Europe, thus highlighting the implications of alternative fertility levels over the coming years. We will also discuss trade-offs between fertility and immigration, and the phenomenon that Europe now has entered a period of negative momentum of population growth.

Localization and significance of molecular chaperones, heat shock protein 1, and tumor rejection antigen gp96 in the male reproductive tract and during capacitation and acrosome reaction

Publisher: Oxford University Press (OUP)

Date: 02-2005

DOI: 10.1095/BIOLREPROD.104.034470

Identification and characterization of a novel splice variant of mouse and rat cytochrome b5/cytochrome b5 reductase

Publisher: Elsevier BV

Date: 03-2004

DOI: 10.1016/J.YGENO.2003.08.020

Analysis of the mechanism by which calcium negatively regulates the tyrosine phosphorylation cascade associated with sperm capacitation

Publisher: The Company of Biologists

Date: 15-01-2004

DOI: 10.1242/JCS.00842

Abstract: The capacitation of mammalian spermatozoa involves the activation of a cAMP-mediated signal transduction pathway that drives tyrosine phosphorylation via mechanisms that are unique to this cell type. Controversy surrounds the impact of extracellular calcium on this process, with positive and negative effects being recorded in independent publications. We clearly demonstrate that the presence of calcium in the external medium decreases tyrosine phosphorylation in both human and mouse spermatozoa. Under these conditions, a rise in intracellular pH was recorded, however, this event was not responsible for the observed changes in phosphotyrosine expression. Rather, the impact of calcium on tyrosine phosphorylation in these cells was associated with an unexpected change in the intracellular availability of ATP. Thus, the ATP content of both human and mouse spermatozoa fell significantly when these cells were incubated in the presence of external calcium. Furthermore, the removal of glucose, or addition of 2-deoxyglucose, decreased ATP levels within human spermatozoon populations and induced a corresponding decline in phosphotyrosine expression. In contrast, the mitochondrial inhibitor rotenone had no effect on either ATP levels or tyrosine phosphorylation. Addition of the affinity-labeling probe 8-N3 ATP confirmed our prediction that spermatozoa have many calcium-dependent ATPases. Moreover, addition of the ATPase inhibitor thapsigargin, increased intracellular calcium levels, decreased ATP and suppressed tyrosine phosphorylation. Based on these findings, the present study indicates that extracellular calcium suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP, and not by activating tyrosine phosphatases or inhibiting tyrosine kinases as has been previously suggested.

Investigation of the expression and functional significance of the novel mouse sperm protein, a disintegrin and metalloprotease with thrombospondin type 1 motifs number 10 (ADAMTS10)

Publisher: Wiley

Date: 28-03-2012

DOI: 10.1111/J.1365-2605.2011.01235.X

Abstract: Fertilization represents the culmination of a series of complex interactions between male and female gametes. Despite advances in our understanding, the precise molecular mechanisms underlying these fundamental interactions remain largely uncharacterized. There is however growing recognition that this process requires the concerted action of multiple sperm receptors that possess affinity for complementary zona pellucida ligands and those that reside on the surface of the oolemma. Among the candidate sperm proteins that have been implicated in fertilization, those belonging to the ADAM (a disintegrin and metalloprotease) family of proteases have received considerable attention. The focus of the studies described herein has been the characterization of a closely related member of this protease family, ADAMTS10 (a disintegrin and metalloprotease with thrombospondin type 1 motifs number 10). We have demonstrated that ADAMTS10 is expressed during the later stages of mouse spermatogenesis and incorporated into the acrosomal domain of developing spermatids. During sperm maturation, the protein appears to be processed before being expressed on the surface of the peri-acrosomal region of the head. Our collective data suggest that, from this position, ADAMTS10 participates in sperm adhesion to the zona pellucida. Indeed, pre-incubation of capacitated spermatozoa with either galardin, a broad spectrum inhibitor of metalloprotease activity, or anti-ADAMTS10 antisera elicited a significant reduction in their ability to engage in zona adhesion. Overall, these studies support the notion that sperm-oocyte interactions involve considerable functional redundancy and identify ADAMTS10 as a novel candidate in the mediation of these fundamentally important events.

Impact of oxidative stress on male and female germ cells: Implications for fertility

Publisher: Bioscientifica

Date: 04-2020

DOI: 10.1530/REP-19-0452

Abstract: Male and female germ lines are vulnerable to oxidative stress. In spermatozoa, such stress triggers a lipid peroxidation cascade that culminates in the generation of electrophilic lipid aldehydes that bind to DNA and a raft of proteins involved in the delivery of functionally competent cells. One set of targets for these aldehydes are the proteins of the mitochondrial electron transport chain. When this interaction occurs, mitochondrial ROS generation is enhanced leading to the sustained generation of oxidative damage in a self-perpetuating cycle. Such damage affects all aspects of sperm function including motility, sperm-egg recognition, acrosomal exocytosis and sperm-oocyte fusion. Oxidative stress in the male germ line also attacks the integrity of sperm DNA with potential impacts on the developmental capacity of embryos and the health and wellbeing of the offspring. Potential pathways of reactive oxygen species (ROS) generation in male germ cells could involve enhanced lipoxygenase activity, activation of NADPH oxidase and/or electron leakage from mitochondria. Similarly, in the female germ line, both the induction of oocyte senescence following ovulation and the deterioration of oocyte quality with maternal age appear to involve the generation of oxidative damage. In this case, the mitochondria appear to be a particularly important source of ROS compromising the viability and fertilizability of the oocyte and interfering with the normal segregation of chromosomes during meiosis. In light of these considerations, antioxidants should have some role to play in the preservation of reproductive function in both men and women however, we still await appropriate trials to test this hypothesis.

Potential importance of transition metals in the induction of DNA damage by sperm preparation media

Publisher: Oxford University Press (OUP)

Date: 19-08-2012

DOI: 10.1093/HUMREP/DEU204

Abstract: What are the mechanisms by which the preparation of spermatozoa on discontinuous density gradients leads to an increase in oxidative DNA damage? The colloidal silicon solutions that are commonly used to prepare human spermatozoa for assisted reproduction technology (ART) purposes contain metals in concentrations that promote free radical-mediated DNA damage. Sporadic reports have already appeared indicating that the use of colloidal silicon-based discontinuous density gradients for sperm preparation is occasionally associated with the induction of oxidative DNA damage. The cause of this damage is however unknown. This study comprised a series of experiments designed to: (i) confirm the induction of oxidative DNA damage in spermatozoa prepared on commercially available colloidal silicon gradients, (ii) compare the levels of damage observed with alterative sperm preparation techniques including an electrophoretic approach and (iii) determine the cause of the oxidative DNA damage and develop strategies for its prevention. The semen s les employed for this analysis involved a cohort of >50 unselected donors and at least three independent s les were used for each component of the analysis. The setting was a University biomedical science laboratory. The major techniques employed were: (i) flow cytometry to study reactive oxygen species generation, lipid peroxidation and DNA damage, (ii) computer-aided sperm analysis to measure sperm movement and (iii) inductively coupled mass spectrometry to determine the elemental composition of sperm preparation media. Oxidative DNA damage is induced in spermatozoa prepared on PureSperm(®) discontinuous colloidal silicon gradients (P < 0.001 versus repeated centrifugation) because this medium contains metals, particularly Fe, Al and Cu, which are known to promote free radical generation in the immediate vicinity of DNA. This damage can be significantly accentuated by reducing agents, such as ascorbate (P < 0.001) and inhibited by selective chelation (P < 0.001). This problem is not confined to PureSperm(®) analysis of additional commercial sperm preparation media revealed that metal contamination is a relatively constant feature of such products. While the presence of metals, particularly transition metals, may exacerbate the levels of oxidative DNA damage seen in human spermatozoa, the significance of such damage has not yet been tested in suitably powered clinical trials. The results explain why the preparation of spermatozoa on discontinuous colloidal silicon gradients can result in oxidative DNA damage. The results are of immediate relevance to the development of safe, effective protocols for the preparation of spermatozoa for ART purposes. The study was funded by the Australian Health and Medical Research Council. One of the authors (R.J.A.) has had a consultantship with a biotechnology company, NuSep, interested in the development of electrophoretic methods of sperm preparation. He has no current financial interest in this area. None of the other authors have a conflict of interest to declare.

Origins and consequences of DNA damage in male germ cells

Publisher: Elsevier BV

Date: 2007

DOI: 10.1016/S1472-6483(10)60676-1

Abstract: DNA damage in the male germline is associated with poor fertilization rates following IVF, defective preimplantation embryonic development, and high rates of miscarriage and morbidity in the offspring, including childhood cancer. This damage is poorly characterized, but is known to involve hypomethylation of key genes, oxidative base damage, endonuclease-mediated cleavage and the formation of adducts with xenobiotics and the products of lipid peroxidation. There are many possible causes of such DNA damage, including abortive apoptosis, the oxidative stress associated with male genital tract infection, exposure to redox cycling chemicals, and defects of spermiogenesis associated with the retention of excess residual cytoplasm. Physical factors such as exposure to radiofrequency electromagnetic radiation or mild scrotal heating can also induce DNA damage in mammalian spermatozoa, although the underlying mechanisms are unclear. Ultimately, resolving the precise nature of the DNA lesions present in the spermatozoa of infertile men will be an important step towards uncovering the aetiology of this damage and developing strategies for its clinical management.

Andrology: Analysis of the ability of three probes targeting the outer acrosomal membrane or acrosomal contents to detect the acrosome reaction in human spermatozoa

Publisher: Oxford University Press (OUP)

Date: 10-1993

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A137910

Abstract: A comparative analysis has been undertaken of the behaviour of probes targeting the outer acrosomal membrane (Arachis hypogaea lectin) or constituents of the acrosomal vesicle (Pisum sativum lectin or monoclonal antibody CRB9) following the induction of the acrosome reaction in human spermatozoa with the ionophore A23187. The results obtained with these two classes of reagent were highly correlated (r = approximately 0.8), although the absolute rates of acrosome reaction were significantly different the probe targeting the outer acrosomal membrane (A. hypogaea) consistently gave higher results than either of the probes targeting the acrosomal contents. Time-dependent studies, employing a double-labelling technique, indicated that this difference was apparent from the earliest time point examined (15 min after A23187 addition) and reflected the more rapid dissipation of the A. hypogaea lectin from the acrosomal region of the cell than either of the probes targeting the acrosomal vesicle. These results indicate that the outer acrosomal membrane is dispersed from acrosome-reacting human spermatozoa more rapidly than certain major constituents of the acrosomal vesicle and have possible implications for the design of diagnostic assays focusing on this aspect of human sperm function.

Influence of Antisperm Antibodies on Human Sperm Function

Publisher: Wiley

Date: 10-1988

DOI: 10.1111/J.1464-410X.1988.TB04367.X

Abstract: Antisperm antibodies have been found in about 8% of men with infertility and in 60 to 80% of patients following vasectomy. In order to investigate the way antibodies influence sperm function we studied serum and seminal plasma from patients with infertility (n = 61) or undergoing vasovasostomy (n = 25). These antisera were characterised to determine their TAT titre, the nature of the target antigens and their capacity to interfere directly with fertilisation. The results indicate that antibodies from both groups of patients exhibit a capacity to stimulate or suppress sperm/oocyte fusion. The proportion of s les showing stimulatory activity was higher (50%) in the vasovasostomised population than in patients with infertility (21%). The remainder of the antisera suppressed sperm/oocyte fusion. There was no correlation between the titre of antisperm antibodies and their capacity to influence sperm function, indicating that it is the nature of the target antigens which is of significance rather than the antibody concentration. Western blot analysis indicated that these antisera targeted a group of sperm surface antigens with molecular weights of 30kD (35,45,66,90 and 115kD). Monoclonal antibodies are now being prepared in order to determine which of these specific components are involved in the suppression of sperm function.

Analysis of sperm function in Kartagener's syndrome

Publisher: Elsevier BV

Date: 11-1983

DOI: 10.1016/S0015-0282(16)47434-0

Abstract: Adaptation allows neurons to respond to a wide range of stimulus intensities. However, it also leads to ambiguity as the representation of the external world depends on the context. We recorded neurons from Wistar rats' brainstem nuclei belonging to two major somatosensory pathways (lemniscal and paralemniscal) and explored the way in which they encode noisy stimuli under different contexts. We found that although their unadapted intensity-response curves are very similar, the adapted curves of the two pathways are distinctively different as they are optimized for encoding different intensity ranges. Lemniscal neurons most faithfully encoded stimuli when the background intensity was high, whereas paralemniscal cells best encoded stimuli under low intensity context. Intracellular recordings indicate that these differences emerge already at the synaptic level. We suggest that the two pathways synergistically improve the ability of this system to encode a wide range of intensities during natural stimulation, potentially reducing the inherent ambiguity of adaptive coding.

Phosphorylation of Izumo1 and Its Role in Male Infertility

Publisher: Medknow

Date: 2015

DOI: 10.4103/1008-682X.156119

Recombinant Human Zona Pellucida Protein ZP3 Produced by Chinese Hamster Ovary Cells Induces the Human Sperm Acrosome Reaction and Promotes Sperm-Egg Fusion

Publisher: Oxford University Press (OUP)

Date: 10-1994

DOI: 10.1095/BIOLREPROD51.4.607

Abstract: Studies on the role of specific molecules in the human fertilization process and additional assessments of potential applications for these proteins are h ered by the limited amount of available biological material. However, this drawback might be circumvented by the recent cloning of several gamete-specific genes, which opens possibilities for the production of recombinant proteins. By use of cDNA and genomic DNA fragments of the human ZP3 gene, which encodes a major constituent of the zona pellucida surrounding the oocyte, a 2.7-kb minigene was constructed containing the natural third and fourth introns of the gene and a truncated intron between exons 2 and 3. This ZP3 DNA was transfected to Chinese hamster ovary cells, and a single-cell clone producing the recombinant ZP3 protein (recZP3) was generated. Western blot analysis of culture medium from these cells showed that recZP3 has a molecular mass +/- 5 kDa smaller than that of natural ZP3. Under reducing conditions, it migrates at an apparent molecular mass of 55-60 kDa. RecZP3 induced the sperm acrosome reaction and promoted fusion of human spermatozoa with zona-free hamster oocytes, indicating that the recombinant protein is biologically active. RecZP3 provides an attractive tool for studying the initial stage of the human fertilization process. Furthermore, it might have clinical applications in the development of diagnostic tests for male infertility and serve as target antigen in the design of contraceptive vaccines.

The effects of radiofrequency electromagnetic radiation on sperm function

Publisher: Bioscientifica

Date: 12-2016

DOI: 10.1530/REP-16-0126

Abstract: Mobile phone usage has become an integral part of our lives. However, the effects of the radiofrequency electromagnetic radiation (RF-EMR) emitted by these devices on biological systems and specifically the reproductive systems are currently under active debate. A fundamental hindrance to the current debate is that there is no clear mechanism of how such non-ionising radiation influences biological systems. Therefore, we explored the documented impacts of RF-EMR on the male reproductive system and considered any common observations that could provide insights on a potential mechanism. Among a total of 27 studies investigating the effects of RF-EMR on the male reproductive system, negative consequences of exposure were reported in 21. Within these 21 studies, 11 of the 15 that investigated sperm motility reported significant declines, 7 of 7 that measured the production of reactive oxygen species (ROS) documented elevated levels and 4 of 5 studies that probed for DNA damage highlighted increased damage due to RF-EMR exposure. Associated with this, RF-EMR treatment reduced the antioxidant levels in 6 of 6 studies that discussed this phenomenon, whereas consequences of RF-EMR were successfully ameliorated with the supplementation of antioxidants in all 3 studies that carried out these experiments. In light of this, we envisage a two-step mechanism whereby RF-EMR is able to induce mitochondrial dysfunction leading to elevated ROS production. A continued focus on research, which aims to shed light on the biological effects of RF-EMR will allow us to test and assess this proposed mechanism in a variety of cell types.

Is there a role for immunocontraception?

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1016/J.MCE.2010.04.004

Abstract: The world's population is continuing to grow at an alarming rate and yet no novel methods of contraception have been introduced since 1960s. The paucity of our current contraceptive armoury is indicated by the 46 million abortions that are performed each year, largely in developing countries where population growth is greatest. Thus, whatever new forms of fertility control we develop for the next millennium, the particular needs of developing countries should be borne in mind. Contraceptive vaccines have the potential to provide safe, effective, prolonged, reversible protection against pregnancy in a form that can be easily administered in the Third World. In this review we consider the contraceptive targets that might be pursued, how vaccines might be engineered and the problems generated by inter-in idual variations in antibody titre. We conclude that the specifications for a safe, effective, reversible vaccine are more likely to be met in animals than man.

The mouse sperm proteome characterized via IPG strip prefractionation and LC-MS/MS identification

Publisher: Wiley

Date: 04-2008

DOI: 10.1002/PMIC.200701020

Abstract: Proteomic profiling of the mouse spermatozoon has generated a unique and valuable inventory of candidates that can be mined for potential contraceptive targets and to further our understanding of the PTMs that regulate the functionality of this highly specialized cell. Here we report the identification of 858 proteins derived from mouse spermatozoa, 23 of which demonstrated testis only expression. The list contained many proteins that are known constituents of murine spermatozoa including Izumo, Spaca 1, 3, and 5, Spam 1, Zonadhesin, Spesp1, Smcp, Spata 6, 18, and 19, Zp3r, Zpbp 1 and 2, Spa17, Spag 6, 16, and 17, CatSper4, Acr, Cylc2, Odf1 and 2, Acrbp, and Acrv1. Certain protein families were highly represented in the proteome. For ex le, of the 42 gene products classified as proteases, 26 belonged to the 26S-proteasome. Of the many chaperones identified in this proteome, eight proteins with a TCP-1 domain were found, as were seven Rab guanosine triphosphatases. Finally, our list yielded three putative seven-transmembrane proteins, two of which have no known tissue distribution, an extragenomic progesterone receptor and three unique testis-specific kinases all of which may have some potential in the future regulation of male fertility.

Development of a novel electrophoretic system for the isolation of human spermatozoa

Publisher: Oxford University Press (OUP)

Date: 14-04-2005

DOI: 10.1093/HUMREP/DEI024

Abstract: Optimization of assisted conception outcomes involves the development of rapid, safe, effective techniques for the isolation of functional human spermatozoa free from significant DNA damage. In this study we describe a novel electrophoretic sperm isolation technique that achieves these objectives. The separation system consisted of a cassette comprising two 400 mul chambers separated by a polycarbonate filter containing 5 micromol/l pores and bounded by a 15 kDa polyacrylamide membrane to allow the free circulation of buffer. Semen was introduced into one chamber, current applied (75 mA at variable voltage) and within seconds a purified suspension of spermatozoa could be collected from the adjacent chamber. These cells were assessed for their count, viability, motility, morphology and DNA integrity. The suspensions generated by the electrophoretic separation technique contained motile, viable, morphologically normal spermatozoa and exhibited low levels of DNA damage. Moreover, these cell suspensions were free from contaminating cells, including leukocytes. The technique was comparable to discontinuous gradient centrifugation except that it took a fraction of the time and generated cells with significantly less DNA damage. Electrophoretic separation represents a highly effective, novel approach for the isolation of spermatozoa for assisted conception purposes.

Oxidative stress and male reproductive biology

Publisher: CSIRO Publishing

Date: 2004

DOI: 10.1071/RD03089

Abstract: Spermatozoa were the first cell type in which the cellular generation of reactive oxygen was demonstrated. This activity has now been confirmed in spermatozoa from all mammalian species examined including the rat, mouse, rabbit, horse, bull and human being. Under physiological circumstances, cellular redox activity is thought to drive the cAMP-mediated, tyrosine phosphorylation events associated with sperm capacitation. In addition to this biological role, human spermatozoa also appear to suffer from oxidative stress, with impacts on the normality of their function and the integrity of their nuclear and mitochondrial DNA. Recent studies have helped to clarify the molecular basis for the intense redox activity observed in defective human spermatozoa, the nature of the subcellular structures responsible for this activity and possible mechanisms by which oxidative stress impacts on these cells. Given the importance of oxidative damage in the male germ line to the origins of male infertility, early pregnancy loss and childhood disease, this area of sperm biochemistry deserves attention from all those interested in improved methods for the diagnosis, management and prevention of male-mediated reproductive failure.

Lessons learned in Andrology: revelations on a road less traveled

Publisher: Wiley

Date: 20-08-2015

DOI: 10.1111/ANDR.12087

Analysis of sperm separation protocols for isolating cryopreserved human spermatozoa

Publisher: Bioscientifica

Date: 31-03-2023

DOI: 10.1530/RAF-22-0133

Abstract: Sperm cryopreservation is a valuable tool for the long-term preservation of male fertility. Thus, determining the optimal technique for isolating spermatozoa post-thaw is vital to ensure recovery of the highest quality spermatozoa with minimal iatrogenic damage. This not only enhances the chances of successful conception but also reduces the risk of genetic damage in the embryo. To address this issue, human semen s les were cryopreserved using a slow freezing protocol and Quinn's Advantage™ Sperm Freeze medium. The s les were subsequently thawed and subjected to three types of sperm isolation procedures: direct swim-up, density gradient centrifugation, and electrophoretic separation using the Felix™ device. Cryopreservation led to the anticipated loss of sperm motility and vitality in association with increases in lipid peroxidation and DNA damage. Following sperm selection, all three isolation techniques resulted in an increase in sperm motility which was particularly evident with the swim-up and Felix™ procedures. The latter also significantly improved sperm vitality. There were no differences between sperm separation techniques with respect to morphology, and mitochondrial reactive oxygen species generation remained essentially unchanged when cell vitality was taken into account. By contrast, major differences were observed in DNA integrity and lipid aldehyde formation, where Felix™ isolated cells exhibiting significantly less DNA damage than the other isolation procedures as well as lower levels of 4-hydroxynonenal formation. Electrophoretic sperm isolation, therefore, offers significant advantages over alternative separation strategies, in terms of the quality of the gametes isolated and the time taken to achieve the isolation. Long-term storage of sperm is vital to assisted reproductive technology because it permits the preservation of fertility that might be compromised as a result of factors such as chemotherapy or vasectomy. This goal can be achieved via cryopreservation – the freezing of cells to −196°C. When the sperm are subsequently required for conception, they must be carefully separated from the cryopreservation medium in a manner that maximizes the chances of successful conception and minimizes the risk of genetic defects in the offspring. In this paper, three isolation techniques were compared for their ability to separate ideal sperm from semen and media following cryopreservation. It was found that cryopreservation led to lower levels of motility and vitality and created higher levels of DNA and cell membrane damage. Of the three techniques compared, only cells separated on the basis of their size and electric charge (electrophoretic isolation) exhibited significantly lower levels of DNA fragmentation.

Analysis of the relationship between reactive oxygen species production and leucocyte infiltration in fractions of human semen separated on Percoll gradients

Publisher: Wiley

Date: 12-1990

DOI: 10.1111/J.1365-2605.1990.TB01051.X

Abstract: We have shown previously that the reactive oxygen species generated by washed human ejaculates originate from cells which can be isolated in the low density region of discontinuous Percoll gradients. In this study we have used a simplified two-step (40/80%) Percoll gradient to separate human ejaculates (n = 109) into two populations of spermatozoa, exhibiting either a high or a low capacity for reactive oxygen species generation. We have then examined the relationships between this activity and other properties of the isolated fractions, with particular emphasis on the presence of leucocytes, which we have quantified using a monoclonal antibody directed against the common leucocyte antigen. The low-density cells recovered from the 40%/80% interface of the Percoll gradients, differed from the high-density fraction in exhibiting significantly reduced sperm motility, poorer sperm morphology, and a considerably enhanced capacity for reactive oxygen species production (P less than 0.001). In six cases the elevated levels of reactive oxygen species generation were associated with leucocyte concentrations in excess of 1 x 10(6) per 10(7) sperm, suggesting that leucocytes enter the seminal compartment in an activated, oxygen-radical generating, state. However, in the majority of cases exhibiting high levels of reactive oxygen species production, leucocyte numbers were low or absent and the semen profiles were unremarkable, except that seminal sperm concentrations tended to be low. These results suggest that the oxidative stress responsible for defective sperm function involves reactive oxygen species originating from two sources the sperm and infiltrating leucocytes.

Swim-up of tammar wallaby (Macropus eugenii) spermatozoa in Biggers, Whitter and Whittingham (BWW) medium: maximisation of sperm motility, minimisation of impairment of sperm metabolism and induction

Publisher: CSIRO Publishing

Date: 2017

DOI: 10.1071/RD15152

Abstract: A variety of media were compared for their ability to sustain the motility of tammar wallaby spermatozoa over an 8-h period following swim–up from coagulated semen. The study demonstrated that a modified Tyrode’s solution, Biggers, Whitter and Whittingham medium (BWW) was significantly better than any of the other assessed media in supporting wallaby sperm motility. After 8 h of incubation in BWW, motility was maintained at 79.3 ± 9.3%, with 77.0 ± 10.4% rapid and 65.7 ± 8.7% progressively motile spermatozoa. By contrast, motility was % at the same 8-h time point in all of the other media assessed. After 2 h of incubation in BWW, tammar spermatozoa consumed more oxygen than their counterparts in PBS (52.0 ± 2.7 vs 75.0 ± 6.6 μL per 108 spermatozoa per 2 h P 0.001). Motility was not enhanced in any of these media by the addition of 5 mM N-acetyl-D-glucosamine, the major energy substrate in wallaby semen. However, addition of dibutyryl cAMP and pentoxifylline in BWW resulted in the extremely rapid induction of hyperactivated motility in the entire sperm population. This burst of hyperactivated motility was entirely dependent on calcium in BWW and significantly inhibited by calmidazolium, a calmodulin inhibitor. A set of computer-assisted sperm analysis parameters were identified that permitted the accurate quantification of hyperactivation rates in this species. This is the first comparative analysis of media for harvesting and incubating marsupial spermatozoa and the first record of hyperactivated motility in any marsupial species.

GLIPR1L1 is an IZUMO-binding protein required for optimal fertilization in the mouse

Publisher: Springer Science and Business Media LLC

Date: 31-10-2019

DOI: 10.1186/S12915-019-0701-1

Abstract: The sperm protein IZUMO1 (Izumo sperm-egg fusion 1) and its recently identified binding partner on the oolemma, IZUMO1R, are among the first ligand-receptor pairs shown to be essential for gamete recognition and adhesion. However, the IZUMO1-IZUMO1R interaction does not appear to be directly responsible for promoting the fusion of the gamete membranes, suggesting that this critical phase of the fertilization cascade requires the concerted action of alternative fusogenic machinery. It has therefore been proposed that IZUMO1 may play a secondary role in the organization and/or stabilization of higher-order heteromeric complexes in spermatozoa that are required for membrane fusion. Here, we show that fertilization-competent (acrosome reacted) mouse spermatozoa harbor several high molecular weight protein complexes, a subset of which are readily able to adhere to solubilized oolemmal proteins. At least two of these complexes contain IZUMO1 in partnership with GLI pathogenesis-related 1 like 1 (GLIPR1L1). This interaction is associated with lipid rafts and is dynamically remodeled upon the induction of acrosomal exocytosis in preparation for sperm adhesion to the oolemma. Accordingly, the selective ablation of GLIPR1L1 leads to compromised sperm function characterized by a reduced ability to undergo the acrosome reaction and a failure of IZUMO1 redistribution. Collectively, this study characterizes multimeric protein complexes on the sperm surface and identifies GLIPRL1L1 as a physiologically relevant regulator of IZUMO1 function and the fertilization process.

Sperm selection for assisted conception

Publisher: Cambridge University Press

Date: 2011

DOI: 10.1017/CBO9780511894756.023

Loss of Nuclear/DNA Integrity in Mouse Epididymal Spermatozoa after Short-Term Exposure to Low Doses of Dibutyl Phthalate or Bisphenol AF and Its Mitigation by Oral Antioxidant Supplementation

Publisher: MDPI AG

Date: 05-05-2023

DOI: 10.3390/ANTIOX12051046

Abstract: Routine exposure to chemicals omnipresent in the environment, particularly the so-called endocrine-disrupting chemicals (EDCs), has been associated with decreased sperm quality and increased anomalies in testis. The decline in semen quality and testicular abnormalities have been attributed to the disruption of endocrine signaling as well as oxidative stress. The present study set out to examine the effect of short-term exposure of two common EDCs widely used in the plastic industry: Dibutyl Phthalate (DBP) and Bisphenol AF (BPAF). Our research objective was to focus on the post-testicular compartment of the epididymis, where spermatozoa acquire their functional capacity and are stored. The data obtained indicated no significant effect for either chemicals on sperm viability, motility or acrosome integrity. Neither of the EDCs had a noticeable effect on the structures of the testis and epididymis. However, substantial impact on the integrity of the sperm nucleus and DNA structure was evidenced by a significant increase in nuclear decondensation and DNA base oxidation. The damage observed was postulated to arise from the pro-oxidant properties of the EDCs generating excess of reactive oxygen species (ROS) and triggering a state of oxidative stress. This hypothesis was confirmed when the observed damage was largely blocked by co-administering EDCs with an evidenced-based antioxidant formulation.

Reactive oxygen species: Friend or foe

Publisher: Cambridge University Press

Date: 06-04-2006

DOI: 10.1017/CBO9780511545115.008

Sperm DNA fragmentation abnormalities in men from couples with a history of recurrent miscarriage

Publisher: Wiley

Date: 23-07-2015

DOI: 10.1111/AJO.12373

Abstract: Previous studies have described an association between sperm with DNA damage and a history of recurrent miscarriage (RM), although it is not clear whether there is benefit in screening for sperm DNA fragmentation and to what extent DNA fragmentation impacts upon RM. To identify what proportion of couples experiencing RM are affected by DNA fragmentation abnormalities. In this retrospective study, between 2008 and 2013, couples with a history of recurrent miscarriage (≥3 first trimester miscarriages) were investigated comprehensively for known causes (karyotype, uterine, antiphospholipid syndrome, thrombophilia) and also by semen analysis, including DNA fragmentation [sperm chromatin structure analysis (SCSA)]. Statistical analysis was performed on SPSS software with significance taken as P < 0.05. There were 108 couples with a median sperm DNA fragmentation index (DFI) of 9.50%. Normal levels were found in 70.5% of men (DFI 30%). Couples with otherwise unexplained recurrent miscarriage had significantly higher DFI than those with other causes identified on routine screening (P = 0.012). In couples experiencing RM, 30% (32/108) of men had sperm with high levels of DNA fragmentation (DFI > 15%). This may be a contributing factor to the clinical syndrome of RM, and future clinical trials of therapies for these couples are warranted.

Application of Poisson distribution theory to the zona-free hamster oocyte penetration test to assess sperm function of men with asthenozoospermia

Publisher: Bioscientifica

Date: 05-1986

DOI: 10.1530/JRF.0.0770067

Abstract: Assessments of the penetrating potential of human spermatozoa were carried out using the zona-free hamster oocyte penetration test on 4 groups of subjects exhibiting normal fertility, idiopathic asthenozoospermia(less than 40% motility), asthenozoospermia associated with varicocele and oligoaesthenozoospermia (less than 20 X 10(6) spermatozoa/ml and less than 40% motility). When the Poisson model was used to correct the results of the in-vitro penetration experiments for differences in motile sperm concentration, significant differences were apparent between the normal fertile controls and all 3 categories of asthenozoospermic patient. Furthermore, the penetrating ability of the motile spermatozoa from patients presenting with a varicocele or oligoaesthenozoospermia was significantly less than that for the group in which asthenozoospermia was the only detectable defect. These results emphasize the practical significance of the Poisson model in the analysis of male fertility and demonstrate that the asthenozoospermic condition is associated with a significant reduction in the fertilizing potential of the motile spermatozoa.

Patterns of MTT reduction in mammalian spermatozoa

Publisher: Bioscientifica

Date: 09-2020

DOI: 10.1530/REP-20-0205

Abstract: MTT is widely used in biology as a probe for cell viability by virtue of its ability to generate deposits of insoluble formazan at sites of intense oxidoreductase activity. This response is generally held to reflect mitochondrial redox activity however, extra-mitochondrial MTT reduction has also been recorded in certain cell types. Given this background, we set out to determine the major sites of formazan deposition in mammalian spermatozoa. In the mouse, most MTT reduction took place within the extensive mitochondrial gyres, with a single minor site of formazan deposition on the sperm head. By contrast, human spermatozoa generally displayed small disorganized midpieces exhibiting moderate MTT reduction activity accompanied by a major extra-mitochondrial formazan deposit on various locations in the sperm head from the neck to the anterior acrosome. Equine spermatozoa presented a combination of these two patterns, with major formazan deposition in the mitochondria accompanied by an extra-mitochondrial formazan deposit in around 20% of cells. The functionality of human spermatozoa was positively associated with the presence of an extra-mitochondrial formazan granule. Subsequent studies indicated that this extra-mitochondrial activity was suppressed by the presence of diphenylene iodonium, zinc, 2-deoxyglucose, co-enzyme Q, an SOD mimetic and NADPH oxidase inhibitors. We conclude that the pattern of MTT reduction to formazan by spermatozoa is species specific and conveys significant information about the relative importance of mitochondrial vs extra-mitochondrial redox activity that, in turn, defines the functional qualities of these cells.

Fertilization stimulates 8-hydroxy-2'-deoxyguanosine repair and antioxidant activity to prevent mutagenesis in the embryo

Publisher: Elsevier BV

Date: 10-2015

DOI: 10.1016/J.YDBIO.2015.07.024

Abstract: Oxidative DNA damage harbored by both spermatozoa and oocytes at the time of fertilization must be repaired prior to S-phase of the first mitotic ision to reduce the risk of transversion mutations occurring in the zygote and subverting the normal patterns of cell differentiation and development. Of the characterised oxidative DNA lesions, 8-hydroxy-2'-deoxyguanosine (8OHdG) is particularly mutagenic. The current study reveals for the first time a marked acceleration of 8OHdG repair in the mouse oocyte/zygote by the base excision repair (BER) pathway following fertilization. Specifically, fertilization initiates post-translational modification to BER enzymes such as OGG1 and XRCC1, causing nuclear localisation and accelerated 8OHdG excision. Additionally, both the nuclear and mitochondrial genomes appear to benefit from increased protection against further 8OHdG formation by a fertilization-associated increase in glutathione peroxidase activity. The major limitation of the characterised 8OHdG repair system is the relatively low level of OGG1 expression in the oocyte, in contrast to the male germ line where it is the only constituent of the BER pathway. The male and female germ lines therefore collaborate in the repair of oxidative DNA damage, and oocytes are vulnerable to high levels of 8OHdG being carried into the zygote by the fertilizing spermatozoon.

Use of Titanium Dioxide To Find Phosphopeptide and Total Protein Changes During Epididymal Sperm Maturation

Publisher: American Chemical Society (ACS)

Date: 27-01-2011

DOI: 10.1021/PR1007224

Abstract: Although the overall performance of modern mass spectrometers has increased, proteomic analysis of complex s les still requires prefractionation either at the protein or peptide level to allow for in-depth analysis of normal cellular function. Here, we report a novel way to identify protein changes occurring during sperm development through the epididymis. Phosphopeptides were first enriched from either the rat caput or caudal regions of the epididymides using TiO(2), and the profiles then quantitatively compared. We show that 77 TiO(2)-enriched peptides become significantly modified in the epididymis, equating to 53 proteins. Through the use of immunoblot analysis, we confirmed that three proteins, ornithine-decarboxylase antizyme 3, heat-shock protein 90α, and testis-lipid binding protein, undergo major protein loss during epididymal passage. Many other proteins, including t-complex protein 10 and Spata18 show testis unique expression, appear to undergo phosphorylation during this same time frame. These data provide mechanistic insight into the means by which spermatozoa acquire functionality during epididymal transit.

The senescence-accelerated mouse prone 8 as a model for oxidative stress and impaired DNA repair in the male germ line

Publisher: Bioscientifica

Date: 09-2013

DOI: 10.1530/REP-13-0186

Abstract: The discovery of a truncated base excision repair pathway in human spermatozoa mediated by OGG1 has raised questions regarding the effect of mutations in critical DNA repair genes on the integrity of the paternal genome. The senescence-accelerated mouse prone 8 (SAMP8) is a mouse model containing a suite of naturally occurring mutations resulting in an accelerated senescence phenotype largely mediated by oxidative stress, which is further enhanced by a mutation in the Ogg1 gene, greatly reducing the ability of the enzyme to excise 8-hydroxy,2′-deoxyguanosine (8OHdG) adducts. An analysis of the reproductive phenotype of the SAMP8 males revealed a high level of DNA damage in caudal epididymal spermatozoa as measured by the alkaline Comet assay. Furthermore, these lesions were confirmed to be oxidative in nature, as demonstrated by significant increases in 8OHdG adduct formation in the SAMP8 testicular tissue ( P .05) as well as in mature spermatozoa ( P .001) relative to a control strain (SAMR1). Despite this high level of oxidative DNA damage in spermatozoa, reactive oxygen species generation was not elevated and motility of spermatozoa was found to be similar to that for the control strain with the exception of progressive motility, which exhibited a slight but significant decline with advancing age ( P .05). When challenged with Fenton reagents (H 2 O 2 and Fe 2 + ), the SAMP8 spermatozoa demonstrated a highly increased susceptibility to formation of 8OHdG adducts compared with the controls ( P .001). These data highlight the role of oxidative stress and OGG1-dependent base excision repair mechanisms in defining the genetic integrity of mammalian spermatozoa.

The biological significance of detergent-resistant membranes in spermatozoa

Publisher: Elsevier BV

Date: 12-2009

DOI: 10.1016/J.JRI.2009.06.258

Abstract: As mammalian spermatozoa ascend the female reproductive tract, they acquire the ability to fertilize an oocyte via a complex cascade of biophysical and biochemical changes collectively know as 'capacitation'. In virtually all species studied, capacitation is accompanied by dramatic remodeling of the surface architecture, in order to render spermatozoa competent to recognize the oocyte and initiate fertilization. Although the fundamental mechanisms that underpin the dynamic redistribution of sperm surface proteins are poorly understood, recent evidence indicates that this process may be facilitated, at least in part, by specialized membrane microdomains or lipid rafts. This notion is consistent with numerous demonstrations that lipid rafts contain a number of putative zona pellucida receptors, undergo a marked capacitation-associated lateral migration to the apical region of the sperm head, and possess the ability to selectively bind to the zona pellucida of unfertilized, but not fertilized oocytes. Accordingly, this review aims to cover the latest insights into sperm lipid raft research and considers the evidence that these microdomains serve as platforms for the assembly of key recognition molecules on the sperm surface during capacitation.

Falling sperm counts twenty years on: where are we now?

Publisher: Medknow

Date: 28-01-2013

DOI: 10.1038/AJA.2012.167

Exposure of spermatozoa to dibutyl phthalate induces abnormal embryonic development in a marine invertebrate Galeolaria caespitosa (Polychaeta: Serpulidae)

Publisher: Elsevier BV

Date: 10-2017

DOI: 10.1016/J.AQUATOX.2017.08.008

Abstract: In this study, we have investigated the impact of dibutyl phthalate (DBP) on early embryogenesis in a sessile marine invertebrate, Galeolaria caespitosa. DBP was found to induce sperm dysfunction as well as impaired and defective embryogenesis characterised by a particular pattern of abnormality. Thus, after the first cleavage, one blastomere in these abnormal embryos was able to carry out further mitoses, while the other arrested. Analysis of microtubules, chromosomes and actin filaments demonstrated that the mitotic spindles in the abnormal embryos were irregularly bent, shortened and unable to anchor to the cortex, resulting in the defective segregation of chromosomes. Within the non- iding blastomeres, karyokinesis was found to continue at a slow pace as indicated by the presence of multiple sets of abnormal mitotic spindles. However, cytokinesis had been disrupted in these arrested cells due to a failure to assemble the contractile actin ring, as a result of which one pole of the embryos remained as one large, un ided cell. DBP was found to suppress the activity of superoxide dismutase in spermatozoa and, in association with this change, DBP-treated cells experienced oxidative stress as indicated by the presence of lipid aldehydes, such as 4-hydroxynonenal (4-HNE) in the sperm acrosome and neck. Adduction of lipid aldehydes at the level of the acrosome would be expected to impede the acrosome reaction and account for the significant decrease in fertilisation rates. 4-HNE generated as a consequence of lipid peroxidation in the sperm neck resulted in alkylation of the sperm centrioles. Such paternally damaged centrioles were inherited by the embryos and disrupted cytoskeletal protein organisation during early cleavage, generating the observed abnormalities in embryonic development. This research emphasises the vulnerability of spermatozoa to oxidative damage and highlights novel potential mechanisms for reproductive toxicity involving the alkylation of subcellular structures in spermatozoa by lipid aldehydes.

Prospects for immunocontraception in feral horse population control: exploring novel targets for an equine fertility vaccine

Publisher: CSIRO Publishing

Date: 2016

DOI: 10.1071/RD14280

Abstract: Feral horses populate vast land areas and often induce significant ecological and economic damage throughout the landscape. Non-lethal population control methods are considered favourable in light of animal welfare, social and ethical considerations however, no single effective, safe and species-specific contraceptive agent is currently available for use in free-ranging wild and feral horses. This review explores aspects of equine reproductive physiology that may provide avenues for the development of specific and long-lasting immunocontraceptive vaccines and some of the novel strategies that may be employed to facilitate appropriate antigen discovery in future research. Potential antigen targets pertaining to spermatozoa, the ovary and oocyte, as well as the early conceptus and its associated factors, are reviewed in the context of their suitability for immunocontraceptive vaccine development.

Biological and clinical significance of DNA damage in the male germ line

Publisher: Wiley

Date: 05-01-2009

DOI: 10.1111/J.1365-2605.2008.00943.X

Abstract: DNA damage is a common feature of human spermatozoa with purported links to poor rates of conception, impaired embryonic development, an increased incidence of miscarriage and the appearance of various kinds of morbidity in the offspring including childhood cancer. However, difficulties in interpretation arise, because these associations are not consistently observed across all data sets. Such inconsistency reflects the inherent complexity of the reproductive process, large variations in s le size, differences in patient selection, inadequate study design as well as inter-in idual differences in the type of DNA damage being detected and the effectiveness of repair mechanisms in the oocyte. This review considers the type, source and measurement of DNA damage in human spermatozoa. It also addresses the clinical utility of the information generated in such studies, and highlights areas where further research is needed to bridge the gap between an intriguing biological phenomenon and the evidence-based clinical management of male patients characterized by high levels of DNA damage in their spermatozoa.

Properties of intact and univalent (Fab) antibodies raised against isolated, solubilized, mouse zonae pellucidae

Publisher: Bioscientifica

Date: 09-1982

DOI: 10.1530/JRF.0.0660327

Abstract: Intact and univalent antibodies were prepared against mechanically isolated mouse zonae pellucidae solubilized in a variety of ways (heat, low pH, SDS, urea and trypsin). The antisera bound avidly and specifically to solubilized iodinated zona antigens and the intact zona structure. When the concentrations of immunoreactive Fab material in the intact and univalent antibody preparations were equalized and compared for their ability to block the sperm-binding stage of fertilization, only the intact gamma-globulin preparations possessed antifertility activity. These results indicate that antibodies raised against intact solubilized zonae pellucidae block fertilization by cross-linking antigens on the outer zona surface, thereby indirectly masking the sperm receptor sites. The integrity of these surface components did not appear to be affected by solubilization procedures that disrupt non-covalent bonds (heating, low pH, SDS and urea) although they did appear to be adversely affected by trypsin treatment. None of the antisera tested contained antibodies directed against the sperm receptor site indicating that these critical components lack immunogenicity.

New methods for the regulation of implantation

Publisher: Elsevier BV

Date: 09-1977

DOI: 10.1016/0010-7824(77)90022-1

Phosphoinositide 3-kinase signalling pathway involvement in a truncated apoptotic cascade associated with motility loss and oxidative DNA damage in human spermatozoa

Publisher: Portland Press Ltd.

Date: 27-05-2011

DOI: 10.1042/BJ20110114

Abstract: Human spermatozoa are characterized by poor functionality and abundant DNA damage that collude to generate the high incidences of male infertility and miscarriage seen in our species. Although apoptosis has been suggested as a possible cause of poor sperm quality, the ability of these cells to enter an apoptotic state and the factors that might trigger such an event are unresolved. In the present study we provide evidence that the commitment of these cells to apoptosis is negatively regulated by PI3K (phosphoinositide 3-kinase)/AKT. If PI3K activity is inhibited, then spermatozoa default to an apoptotic cascade characterized by rapid motility loss, mitochondrial reactive oxygen species generation, caspase activation in the cytosol, annexin V binding to the cell surface, cytoplasmic vacuolization and oxidative DNA damage. However, the specialized physical architecture of spermatozoa subsequently prevents endonucleases activated during this process from penetrating the sperm nucleus and cleaving the DNA. As a result, DNA fragmentation does not occur as a direct result of apoptosis in spermatozoa as it does in somatic cells, even though oxidative DNA adducts can clearly be detected. We propose that this unusual truncated apoptotic cascade prepares spermatozoa for silent phagocytosis within the female tract and prevents DNA-damaged spermatozoa from participating in fertilization.

Contraceptive vaccine development

Publisher: Elsevier BV

Date: 07-1990

DOI: 10.1016/0014-2999(90)91256-B

The Search for a Topical Dual Action Spermicide/Microbicide

Publisher: Bentham Science Publishers Ltd.

Date: 03-2007

DOI: 10.2174/092986707780090972

Abstract: There is an urgent clinical need to research novel methods of fertility control that are also protective against sexually transmitted diseases (STDs) such as the human immunodeficiency virus (HIV) or Chlamydia. The most obvious way to generate such a dual-purpose contraceptive method would be to develop safe, effective spermicides that were also active against a wide range of pathogenic organisms. The currently available formulations such as nonoxynol-9, gramicidin and benzalkonium chloride are effective spermicides but are toxic to the vaginal epithelium and do not provide protection against STDs. Over 60 agents are in clinical trials as potentially safer topical spermicides and/or microbicides. Compounds that have reached this stage of development include acid buffers, detergents, dendrimers, non-nucleoside reverse transcriptase inhibitors and anionic polymers. In addition, a number of potential spermicides/microbicides are the subject of preclinical investigation, including beta-cyclodextrin, cyanovirin, porphyrins, cyclotriazadisulfonamides, dermaseptins, short-interfering RNA (siRNA) and HIV antibodies. The chemical principles underlying these disparate approaches and potential avenues for future investigation are discussed.

Protein patterns of human uterine flushings collected at various stages of the menstrual cycle

Publisher: Bioscientifica

Date: 07-1978

DOI: 10.1530/JRF.0.0530343

Abstract: Uterine flushings and plasma collected from normal, parous women at various stages of the menstrual cycle were subjected to gel electrophoresis. The protein profiles of the flushings differed in many instances from the plasma pattern by the presence of between one and eleven non-plasma proteins. The distribution of the major non-plasma proteins during the menstrual cycle was not statistically significant but that of a pretransferrin, observed in uterine flushings and in peritoneal fluid, was significant. However, the appearance of this protein band could not be related to a particular phase of the cycle, and hence to a specific hormonal condition.

A survey of semen donation: Phase II the view of the donors

Publisher: Oxford University Press (OUP)

Date: 10-1995

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A135786

Abstract: Children are particularly impressionable and at risk during a global public health crisis, making it essential to examine their unique perspectives. To hear and understand sub-Saharan African children's experiences with the COVID-19 pandemic, we conducted an exploratory qualitative analysis based on interviews with 51 children, ages 9 to 13, from Nigeria, Tanzania, and Sierra Leone. Applying the organization of Bronfenbrenner's ecological systems theory, we reveal how COVID-19 affected children's daily lives and domestic challenges, schooling and neighborhood issues, media use (and its relationship to knowledge and fear of the disease), perceptions of the country, and government response, and thoughts of religion and hope. Children's responses dif-fered greatly, but patterns emerged across sex, age, household size, religion, and country. This study offers guidance and recommendations for meeting the needs of children, especially in times of crisis.

Relative ability of modified versions of hamster oocyte penetration test, incorporating hyperosmotic medium or the ionophore A23187, to predict IVF outcome

Publisher: Oxford University Press (OUP)

Date: 04-1987

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A136518

Abstract: This study was designed to assess the relationship between IVF outcome and the results obtained with two modified versions of the zona-free hamster oocyte penetration test in which the spermatozoa were pre-incubated with either hyperosmotic medium or the alent cation ionophore A23187. When the former system was used, a poor correlation with IVF outcome was observed. S les screened prior to IVF exhibited a 60% false negative rate (failed penetration test, successful IVF), while for those assessed concurrently with IVF, the equivalent figure was 85.7%. Addition of A23187 optimized the penetration system giving higher levels of sperm--oocyte fusion and a more accurate prediction of the capacity of the spermatozoa to fertilize human ova in vitro. With this system the false negative rate was 4.3% for screened s les and 0% for those assessed simultaneously with IVF. These results suggest that the A23187-enhanced system may be of value as a screening criterion for IVF.

Superoxide dismutase in human sperm suspensions: Relationship with cellular composition, oxidative stress, and sperm function

Publisher: Elsevier BV

Date: 1996

DOI: 10.1016/0891-5849(96)00119-0

Abstract: Sensitive techniques have been developed for monitoring superoxide dismutase (SOD) activities in human sperm preparations. In contradiction to the protective role normally assigned to SOD, populations of defective spermatozoa recovered from the low density region of Percoll gradients were found to have three times more SOD than functionally competent preparations pelleting in high density Percoll. SOD activity was negatively correlated with the movement characteristics of human spermatozoa and their capacity for oocyte fusion, and positively associated with the induction of peroxidative damage. SOD activity was also highly correlated with other markers of the cytoplasmic space, creatine kinase (CK), and glucose-6-phosphate dehydrogenase (G-6-PDH). We conclude that while SOD may play a physiological role in maintaining a balance between O2.- and H2O2, high levels of this enzyme are associated with impaired sperm function because (a) the human spermatozoon is highly susceptible to the cytotoxic effects of H2O2, (b) O2.- is an important mediator of normal sperm function, and (c) high SOD activities reflect errors in spermatogenesis associated with germ cell exfoliation and the retention of excess residual cytoplasm by the spermatozoa.

A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtai

Publisher: Bioscientifica

Date: 2004

DOI: 10.1530/REP.1.00073

Abstract: The phosphorylation of tyrosine residues in cellular proteins is a major signal transduction event during sperm capacitation. In this study protein phosphorylation was monitored using a fluorescein isothiocyanate (FITC)-labeled antiphosphotyrosine monoclonal antibody and a flow cytometric procedure optimized for sperm. Using this technique, the correlation between tyrosine phosphorylation and sperm capacitation was examined in two marsupial species, the brushtail possum and the tammar wallaby and compared with that of ram spermatozoa. The levels of tyrosine phosphorylation in sperm from all three species were increased by the addition of cyclic AMP (cAMP) and vandate, a phosphotyrosine phosphatase inhibitor and were decreased by the addition of the phosphotyrosine kinase inhibitor, staurosporine. Oviductal conditioned media (CM) induced a progressive increase in tyrosine phosphorylation in both marsupial species and also induced morphological transition from a streamlined to a ‘T’-shape configuration in brushtail possum spermatozoa but not in tammar wallaby spermatozoa. Transition to the ‘T’-shape orientation associated with capacitation in marsupial spermatozoa was observed by 2 h of incubation in both species when tyrosine phosphorylation was increased by higher levels of cAMP i.e. 5 mM dibutyryl cAMP plus 3 mM pentoxyphylline. Thus the tyrosine phosphorylation trigger with CM may differ in these two marsupial species. Ram sperm tyrosine phosphorylation could be increased by addition of lower levels of cAMP (1 mM). These results support the finding that tyrosine phosphorylation is associated with sperm capacitation in marsupials. Similar results were obtained by using SDS PAGE/Western blot analysis of tyrosine phosphorylation in the brushtail possum spermatozoa. The specificity, efficiency and sensitivity of the procedure described here make it applicable for routine assessment of capacitation in large numbers of s les and in other species.

Cloning and characterization of the human sperm receptor ligand ZP3: Evidence for a second polymorphic allele with a different frequency in the caucasian and Japanese populations

Publisher: Elsevier BV

Date: 12-1992

DOI: 10.1016/S0888-7543(05)80130-2

Abstract: The human ZP3 gene, encoding the glycoprotein responsible for sperm-egg recognition, has been cloned using mouse ZP3 DNA as a probe. Genomic and cDNA cloning revealed eight exons, spread over 18 kb, encoding a protein of 424 amino acids with a 67% homology to mouse and hamster ZP3. Southern blotting, gene cloning, and sequence analysis were used to show that ZP3 is not a single-copy gene and that the human genome contains a second polymorphic locus which, due to an extra G residue in exon 8, has the potential to encode a truncated protein of 372 amino acids. Direct sequence analysis of polymerase chain reaction- lified exon 8 DNA of 56 in iduals of various human populations revealed three different sequence patterns: one containing only ZP3-424-coding sequences and two containing ZP3-424- and ZP3-372-coding DNA. The distribution of these three sequence patterns is significantly different between the Caucasian and Japanese populations, as indicated by ZP3-372 allele frequencies of 69 and 21%, respectively. Isolation of ZP3-424 and ZP3-372 cDNAs suggests that both loci represent functional transcription units. Therefore, it is hypothesized that throughout the human population during oogenesis ZP3 is translated from mRNAs derived from two to four transcription units. Provided that ZP3-372 mRNA is translated in vivo, corresponding differences in ZP3-372 protein levels might have an impact on human zona pellucida composition.

The spermostatic and microbicidal actions of quinones and maleimides: Toward a dual-purpose contraceptive agent

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 31-03-2009

DOI: 10.1124/MOL.108.053645

Abstract: There is an urgent need to develop safe, effective, dual-purpose contraceptive agents that combine the prevention of pregnancy with protection against sexually transmitted diseases. Here we report the identification of a group of compounds that on contact with human spermatozoa induce a state of "spermostasis," characterized by the extremely rapid inhibition of sperm movement without compromising cell viability. These spermostatic agents were more active and significantly less toxic than the reagent in current clinical use, nonoxynol 9, giving therapeutic indices (ratio of spermostatic to cytotoxic activity) that were orders of magnitude greater than this traditional spermicide. Although certain compounds could trigger reactive oxygen species generation by spermatozoa, this activity was not correlated with spermostasis. Rather, the latter was associated with alkylation of two major sperm tail proteins that were identified as A Kinase-Anchoring Proteins (AKAP3 and AKAP4) by mass spectrometry. As a consequence of disrupted AKAP function, the abilities of cAMP to drive protein kinase A-dependent activities in the sperm tail, such as the activation of SRC and the consequent stimulation of tyrosine phosphorylation, were suppressed. Furthermore, analysis of microbicidal activity using Chlamydia muridarum revealed powerful inhibitory effects at the same low micromolar doses that suppressed sperm movement. In this case, the microbicidal action was associated with alkylation of Major Outer Membrane Protein (MOMP), a major chlamydial membrane protein. Taken together, these results have identified for the first time a novel set of cellular targets and chemical principles capable of providing simultaneous defense against both fertility and the spread of sexually transmitted disease.

Human spermatozoa: revelations on the road to conception

Publisher: Faculty Opinions Ltd

Date: 10-2013

DOI: 10.12703/P5-39

Predicting the fertilizing potential of human sperm suspensions in vitro: Importance of sperm morphology and leukocyte contamination

Publisher: Elsevier BV

Date: 06-1995

DOI: 10.1016/S0015-0282(16)57614-6

Abstract: To determine the relationships between sperm function tests and fertilization of human oocytes in vitro. Analysis of infertile patients undergoing IVF therapy. Diagnostic Andrology Laboratory and Assisted Conception Service. Forty-one couples who underwent IVF-ET therapy were studied. None. The ability of human spermatozoa to achieve fertilization in vitro was examined in relation to numerous criteria of semen quality, including the conventional semen profile, the computer-aided assessment of sperm movement, ionophore-induced acrosome reaction, acridine orange staining, sperm morphology, and chemiluminescent signals induced by phorbol ester and N-formyl-methionyl-leucyl-phenylalanine (FMLP). Significant correlations were observed between fertilization rates and several attributes of the sperm preparations, including elements of sperm function (acrosome reaction), movement (percentage motile, hyperactivation, the litude of lateral sperm head displacement), morphology (normal morphology, midpiece defects, multiple anomalies index), nuclear normality (acridine orange staining), and reactive oxygen species generation (chemiluminescence induced by phorbol ester and FMLP). In a stepwise multiple regression analysis, an accurate prediction of fertilization rates was obtained using a multiple regression equation incorporating six variables of which sperm morphology and FMLP-induced chemiluminescence were the most informative. A set of criteria have been identified that accurately predict the fertilizing potential of human sperm suspensions in vitro and that place particular emphasis on sperm morphology and the degree of leukocyte contamination.

The impact of oxidative stress on chaperone-mediated human sperm-egg interaction

Publisher: Oxford University Press (OUP)

Date: 07-09-2015

DOI: 10.1093/HUMREP/DEV214

Abstract: How does oxidative stress impact upon human sperm-egg interaction and in particular the formation of zona pellucida-receptor complexes on the sperm surface? Oxidative stress during human sperm capacitation resulted in the chemical alkylation of the molecular chaperone heat shock protein A2 (HSPA2), a concomitant reduction in surface expression of the zona pellucida-receptor arylsulphatase A (ARSA) and a severe loss of zona pellucida binding ability. An inability to bind to the zona pellucida is commonly encountered in the defective spermatozoa generated by male infertility patients however, the underlying mechanisms remain unresolved. Recent studies have revealed that zona pellucida binding is mediated by molecular chaperones, particularly HSPA2, that facilitate the formation of multimeric zona pellucida-receptor complexes on the surface of mammalian spermatozoa during capacitation. Spermatozoa were collected from healthy normozoospermic donors (n = 15). Low levels of oxidative stress were induced in populations of non-capacitated spermatozoa by a 1 h treatment with 4-hydroxynonenal (4HNE) or hydrogen peroxide (H2O2) and then these insults were removed and cells were capacitated for 3 h. Motility, membrane fluidity, protein tyrosine phosphorylation and lipid raft distribution were evaluated after sperm capacitation to determine the impact of oxidative stress on this process. The surface expression of ARSA and sperm adhesion molecule 1 (SPAM1) was observed using fluorescence microscopy, and the ability of treated cells to interact with homologous human zonae pellucidae was assessed through gamete co-incubation. Proximity ligation was used to evaluate the state of the HSPA2-laden zona pellucida-receptor complex and an immunoprecipitation approach was taken to establish the chemical alkylation of HSPA2 by the cytotoxic lipid aldehyde 4HNE. The validity of these findings was then tested through treatment of oxidatively stressed cells with the nucleophile penicillamine in order to scavenge lipid aldehydes and limit their ability to interact with HSPA2. All experiments were performed on s les pooled from two or more donors per replicate, with a minimum of three replicates. The oxidative treatments employed in this study did not influence sperm motility or capacitation-associated changes in membrane fluidity, tyrosine phosphorylation and lipid raft redistribution. However, they did significantly impair zona pellucida binding compared with the capacitated control (P < 0.01). The reduction in zona pellucida binding was associated with the impaired surface expression (P < 0.02) of a zona pellucida-receptor complex comprising HSPA2, SPAM1 and ARSA. Proximity ligation and immunoprecipitation assays demonstrated that impaired zona pellucida binding was, in turn, associated with the chemical alkylation of HSPA2 with 4HNE and the concomitant disruption of this zona pellucida-receptor complex. The use of penicillamine enabled a partial recovery of ARSA surface expression and zona pellucida adherence in H2O2-treated cells. These data suggest that the ability of low levels of oxidative stress to disrupt sperm function is mediated by the production of lipid aldehydes as a consequence of lipid peroxidation and their adduction to the molecular chaperone HSPA2 that is responsible for co-ordinating the assembly of functional zona pellucida-receptor complexes during sperm capacitation. While these results extend only to one particular zona pellucida-receptor complex, we postulate that oxidative stress may more broadly impact upon sperm surface architecture. In this light, further study is required to assess the impact of oxidative stress on additional HSPA2-laden protein complexes. These findings link low levels of oxidative stress to a severe loss of sperm function. In doing so, this work suggests a potential cause of male infertility pertaining to a loss of zona pellucida recognition ability and will contribute to the more accurate diagnosis and treatment of such conditions.

Structure of the O‐linked carbohydrate chains of porcine zona pellucida glycoproteins

Publisher: Wiley

Date: 04-1994

DOI: 10.1111/J.1432-1033.1994.TB18762.X

Abstract: The N-linked carbohydrate chains of porcine zona pellucida glycoproteins were released by digestion with peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase F and subsequently separated from the O-glycoprotein by gel-permeation chromatography on Bio-Gel P-100. The O-linked carbohydrate chains were released from the O-glycoprotein by alkaline borohydride treatment. Fractionation of the extremely heterogeneous mixture of O-linked oligosaccharide alditols was achieved by a combination of chromatographic techniques comprising gel-permeation chromatography on Bio-Gel P-4 and P-6, anion-exchange FPLC on Mono Q, and high-pH anion-exchange chromatography on CarboPac PA-1. The primary structures of 32 O-glycans were determined by one- and two-dimensional 1H-NMR spectroscopy. The major part of the analyzed compounds contain a combination of the structural elements Gal beta 1-4GlcNAc beta 1-3Gal beta 1-3GalNAc-ol, Gal beta 1-4(6SO4-)GlcNAc, and alpha 2-3-linked Neu5Gc or Neu5Ac. This series of compounds has the following structure, where n = 0 to > 6: [Neu5Gc/Ac alpha2-3]0-1[Gal beta 1-4(6SO4-)GlcNAc beta 1-3]nGal beta 1-4GlcNAc beta 1-3Gal Beta 1-3GalNAc-ol. In addition, smaller compounds were identified in which the Gal beta 1-3GalNAc-ol core is substituted by Neu5Gc/Ac alpha 2-6-linked to GalNAc-ol and/or Neu5Gc/Ac alpha 2-3-linked to Gal. Furthermore, oligosaccharides were obtained in which the distribution of 6-O-sulfated GlcNAc residues differs from that in the above-mentioned general structure, and a small portion of the oligosaccharides has the GlcNAc beta 1-3GalNAc-ol core structure. Analysis of the endo-beta-galactosidase digests of pools of N- and O-glycans indicated that the two types of oligosaccharides contain qualitatively similar poly(N-acetyllactosamine) chains. In the case of the N-linked carbohydrate chains, multiple branching of the core structures occurs, resulting in an even larger heterogeneity than observed for the O-linked carbohydrate chains.

Detection of Ehrlichia platys in dogs in Australia

Publisher: Wiley

Date: 08-2001

DOI: 10.1111/J.1751-0813.2001.TB10747.X

Abstract: To describe the detection of Ehrlichia platys in free-roaming dogs in Central Australia. Blood s les were collected from four dogs and examined for bacterial 16S ribosomal DNA using Polymerase Chain Reaction (PCR)-based assays. The three positive s les obtained were then sequenced and identification of the PCR product carried out. As a result of all three s les being identical to or closely related to part of the 16S rRNA gene of E. platys, blood s les were subsequently obtained from a further 24 dogs. These s les were screened using a PCR-assay to determine the presence of Ehrlichia DNA using genus-specific primers. The positive s les obtained from the screening process were then subjected to a further PCR-assay using E. platys specific primers. Of 28 dogs s led, Ehrlichia DNA was detected in the blood of 13 dogs. Sequencing of the licons obtained indicated a high homology with the 16S rRNA gene for E. platys. When the E. platys-specific PCR was performed for 10 of those dogs, the 678 bp product obtained from the PCR lification confirmed the identification as part of the 16S rRNA gene of E. platys in all 10 dogs. This study reports for the first time Ehrlichia carriage by dogs in Australia. It also indicates the usefulness of the PCR technique in rapidly and accurately identifying diseases that are otherwise difficult to detect. By using universal primers directed against bacterial 16S ribosomal DNA and sequencing analysis, the detection of potentially pathogenic Ehrlichia organisms that had not previously been found in Australia has been made possible.

Recent Developments in Stallion Semen Preservation

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1016/J.JEVS.2016.06.006

Identification of Cytochrome P450-Reductase as the Enzyme Responsible for NADPH-Dependent Lucigenin and Tetrazolium Salt Reduction in Rat Epididymal Sperm Preparations1

Publisher: Oxford University Press (OUP)

Date: 07-2004

DOI: 10.1095/BIOLREPROD.104.027748

The predictability of subnormal penetrating capacity of sperm in cases of unexplained infertility

Publisher: Wiley

Date: 06-1983

DOI: 10.1111/J.1365-2605.1983.TB00534.X

Abstract: Multivariate discriminant analysis has been used to determine the predictability of sub-normal penetrating capacity in cases of unexplained infertility. The product of this analysis was the identification of 7 discriminating variables, all of which described various aspects of the post-capacitation movement characteristics exhibited by the spermatozoa, omitting all reference to the conventional parameters of semen quality. On the basis of these discriminating variables, 90.9% of s les exhibiting penetration rates within the normal range were correctly predicted to be functionally competent. The same variables correctly identified only 60% of s les with impaired penetrating capacity, indicating that in the remaining 40%, defects are present in the spermatozoa which are not reflected in their motility patterns.

Polyunsaturated Fatty Acids in Male and Female Reproduction1

Publisher: Oxford University Press (OUP)

Date: 08-2007

DOI: 10.1095/BIOLREPROD.107.060558

Abstract: In Westernized societies, average consumption of n-6 polyunsaturated fatty acids (PUFAs) far exceeds nutritional requirements. The ratio of n-6 to n-3 PUFAs is generally >10:1 whereas on a primitive human diet it was closer to 1:1. Diets fed to intensively farmed livestock have followed a similar trend. Both n-6 and n-3 PUFAs can influence reproductive processes through a variety of mechanisms. They provide the precursors for prostaglandin synthesis and can modulate the expression patterns of many key enzymes involved in both prostaglandin and steroid metabolism. They are essential components of all cell membranes. The proportions of different PUFAs in tissues of the reproductive tract reflect dietary consumption. PUFA supplements (particularly n-3 PUFAs in fish oil) are promoted for general health reasons. Fish oils may also benefit fertility in cattle and reduce the risk of preterm labor in women, but in both cases current evidence to support this is inconclusive. Gamma-linolenic acid containing oils can alter the types of prostaglandins produced by cells in vitro, but published data to support claims relating to effects on reproductive health are lacking. Spermatozoa require a high PUFA content to provide the plasma membrane with the fluidity essential at fertilization. However, this makes spermatozoa particularly vulnerable to attack by reactive oxygen species, and lifestyle factors promoting oxidative stress have clear associations with reduced fertility. Adequately powered trials that control for the ratios of different PUFAs consumed are required to determine the extent to which this aspect of our diets does influence our fertility.

DNA damage in testicular germ cells and spermatozoa. When and how is it induced? How should we measure it? What does it mean?

Publisher: Wiley

Date: 16-01-2023

DOI: 10.1111/ANDR.13375

Abstract: This review surveys the causes and consequences of DNA damage in the male germ line from spermatogonial stem cells to fully differentiated spermatozoa. Within the stem cell population, DNA integrity is well maintained as a result of excellent DNA surveillance and repair however, a progressive increase in background mutation rates does occur with paternal age possibly as a result of aberrant DNA repair as well as replication error. Once a germ cell has committed to spermatogenesis, it responds to genetic damage via a range of DNA repair pathways or, if this process fails, by the induction of apoptosis. When fully‐differentiated spermatozoa are stressed, they also activate a truncated intrinsic apoptotic pathway which results in the activation of nucleases in the mitochondria and cytoplasm however, the physical architecture of these cells prevents these enzymes from translocating to the nucleus to induce DNA fragmentation. Conversely, hydrogen peroxide released from the sperm midpiece during apoptosis is able to penetrate the nucleus and induce DNA damage. The base excision repair pathway responds to such damage by cleaving oxidized bases from the DNA, leaving abasic sites that are alkali‐labile and readily detected with the comet assay. As levels of oxidative stress increase and these cells enter the perimortem, topoisomerase integrated into the sperm chromatin becomes activated by SUMOylation. Such activation may initially facilitate DNA repair by reannealing double strand breaks but ultimately prepares the DNA for destruction by nucleases released from the male reproductive tract. The abasic sites and oxidized base lesions found in live spermatozoa are mutagenic and may increase the mutational load carried by the offspring, particularly in the context of assisted conception. A variety of strategies are described for managing patients expressing high levels of DNA damage in their spermatozoa, to reduce the risks such lesions might pose to offspring health.

Analysis of chaperone proteins associated with human spermatozoa during capacitation

Publisher: Oxford University Press (OUP)

Date: 26-06-2007

DOI: 10.1093/MOLEHR/GAM043

Abstract: Mammalian spermatozoa must undergo a post-ejaculatory period of maturation, known as capacitation, before they can engage in the process of fertilization. Studies in the mouse have established that capacitation facilitates sperm-zona recognition via mechanisms that involve the appearance of tyrosine phosphorylated chaperone proteins on the sperm surface overlying the acrosome, the site of sperm-zona recognition. In this study, we examined whether a similar relationship existed between the tyrosine phosphorylation events associated with capacitation and sperm-zona interaction in human spermatozoa. These studies confirmed that capacitation is associated with an increase in both sperm-zona binding and an increase in tyrosine phosphorylation over the sperm tail. However, we could not detect the surface expression of phosphotyrosine residues over the sperm head, as observed with murine spermatozoa. Moreover, although we could clearly detect a number of chaperone proteins in human spermatozoa including HSPE1, DNAJB1, HSPD1, HSPA1A, HSPCA, HSPH1, HSPA5 and TRA1, none of these molecules were expressed on the sperm surface. On the basis of these results, it is unlikely that these proteins play an active role in the remodeling of the sperm surface during capacitation. We conclude that strong species-specific differences exist in the molecular mechanisms that drive sperm-egg recognition and that alternative, chaperone-independent, mechanisms must underpin sperm-zona interaction in the human.

The role of proteomics in understanding sperm cell biology

Publisher: Wiley

Date: 30-12-2007

DOI: 10.1111/J.1365-2605.2007.00851.X

Abstract: The advent of enhanced methods for the pre-fractionation of proteins, improved high speed, high resolution, high sensitivity mass spectrometry hardware and superior informatics/bioformatics software is heralding a new era in our capacity to analyse the proteomic composition of human spermatozoa. Parallel improvements in our capacity to compare proteomic profiles from spermatozoa in different functional states (immature vs. mature, uncapacitated vs. capacitated, normal vs. defective) is also helping to define which specific elements of the proteome are of functional significance. The ultimate aim of such studies will be to integrate the proteome with the sperm metabolome so that we shall not only understand the cascade of post-translational modifications (e.g., phosphorylation, glycosylation, proteolytic cleavage) involved in generating a functional spermatozoon but also determine how these physiological changes are brought about. This fundamental information will then create a basis for identifying key points in the post-testicular maturation of spermatozoa that might be targeted for contraceptive purposes or implicated in the defective sperm function observed in a significant proportion of infertile males.

Role of environmental factors in timing the onset and progression of puberty

Publisher: Wiley

Date: 02-2006

DOI: 10.1111/J.1365-2605.2005.00680.X

Identification of the molecular chaperone, heat shock protein 1 (chaperonin 10), in the reproductive tract and in capacitating spermatozoa in the male mouse

Publisher: Oxford University Press (OUP)

Date: 06-2008

DOI: 10.1095/BIOLREPROD.107.066860

Abstract: Mammalian spermatozoa must undergo epididymal maturation in the male reproductive tract and capacitation in the female tract before acquiring the ability to fertilize an oocyte. Previous studies from our laboratory have demonstrated a causal relationship between capacitation-associated surface phosphotyrosine expression and the ability of mouse spermatozoa to recognize the oocyte and engage in sperm-zona pellucida interaction. Our previous analyses of the surface phosphoproteome of capacitated murine spermatozoa identified two molecular chaperones, heat shock protein (HSP) D1 and HSP90B1, with well-characterized roles in protein folding and the assemblage of multimeric protein complexes. The expression of these chaperones was restricted to the rostral aspect of the sperm head, in an ideal position to mediate sperm-zona pellucida interaction. Herein, we report the characterization of an additional chaperone in this location, HSPE1 (chaperonin 10 HSP10). This chaperone was identified using a coimmunoprecipitation strategy employing HSPD1 as bait. The putative interaction between HSPE1 and HSPD1 was supported by reciprocal immunoprecipitation and colocalization studies, which demonstrated the coordinated appearance of both proteins on the surface of the sperm head during capacitation. However, the surface exposure of the protein was lost upon induction of acrosomal exocytosis, as would be expected of a protein potentially involved in sperm-zona pellucida interaction. Collectively, these data invite speculation that a number of molecular chaperones are involved in modification of the sperm surface during capacitation to render these cells functionally competent to engage the process of fertilization.

Not every sperm is sacred; a perspective on male infertility

Publisher: Oxford University Press (OUP)

Date: 13-03-2018

DOI: 10.1093/MOLEHR/GAY010

Abstract: This article is a personal perspective on male infertility, a condition that is not only extremely prevalent but also a major reason for couples to resort to ART. The introduction of ICSI as a form of facilitated fertilization had a revolutionary impact on our capacity to treat cases of male infertility associated with severely compromised semen quality. However, the widespread use of this technique is also thought to pose risks in terms of the incidence of miscarriage, the health and wellbeing of the offspring and perpetuation of the infertile phenotype into future generations. Furthermore, the advent of ICSI curtailed intellectual interest in the underlying aetiology of male infertility or the development of non-invasive therapeutic strategies that target the male patient rather than the physical deployment of his gametes. As a consequence, progress on elucidating the pathological mechanisms responsible for male infertility has been extremely slow. Genetic and/or epigenetic defects are certainly involved in many cases and may involve mutations/splicing defects affecting the integrity of the testicular RNA profile, as well as the overall kinetics of the transcription process. In addition, spermatogenesis is disrupted by a variety of factors (age, smoking, obesity) many of which are thought to influence fertility and the integrity of sperm DNA through the creation of oxidative stress. Determining the relative contributions of oxidative stress and genetic/epigenetic mutations to the aetiology of male infertility will be a major focus for future research in this important but neglected area.

Oxidative stress, spermatozoa and leukocytic infiltration: Relationships forged by the opposing forces of microbial invasion and the search for perfection

Publisher: Elsevier BV

Date: 11-2013

DOI: 10.1016/J.JRI.2013.06.005

Abstract: This review addresses the complex relationships that exist between spermatozoa and the immune system and highlights the role of oxidative stress in regulating the direction and functional relevance of these interactions. Spermatozoa are potentially antigenic however, in the testes and epididymis these cells are sequestered behind physical barriers and benefit from a tolerogenic state generated through the mediation of indoleamine dioxygenase. In the female there are no such barriers however, inseminated spermatozoa are protected by the concomitant presence of seminal plasma. The latter possesses immunosuppressive properties, a powerful array of antioxidants and cytokines that modulate the immunological response to semen deposition. Subsequent to insemination, leukocytic infiltration of the female tract occurs to facilitate the removal of millions of residual moribund and senescent spermatozoa, while allowing the most competent cells to ascend to the site of fertilization. The post-insemination phagocytosis of non-viable spermatozoa is 'silent' in the sense that no reactive oxygen species (ROS) or pro-inflammatory cytokines are generated. The silent phagocytosis of senescent spermatozoa is a response to markers, such as phosphatidylserine, which are expressed on the surface of spermatozoa as they engage in the intrinsic apoptotic cascade. By contrast, infection can bring fully activated leukocytes into the male reproductive tract that are actively generating ROS and releasing pro-inflammatory cytokines. Such free-radical-generating leukocytes have the potential to seriously damage the functionality of spermatozoa as well as the integrity of their DNA, particularly in vitro, when these cells are devoid of the antioxidant protection afforded by seminal plasma.

Evaluation of the Contraceptive Potential of Recombinant Human ZP3 and Human ZP3 Peptides in a Primate Model: Their Safety and Efficacy

Publisher: Wiley

Date: 09-1998

DOI: 10.1111/J.1600-0897.1998.TB00413.X

Abstract: The unique recognition events that result in the avid binding of mammalian spermatozoa to the surface of the zona pellucida (ZP) are being exploited in the development of contraceptive vaccines. In this study, the safety and efficacy of a vaccination strategy based on the induction of active immunity against purified, glycosylated, recombinant human ZP3 (rhZP3) has been evaluated in a primate model, Callithrix jacchus. Long-term infertility was established after immunization with rhZP3 and the resulting immune sera reacted with rhZP3 on an enzyme-linked immunosorbent assay (ELISA) and immunolocalized exclusively to the outer surface of native ZP on marmoset ovarian sections. However, this contraceptive effect was inevitably associated with the eventual appearance of an ovarian pathology characterized by a depletion of primordial follicles. In an attempt to circumvent this side effect, human ZP3 (hZP3) was epitope mapped and four continuous, immunodominant B-cell epitopes (hZP3(45-64), hZP3(93-110), hZP3(172-190) and hZP3(341-360) were evaluated for contraceptive efficacy in vivo. Using peptide-tetanus toxoid (TT) conjugates to enhance immunogenicity, antipeptide antibodies were raised against these immunogens, which also cross-reacted with rhZP3 on ELISA. In addition, antibodies against hZP3(45-64) and hZP3(172-190) recognized native ZP on marmoset ovarian sections when a microwave technique was used to enhance epitope presentation. No ovarian pathology was observed after the long-term administration of these peptide immunogens, and fertility was suppressed when compared with TT controls but could not be correlated to the antibody titer. Clearly, further research is required to identify optimal B-cell epitopes that will reliably induce infertility, free from any ovarian pathology.

Capacitation in the presence of methyl-β-cyclodextrin results in enhanced zona pellucida-binding ability of stallion spermatozoa

Publisher: Bioscientifica

Date: 02-2014

DOI: 10.1530/REP-13-0393

Abstract: While IVF has been widely successful in many domesticated species, the development of a robust IVF system for the horse remains an elusive and highly valued goal. A major impediment to the development of equine IVF is the fact that optimised conditions for the capacitation of equine spermatozoa are yet to be developed. Conversely, it is known that stallion spermatozoa are particularly susceptible to damage arising as a consequence of capacitation-like changes induced prematurely in response to semen handling and transport conditions. To address these limitations, this study sought to develop an effective system to both suppress and promote the in vitro capacitation of stallion spermatozoa. Our data indicated that the latter could be achieved in a bicarbonate-rich medium supplemented with a phosphodiesterase inhibitor, a cyclic AMP analogue, and methyl-β-cyclodextrin, an efficient cholesterol-withdrawing agent. The populations of spermatozoa generated under these conditions displayed a number of hallmarks of capacitation, including elevated levels of tyrosine phosphorylation, a reorganisation of the plasma membrane leading to lipid raft coalescence in the peri-acrosomal region of the sperm head, and a dramatic increase in their ability to interact with heterologous bovine zona pellucida (ZP) and undergo agonist-induced acrosomal exocytosis. Furthermore, this functional transformation was effectively suppressed in media devoid of bicarbonate. Collectively, these results highlight the importance of efficient cholesterol removal in priming stallion spermatozoa for ZP binding in vitro .

Non-surgical sterilisation methods may offer a sustainable solution to feral horse (Equus caballus) overpopulation

Publisher: CSIRO Publishing

Date: 2017

DOI: 10.1071/RD16200

Abstract: Feral horses are a significant pest species in many parts of the world, contributing to land erosion, weed dispersal and the loss of native flora and fauna. There is an urgent need to modify feral horse management strategies to achieve public acceptance and long-term population control. One way to achieve this is by using non-surgical methods of sterilisation, which are suitable in the context of this mobile and long-lived species. In this review we consider the benefits of implementing novel mechanisms designed to elicit a state of permanent sterility (including redox cycling to generate oxidative stress in the gonad, random peptide phage display to target non-renewable germ cells and the generation of autoantibodies against proteins essential for conception via covalent modification) compared with that of traditional immunocontraceptive approaches. The need for a better understanding of mare folliculogenesis and conception factors, including maternal recognition of pregnancy, is also reviewed because they hold considerable potential in providing a non-surgical mechanism for sterilisation. In conclusion, the authors contend that non-surgical measures that are single shot and irreversible may provide a sustainable and effective strategy for feral horse control.

Cyclic AMP-Dependent Tyrosine Phosphorylation in Tammar Wallaby (Macropus eugenii) Spermatozoa

Publisher: Wiley

Date: 2004

DOI: 10.1002/JEZ.A.20020

Abstract: Despite considerable advances in our understanding of the molecular mechanisms regulating eutherian sperm function, there is a paucity of such knowledge for the Metatheria. In eutherian spermatozoa, the attainment of functional competence is associated with a redox-regulated, cAMP-mediated tyrosine phosphorylation cascade, activated during capacitation. In this report we investigate whether tammar wallaby (Macropus eugenii) spermatozoa possess a similar signal transduction pathway. Western blot analysis of phosphotyrosine expression in caudal and ejaculated populations of tammar spermatozoa revealed that elevation of intracellular cAMP levels, but not exposure to oxidants or NADPH, induced a dramatic increase in the overall level of tyrosine phosphorylation. Washed, ejaculated spermatozoa exhibited more pronounced increases in tyrosine phosphorylation than unwashed sperm populations. Localisation of tyrosine phosphorylation by immunocytochemistry showed that phosphotyrosine residues were principally located along the tammar sperm flagellum, and occasionally at a small region of the sperm head, adjacent to the acrosome. Associated with the tyrosine phosphorylation of tammar spermatozoa, was a change in sperm head conformation to a T-shaped orientation, further implying the importance of these pathways to normal tammar sperm function. Redox activity, as detected by lucigenin-dependent chemiluminescence, was stimulated by NADPH in caudal sperm preparations but not ejaculated spermatozoa. However, neither sperm population responded to treatment with NADPH with changes in intracellular cAMP or tyrosine phosphorylation. In conclusion, tammar spermatozoa possess the same cAMP-mediated, tyrosine phosphorylation-dependent signal transduction cascade that has been associated with capacitation in eutherian spermatozoa. However in Metatherian spermatozoa we could find no evidence that this pathway was redox regulated.

Investigation of the role of SRC in capacitation-associated tyrosine phosphorylation of human spermatozoa

Publisher: Oxford University Press (OUP)

Date: 31-01-2008

DOI: 10.1093/MOLEHR/GAN007

Abstract: The process of capacitation is a pre-requisite for mammalian spermatozoa allowing them to gain the ability to fertilize an oocyte. A fundamental part of this mechanism is a dramatic increase in the level of tyrosine phosphorylation. Implicated in this process is a unique cAMP rotein kinase A (PKA)-mediated pathway involving an intermediate PKA-activated tyrosine kinase suggested to be pp60(c-src) (SRC) in the mouse. This study has verified the importance of SRC as a key intermediate kinase in promoting the tyrosine phosphorylation events associated with human sperm capacitation. The presence of SRC in human spermatozoa was confirmed immunocytochemically and the kinase was localized to subcellular domains compatible with a role in tyrosine phosphorylation. Additionally SRC co-immunoprecipitated with PKA and became activated by phosphorylation of the Y416 residue during human sperm capacitation. Furthermore, the suppression of PKA and SRC through the application of specific inhibitors led to a dramatic decrease in tyrosine phosphorylation. However, although the inhibition of PKA was also accompanied by a suppression of sperm motility, SRC inhibition did not induce a similar response.

Age, the environment and our reproductive future: Bonking baby boomers and the future of sex

Publisher: Bioscientifica

Date: 02-2014

DOI: 10.1530/REP-13-0399

Abstract: There has never been a greater need for scientists trained in reproductive science. Most developed countries are witnessing unprecedented rates of recourse to assisted conception sitting cheek-by-jowl with high rates of induced abortion. This article addresses these two incongruous faces of reproductive healthcare. Every year at least 44 million abortions are performed worldwide, many under unsafe and insanitary conditions that carry a significant risk to the lives of women deprived of safe, effective methods for controlling their fertility. Although birth control is a complex issue involving myriad social and political factors, the technical vacuum in this area is significant. Through no fault of the family planning authorities, there have been no radically new methods of fertility control since the oral contraceptive pill was introduced in 1960 and even this contribution to planned parenthood has its roots in the biochemistry of the 1920s and 1930s. Moreover, the pharmaceutical industry has, by and large, turned its back on fundamental research activities in this area. At present, our major investment in reproductive healthcare involves treating ever-increasing numbers of couples with assisted reproductive technologies (ART). However, these treatments are often delivered without critically considering the underlying causes of this condition or seriously contemplating the long-term consequences of the current enthusiasm for such therapy. Significantly, the clinical factors underpinning the commitment of couples to ART include advanced maternal age and a variety of lifestyle factors, such as smoking and obesity, which are known to compromise the developmental potential of the oocyte and DNA integrity in spermatozoa.

Development of peptides for targeting cell ablation agents concurrently to the Sertoli and Leydig cell populations of the testes: an approach to non-surgical sterilization

Publisher: Cold Spring Harbor Laboratory

Date: 18-09-2023

DOI: 10.1101/2023.09.17.558160

The TUNEL assay consistently underestimates DNA damage in human spermatozoa and is influenced by DNA compaction and cell vitality: development of an improved methodology

Publisher: Wiley

Date: 11-01-2011

DOI: 10.1111/J.1365-2605.2009.01042.X

Abstract: The purpose of this study was to evaluate the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay as a method for assessing DNA damage in human spermatozoa. The conventional assay was shown to be insensitive and unresponsive to the DNA fragmentation induced in human and mouse spermatozoa on exposure to Fenton reagents (H₂O₂ and Fe(2+) ). However, both time- and dose-dependent responses could be readily detected if the chromatin was exposed to 2 mm dithiothreitol (DTT) for 45 min prior to fixation. This modified version of the assay significantly enhanced the TUNEL signals generated by subpopulations of spermatozoa isolated on discontinuous Percoll gradients as well as the responses triggered by reagents (arachidonic acid and menadione) that are known to stimulate superoxide anion production by human spermatozoa. DTT exposure also improved the signals detected with chromomycin A₃ (CMA₃), a probe designed to determine the efficacy of chromatin protamination, and enhanced the correlation observed between this criterion of sperm quality and the TUNEL assay. Finally, the output of the TUNEL assay was found to be highly correlated with sperm vitality. The TUNEL methodology was therefore further refined to incorporate a vital stain that covalently bound to intracellular amine groups in non-viable cells. This tag remained associated with the spermatozoa during fixation and processing for the TUNEL assay so that ultimately, both DNA integrity and vitality could be simultaneously assessed in the same flow cytometry assay. The methods described in this article are simple and robust and should facilitate research into the causes of DNA damage in human spermatozoa.

Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: Lipid peroxidation, DNA fragmentation and effectiveness of antioxidants

Publisher: Oxford University Press (OUP)

Date: 06-1998

DOI: 10.1093/HUMREP/13.6.1429

Abstract: Exposure of human spermatozoa to nicotinamide adenine dinucleotide phosphate (NADPH) resulted in the dose dependent generation of reactive oxygen species (ROS) which, at a critical level of intensity, induced lipid peroxidation, DNA damage and a dramatic decline of sperm motility. This system was then used as a model for screening the ability of different antioxidants to combat oxidative stress created through the excessive intracellular generation of toxic oxygen products of metabolism. A variety of antioxidants that has previously been shown to be protective against extracellularly derived oxidants (e.g. superoxide dismutase, catalase, vitamin E, hypotaurine) were ineffective in this system. Albumin, however, could provide complete protection against NADPH induced oxidative stress via mechanisms that did not involve the suppression of the lipid peroxidation cascade but rather the inactivation of lipid peroxides generated during this process. Albumin did not protect against DNA damage induced by NADPH but was extremely effective at preventing DNA fragmentation arising from the suppression of glutathione peroxidase activity with mercaptosuccinate. These studies emphasize that the design of clinically effective antioxidant treatments will depend, critically, upon the source of the oxidative stress. For cases involving excessive intracellular ROS generation, albumin appears to be an important means of neutralizing lipid peroxide-mediated damage to the sperm plasma membrane and DNA.

Definitive Evidence for the Nonmitochondrial Production of Superoxide Anion by Human Spermatozoa

Publisher: The Endocrine Society

Date: 05-2006

DOI: 10.1210/JC.2005-2711

Abstract: Oxidative stress in the male germ line has been associated with poor fertility, impaired embryonic development, miscarriage, and childhood disease. Such stress is known to be associated with the peroxidation of unsaturated fatty acids in the sperm plasma membrane and oxidative DNA damage to both the nuclear and mitochondrial genomes. However, the source of the free radicals responsible for such damage is still unresolved. The objective of this study was to chemically validate the use of dihydroethidium (DHE) as a probe for detecting the generation of superoxide anion by human spermatozoa and to examine the relationship between this activity and defective sperm function. DHE and SYTOX green were used in conjunction with flow cytometry and HPLC to investigate superoxide generation by human spermatozoa. Cause and effect relationships were established using menadione to artificially drive superoxide production by these cells. HPLC, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and spectrofluorometry were used to demonstrate that human spermatozoa generate the superoxide-specific product, 2-hydroxyethidium, from DHE. Spontaneous superoxide production by human spermatozoa was found to originate from a nonmitochondrial source and was inversely correlated with sperm motility. A causative relationship between superoxide generation and sperm function was demonstrated when the pharmacological stimulation of this activity with menadione was shown to result in both severe motility loss and DNA damage. These studies validate a methodology for investigating the origins of oxidative stress in the male germ line and demonstrate, for the first time, the significance of superoxide generation by human spermatozoa in the etiology of this condition.

Evidence for the involvement of PECAM-1 in a receptor mediated signal-transduction pathway regulating capacitation-associated tyrosine phosphorylation in human spermatozoa

Publisher: The Company of Biologists

Date: 15-10-2005

DOI: 10.1242/JCS.02604

Abstract: Mammalian spermatozoa must become `capacitated' in the female reproductive tract before they gain the ability to fertilize the oocyte. The attainment of a capacitated state has been correlated with a number of biochemical changes, the most notable of which is a dramatic increase in the tyrosine phosphorylation status of these cells. Despite its biological importance, the mechanisms responsible for initiating this tyrosine phosphorylation cascade in vivo are unknown. Here, we report that this signalling pathway can be elicited in a rapid, dose-dependent and lectin-specific manner by wheat germ agglutinin (WGA), but none of 18 other lectins assessed. This response was abrogated by prior enzymatic cleavage of either sialic acid or GlcNAc residues from the sperm surface and by treatment with a range of pharmacological inhibitors directed against protein kinase A, protein tyrosine kinases and intermediates including Src. Proteomic analysis of the WGA-binding sites on the sperm surface identified the putative cognate receptor as platelet cell adhesion molecule 1 (PECAM-1/CD31). This conclusion was supported by the following evidence: (i) anti-PECAM-1 antibodies identified a molecule of the correct molecular mass in human spermatozoa, (ii) PECAM-1 could be isolated from a pool of sperm surface proteins using WGA immobilized on a solid phase support, (iii) PECAM-1 and WGA co-localized to the sperm surface and (iv) anti-PECAM-1 antibodies could completely block the ability of WGA to stimulate tyrosine phosphorylation in these cells. Collectively, these data provide the first evidence that a receptor-mediated signal transduction pathway triggers human sperm capacitation and identifies PECAM-1 as the probable initiator of this second messenger cascade.

Effect of human uterine flushings collected at various stages of the menstrual cycle on mouse blastocysts in vitro

Publisher: Bioscientifica

Date: 05-1978

DOI: 10.1530/JRF.0.0530137

Abstract: Mouse blastocysts were cultured in vitro in a defined medium supplemented with uterine flushings (containing 500 microgram protein/ml) obtained from normal women at various stages of the menstrual cycle. With one exception (uterine flushing collected on the last day of a menstrual period) blastocyst hatching and attachment were not impaired by flushings collected before or after ovulation.

Proteolytic degradation of heat shock protein A2 occurs in response to oxidative stress in male germ cells of the mouse

Publisher: Oxford University Press (OUP)

Date: 08-12-2017

DOI: 10.1093/MOLEHR/GAW074

Abstract: Does oxidative stress compromise the protein expression of heat shock protein A2 (HSPA2) in the developing germ cells of the mouse testis? Oxidative stress leads to the modification of HSPA2 by the lipid aldehyde 4-hydroxynonenal (4HNE) and initiates its degradation via the ubiquitin-proteasome system. Previous work has revealed a deficiency in HSPA2 protein expression within the spermatozoa of infertile men that have failed fertilization in a clinical setting. While the biological basis of this reduction in HSPA2 remains to be established, we have recently shown that the HSPA2 expressed in the spermatozoa of normozoospermic in iduals is highly susceptible to adduction, a form of post-translational modification, by the lipid aldehyde 4HNE that has been causally linked to the degradation of its substrates. This modification of HSPA2 by 4HNE adduction dramatically reduced human sperm-egg interaction in vitro. Moreover, studies in a mouse model offer compelling evidence that the co-chaperone BCL2-associated athanogene 6 (BAG6) plays a key role in regulating the stability of HSPA2 in the testis, by preventing its ubiquitination and subsequent proteolytic degradation. Dose-dependent studies were used to establish a 4HNE-treatment regime for primary culture(s) of male mouse germ cells. The influence of 4HNE on HSPA2 protein stability was subsequently assessed in treated germ cells. Additionally, sperm lysates from infertile patients with established zona pellucida recognition defects were examined for the presence of 4HNE and ubiquitin adducts. A minimum of three biological replicates were performed to test statistical significance. Oxidative stress was induced in pachytene spermatocytes and round spermatids isolated from the mouse testis, as well as a GC-2 cell line, using 50-200 µM 4HNE or hydrogen peroxide (H2O2), and the expression of HSPA2 was monitored via immunocytochemistry and immunoblotting approaches. Using the GC-2 cell line as a model, the ubiquitination and degradation of HSPA2 was assessed using immunoprecipitation techniques and pharmacological inhibition of proteasomal and lysosomal degradation pathways. Finally, the interaction between BAG6 and HSPA2 was examined in response to 4HNE exposure via proximity ligation assays. HSPA2 protein levels were significantly reduced compared with controls after 4HNE treatment of round spermatids (P < 0.01) and GC-2 cells (P < 0.001) but not pachytene spermatocytes. Using GC-2 cells as a model, HSPA2 was shown to be both adducted by 4HNE and targeted for ubiquitination in response to cellular oxidative stress. Inhibition of the proteasome with MG132 prevented HSPA2 degradation after 4HNE treatment indicating that the degradation of HSPA2 is likely to occur via a proteasomal pathway. Moreover, our assessment of proteasome activity provided evidence that 4HNE treatment can significantly increase the proteasome activity of GC-2 cells (P < 0.05 versus control). Finally, 4HNE exposure to GC-2 cells resulted in the dissociation of HSPA2 from its regulatory co-chaperone BAG6, a key mediator of HSPA2 stability in male germ cells. While these experiments were performed using a mouse germ cell-model system, our analyses of patient sperm lysate imply that these mechanisms are conserved between mouse and human germ cells. This study suggests a causative link between non-enzymatic post-translational modifications and the relative levels of HSPA2 in the spermatozoa of a specific sub-class of infertile males. In doing so, this work enhances our understanding of failed sperm-egg recognition and may assist in the development of targeted antioxidant-based approaches for ameliorating the production of cytotoxic lipid aldehydes in the testis in an attempt to prevent this form of infertility. Not applicable. This work was supported by the National Health and Medical Research Council of Australia (APP1101953). The authors have no competing interests to declare.

Analysis of Reactive Oxygen Species Generating Systems in Rat Epididymal Spermatozoa1

Publisher: Oxford University Press (OUP)

Date: 10-2001

DOI: 10.1095/BIOLREPROD65.4.1102

Abstract: Epididymal sperm maturation culminates in the acquisition of functional competence by testicular spermatozoa. The expression of this functional state is dependent upon a redox-regulated, cAMP-mediated signal transduction cascade that controls the tyrosine phosphorylation status of the spermatozoa during capacitation. Analysis of superoxide anion (O2(-.)) generation by rat epididymal spermatozoa has revealed a two-component process involving electron leakage from the sperm mitochondria at complexes I and II and a plasma membrane NAD(P)H oxidoreductase. Following incubation in a glucose-, lactate-, and pyruvate-free medium (-GLP), O2(-.) generation was suppressed by 86% and 96% in caput and cauda spermatozoa, respectively. The addition of lactate, malate, or succinate to spermatozoa incubated in medium -GLP stimulated O2(-.) generation. This increase could be blocked by rotenone and oligomycin (R/O) in the presence of malate or lactate but not succinate. Stimulation with all three substrates, as well as spontaneous O2(-.) production in +GLP medium, was blocked by the flavoprotein inhibitor, diphenylene iodonium. Diphenylene iodonium, but not R/O, suppressed NAD(P)H-induced lucigenin-dependent chemiluminescence. This NAD(P)H-dependent enzyme resided in the sperm plasma membrane and its activity was regulated by zinc and uncharacterized cytosolic factors. Reverse transcription-polymerase chain reaction analysis indicated that the sperm NAD(P)H oxidoreductase complex is quite distinct from the equivalent leukocyte system.

Plasma testosterone response to hCG stimulation in the male marmoset monkey (Callithrix jacchus jacchus)

Publisher: Bioscientifica

Date: 03-1983

DOI: 10.1530/JRF.0.0670457

Abstract: In adult animals the intramuscular injection of hCG was followed by a rapid rise in plasma testosterone levels within 2-3 h and at doses of 40 and 80 i.u. hCG this primary response was followed by a second peak of testosterone at 48 h. Prepubertal marmosets also responded to hCG stimulation with a rapid increase in plasma testosterone levels within 3 h, but the magnitude of this peak was lower than that observed in adult animals and no biphasic pattern was observed. In adult and prepubertal animals a second dose of hCG (40 i.u.) administered 24 h after the initial injection failed to produce a further rapid increment in plasma testosterone levels.

Male-mediated developmental defects and childhood disease

Publisher: Cambridge University Press (CUP)

Date: 07-2000

DOI: 10.1017/S0962279900000211

Abstract: Reproductive and developmental toxicology has existed, in some form, at least since the Middle Ages when women commonly used naturally derived abortifacients for birth control. Percival Pott was the first to observe that environmental exposures could detrimentally affect the male reproductive system. He noticed that chimney sweeps exposed to soot developed testicular cancer and infertility at unusually young ages. The formal investigation of male-mediated effects on offspring (germline mutagenesis) began with the pioneering studies of Muller, Hertwig, Snell, Brenneke, and others, who established X-rays as the first identified agent capable of inducing hereditary changes in mice. They showed that litters sired by irradiated males contained fewer pups than controls. Because sperm motility and density were not affected, but chromosome abnormalities were found in fertilized eggs, they concluded that irradiated sperm were the source of the abnormal chromosomes. Since that discovery, the mutagenic effects of radiation on the male germline have been studied extensively (see below). Later, Auerbach & Robson and Bock & Jackson were the first to demonstrate that exposing mice to chemicals decreases their fertility and induces chromosomal abnormalities and other mutations in the male germline. The subsequent five decades of work on chemical mutagens has resulted in a detailed biochemical and genetic characterization of chemical mutagenesis in the male germline and male-mediated developmental problems in laboratory animals (see below).

From past to present

Publisher: Elsevier

Date: 2018

DOI: 10.1016/B978-0-12-812501-4.00003-1

The Complexities of Conception

Publisher: American Association for the Advancement of Science (AAAS)

Date: 07-07-1995

DOI: 10.1126/SCIENCE.7604276

Oxidative damage to sperm DNA: Attack and defense

Publisher: Springer International Publishing

Date: 2019

DOI: 10.1007/978-3-030-21664-1_7

Abstract: Due to its particular "silent" metabolic state, without transcription or translation, and a low level of cytosolic protective activities, mature sperm is a cellular type of aerobic organisms particularly at risk of oxidative damage. Despite the efforts of the male genital tract to treat this problem, a subcellular compartment of the sperm, the nucleus, and consequently, the paternal DNA cannot be effectively protected. There is an accumulation of evidence that oxidative damage to sperm DNA is quite common in male infertilities/subfertilities with potential harmful impacts on reproductive success, including the transgenerational inheritance of a paternal chromosomal lot carrying mutations.

Identification of post-translational modifications that occur during sperm maturation using difference in two-dimensional gel electrophoresis

Publisher: Wiley

Date: 03-2005

DOI: 10.1002/PMIC.200401100

Abstract: Difference in two-dimensional (2-D) gel electrophoresis (DIGE) is a novel method for analyzing up to three s les in one 2-D gel and using the information gained to study post-translational modifications of proteins. We describe the use of DIGE to isolate and characterize those proteins that undergo processing in spermatozoa as they transit the epididymal tract. We find up to 60 protein spots are significantly modified as sperm traverse the epididymis. In this article, we report eight unambiguous protein identifications and demonstrate that one protein, the beta-subunit of the mitochondrial F1-ATPase, is serine-phosphorylated as sperm undergo epididymal maturation. We suggest that phosphorylation of this particular protein in a cAMP-dependent manner may contribute to the mechanisms by which motility is conferred upon spermatozoa.

Paternal obesity, interventions, and mechanistic pathways to impaired health in offspring.

Publisher: S. Karger AG

Date: 2014

DOI: 10.1159/000365026

Abstract: b i Background: /i /b The global rates of male overweight/obesity are rising, approaching 70% of the total adult population in Western nations. Overweight/obesity increases the risk of chronic diseases however, there is increasing awareness that male obesity negatively impacts fertility, subsequent pregnancy, and the offspring health burden. Developmental programming is well defined in mothers however, it is becoming increasingly evident that developmental programming can be paternally initiated and mediated through paternal obesity. b i Key Messages: /i /b Both human and rodent models have established that paternal obesity impairs sex hormones, basic sperm function, and molecular composition. This results in perturbed embryo development and health and an increased subsequent offspring disease burden in both sexes. The reversibility of obesity-induced parental programming has only recently received attention. Promising results in animal models utilizing diet and exercise interventions have shown improvements in sperm function and molecular composition, resulting in restorations of both embryo and fetal health and subsequent male offspring fertility. The direct mode for paternal inheritance is likely mediated via spermatozoa. We propose two main theories for the origin of male obesity-induced paternal programming: (1) accumulation of sperm DNA damage resulting in de novo mutations in the embryo and (2) changes in sperm epigenetic marks (microRNA, methylation, or acetylation) altering the access, transcription, and translation of paternally derived genes during early embryogenesis. b i Conclusions: /i /b Paternal overweight/obesity induces paternal programming of offspring phenotypes likely mediated through genetic and epigenetic changes in spermatozoa. These programmed changes to offspring health appear to be partially restored via diet/exercise interventions in obese fathers preconception, which have been shown to improve aspects of sperm DNA integrity. However, the majority of data surrounding paternal obesity and offspring phenotypes have come from rodent models therefore, we contend that it will be increasingly important to study population-based data to determine the likely mode of inheritance in humans.

The value of adenosine triphosphate (ATP) measurements in assessing the fertilizing ability of human spermatozoa

Publisher: Elsevier BV

Date: 12-1985

DOI: 10.1016/S0015-0282(16)49042-4

Abstract: A study was undertaken to examine the relationship between adenosine triphosphate (ATP) levels and the fertilizing capacity of human spermatozoa in vitro and in vivo. The concentration of ATP in semen was found to be positively correlated with the ability of sperm to fuse with zona-free hamster oocytes. However, it was also demonstrated that a large part of this relationship depends upon the relationship between semen ATP concentrations and sperm number. Measurements of ATP levels in cryostored ejaculates used in an artificial insemination by donor program revealed that such measurements were not able to distinguish fertile from infertile ejaculates. However, among fertile donors, ATP levels did seem to contribute useful information on relative fertility. It is concluded that ATP measurement has a limited role in the laboratory evaluation of sperm function.

Errata

Publisher: Wiley

Date: 08-2000

DOI: 10.1046/J.1365-2605.2000.00253.X

Analysis of Phosphopeptide Changes as Spermatozoa Acquire Functional Competence in the Epididymis Demonstrates Changes in the Post-translational Modification of Izumo1

Publisher: American Chemical Society (ACS)

Date: 28-09-2012

DOI: 10.1021/PR300468M

Abstract: Spermatozoa are functionally inert when they emerge from the testes. Functional competence is conferred upon these cells during a post-testicular phase of sperm maturation in the epididymis. Remarkably, this functional transformation of epididymal spermatozoa occurs in the absence of nuclear gene transcription or protein translation. To understand the cellular mechanisms underpinning epididymal maturation, we have performed a label-free, MS-based, comparative quantification of peptides from caput, corpus and caudal epididymal spermatozoa. In total, 68 phosphopeptide changes could be detected during epididymal maturation corresponding to the identification of 22 modified proteins. Included in this list are the sodium-bicarbonate cotransporter, the sperm specific serine kinase 1, AKAP4 and protein kinase A regulatory subunit. Furthermore, four phosphopeptide changes came from Izumo1, the sperm-egg fusion protein, in the cytoplasmic segment of the protein. 2D-PAGE confirmed that Izumo1 is post-translationally modified during epididymal transit. Interestingly, phosphorylation on Izumo1 was detected on residue S339 in the caput and corpus but not caudal cells. Furthermore, Izumo1 exhibited four phosphorylated residues when spermatozoa reached the cauda, which were absent from caput cells. A model is advanced suggesting that these phospho-regulations are likely to act as a scaffold for the association of adaptor proteins with Izumo1 as these cells prepare for fertilization.

Impact of Epididymal Maturation on the Tyrosine Phosphorylation Patterns Exhibited by Rat Spermatozoa1

Publisher: Oxford University Press (OUP)

Date: 05-2001

DOI: 10.1095/BIOLREPROD64.5.1545

Abstract: As mammalian spermatozoa migrate through the epididymis, they acquire functionality characterized by the potential to express coordinated movement and the competence to undergo capacitation. The mechanisms by which spermatozoa gain the ability to capacitate during epididymal transit are poorly understood. The purpose of this study was to investigate the impact of epididymal maturation on the signal transduction pathways regulating tyrosine phosphorylation, because this process is thought to be central to the attainment of a capacitated state and expression of hyperactivated motility. Western blot and immunocytochemical analyses demonstrated that epididymal maturation in vivo is associated with a progressive loss of phosphotyrosine residues from the sperm head. As cells pass from the caput to the cauda epididymis, tyrosine phosphorylation becomes confined to a narrow band at the posterior margin of the acrosomal vesicle. Epididymal maturation of rat spermatozoa was also associated with an acquired competence to respond to high levels of intracellular cAMP by phosphorylating tyrosine residues on the sperm tail. Immature caput spermatozoa were incapable of exhibiting this response, despite the apparent availability of cAMP and protein kinase A. These findings help to clarify the biochemical changes associated with the functional maturation of spermatozoa during epididymal transit.

L-Carnitine and Pyruvate Are Prosurvival Factors During the Storage of Stallion Spermatozoa at Room Temperature1

Publisher: Oxford University Press (OUP)

Date: 10-2015

DOI: 10.1095/BIOLREPROD.115.131326

Abstract: The spermatozoa of many stallions do not tolerate being cooled, restricting the commercial viability of these animals and necessitating the development of a chemically defined room temperature (RT) storage medium. This study examined the impact of two major modulators of oxidative phosphorylation, pyruvate (Pyr) and L-carnitine (L-C), on the storage of stallion spermatozoa at RT. Optimal concentrations of Pyr (10 mM) and L-C (50 mM) were first identified and these concentrations were then used to investigate the effects of these compounds on sperm functionality and oxidative stress at RT. Mitochondrial and cytosolic reactive oxygen species, along with lipid peroxidation, were all significantly suppressed by the addition of L-C (48 h MitoSOX Red negative: 46.2% vs. 26.1% 48 and 72 h dihydroethidium negative: 61.6% vs. 43.1% and 64.4% vs. 46.9%, respectively 48 and 72 h 4-hydroxynonenal negative: 37.1% vs. 23.8% and 41.6% vs. 25.7%, respectively), while the Pyr + L-C combination resulted in significantly higher motility compared to the control at 72 h (total motility: 64.2% vs. 39.4% progressive motility: 34.2% vs. 15.2%). In addition, supplementation with L-C significantly reduced oxidative DNA damage at 72 h (9.0% vs. 15.6%). To investigate the effects of L-C as an osmolyte, comparisons were made between media that were osmotically balanced with NaCl, choline chloride, or L-C. This analysis demonstrated that spermatozoa stored in the L-C balanced medium had significantly higher total motility (55.0% vs. 39.0%), rapid motility (44.0% vs. 25.7%), and ATP levels (70.9 vs. 12.8 ng/ml) following storage compared with the NaCl treatment, while choline chloride did not significantly improve these parameters compared to the control. Finally, mass spectrometry was used to demonstrate that a combination of Pyr and L-C produced significantly higher acetyl-L-carnitine production than any other treatment (6.7 pg/10(6) spermatozoa vs. control at 4.0 pg/10(6) spermatozoa). These findings suggest that Pyr and L-C could form the basis of a novel, effective RT storage medium for equine spermatozoa.

First recorded pregnancy and normal birth after ICSI using electrophoretically isolated spermatozoa

Publisher: Oxford University Press (OUP)

Date: 13-09-2006

DOI: 10.1093/HUMREP/DEL351

Abstract: DNA damage in the male germ line is associated with poor fertilization and cleavage rates, impaired embryo quality and early pregnancy loss. Given these associations, embryologists are keen to develop techniques that will allow the selection of viable spermatozoa exhibiting low levels of DNA damage for assisted conception purposes. In this article, we describe a novel electrophoretic approach for the rapid isolation of cells possessing little DNA damage. The limits of the method were examined using cryostored and snap-frozen semen s les as well as testicular biopsy material. In addition, clinical utility was demonstrated in a case study involving treatment of a patient exhibiting persistently high levels of DNA damage in his spermatozoa. From a range of difficult starting materials (biopsies, cryostored semen and snap-frozen sperm suspensions), the electrophoretic system rapidly isolated populations of motile, viable, morphologically normal spermatozoa exhibiting high levels of DNA integrity. Clinical application in a couple suffering from long-term infertility associated with extensive DNA damage in the male germ line led to the first human pregnancy following such electrophoretic sperm isolation. The electrophoretic procedure holds promise as a convenient method for the rapid preparation of high-quality spermatozoa for assisted conception purposes.

Improved methods of DNA extraction from human spermatozoa that mitigate experimentally-induced oxidative DNA damage

Publisher: Public Library of Science (PLoS)

Date: 26-03-2018

DOI: 10.1371/JOURNAL.PONE.0195003

Glycerophospholipids protect stallion spermatozoa from oxidative damage in vitro

Publisher: Bioscientifica

Date: 11-08-2021

DOI: 10.1530/RAF-21-0028

Abstract: Stallion sperm membranes comprise a high proportion of polyunsaturated fatty acids, making stallion spermatozoa especially vulnerable to peroxidative damage from reactive oxygen species generated as a by-product of cell metabolism. Membrane lipid replacement therapy with glycerophospholipid (GPL) mixtures has been shown to reduce oxidative damage in vitro and in vivo . The aims of this study were to test the effects of a commercial preparation of GPL, NTFactor ® Lipids, on stallion spermatozoa under oxidative stress. When oxidative damage was induced by the addition of arachidonic acid to stallion spermatozoa, the subsequent addition of GPL reduced the percentage of 4-hydroxynonenal (4-HNE a key end product of lipid peroxidation) positive cells (32.9 ± 2.7 vs 20.9 ± 2.3% P ≤ 0.05) and increased the concentration of 4-HNE within the spent media (0.026 ± 0.003 vs 0.039 ± 0.004 µg/mL P ≤ 0.001), suggesting that oxidized lipids had been replaced by exogenous GPL. Lipid replacement improved several motility parameters (total motility: 2.0 ± 1.0 vs 68.8 ± 2.9% progressive motility: 0 ± 0 vs 19.3 ± 2.6% straight line velocity: 9.5 ± 2.1 vs 50.9 ± 4.1 µm/s curvilinear velocity: 40.8 ± 10 vs 160.7 ± 7.8 µm/s average path velocity: 13.4 ± 2.9 vs 81.9 ± 5.9 µm/s P ≤ 0.001), sperm viability (13.5 ± 2.9 vs 80.2 ± 1.6% P ≤ 0.001) and reduced mitochondrial ROS generation (98.2 ± 0.6 vs 74.8 ± 6.1% P ≤ 0.001). Supplementation with GPL during 17°C in vitro sperm storage over 72 h improved sperm viability (66.4 ± 2.6 vs 78.1 ± 2.9% P ≤ 0.01) and total motility (53 ± 5.6 vs 66.3 ± 3.5% P ≤ 0.05). It is concluded that incubation of stallion spermatozoa with sub-µm-sized GPL micelles results in the incorporation of exogenous GPL into sperm membranes, diminishing lipid peroxidation and improving sperm quality in vitro . Sperm collection and storage is an important step in many artificial insemination and in vitro fertilization regimes for several species, including humans and horses. The sperm membrane, which acts as a protective outer barrier, is made up of fatty acid-containing molecules – called phospholipids. These phospholipids may become damaged by waste products generated by the cell, such as hydrogen peroxide, during non-chilled sperm storage. We aimed to determine if sperm cells were able to repair this membrane damage by supplementing them with phospholipids during non-chilled storage. Sperm was collected from five miniature stallions by artificial vagina, and then supplemented with phospholipids during 72 h sperm storage at 17°C. Our studies show that when stallion sperm are supplemented with phospholipids in vitro , they are able to remove their damaged membrane phospholipids and swap them for undamaged ones, aiding in resistance to cellular waste and improving cell health and potential fertility.

Nitroblue tetrazolium (NBT) assay

Publisher: Elsevier BV

Date: 2018

DOI: 10.1016/J.RBMO.2017.09.005

An important role for B-cell activation factor and B cells in the pathogenesis of Sjögren's syndrome

Publisher: Ovid Technologies (Wolters Kluwer Health)

Date: 09-2007

DOI: 10.1097/BOR.0B013E328277EF4C

Mass spectrometry reveals distinct proteomic profiles in high- And low-quality stallion spermatozoa

Publisher: Bioscientifica

Date: 11-2020

DOI: 10.1530/REP-20-0284

Abstract: The horse breeding industry relies upon optimal stallion fertility. Conventional sperm assessments provide limited information regarding ejaculate quality and are not in idually predictive of fertilizing potential. The aim of this study was to harness mass spectrometry to compare the proteomic profiles of high- and low-quality stallion spermatozoa, with the ultimate goal of identifying fertility biomarker candidates. Extended stallion semen ( n = 12) was fractionated using Percoll density gradients to isolate low-quality and high-quality sperm populations. Motility and morphological assessments were carried out, and proteomic analyses was conducted using UHPLC-MS/MS. High-quality spermatozoa recorded higher total (95.2 ± 0.52% vs 70.6 ± 4.20% P ≤ 0.001) and progressive motilities (43.4 ± 3.42% vs 27.3 ± 4.32% P ≤ 0.05), and a higher proportion of morphologically normal cells (50.2 ± 4.34% vs 38.8 ± 2.72% P ≤ 0.05). In total, 1069 proteins were quantified by UHPLC-MS/MS, of which 22 proteins were significantly more abundant in the high-quality sperm population ( P ≤ 0.05). A-kinase anchor protein 4 (AKAP4) and Hexokinase 1 (HK1) were considered possible biomarker candidates and their differential expression was confirmed by immunoblot. Protein expression was significantly correlated with total (AKAP4 R 2 = 0.38, P ≤ 0.01 HK1 R 2 = 0.46, P ≤ 0.001) and progressive motilities (AKAP4 R 2 = 0.51, P ≤ 0.001 HK1 R 2 = 0.55, P ≤ 0.01), percentage rapid (AKAP4 R 2 = 0.29, P ≤ 0.05 HK1 R 2 = 0.58, P ≤ 0.001), straight-line velocity (HK1 R 2 = 0.50, P ≤ 0.01) and straightness (HK1 R 2 = 0.40, P ≤ 0.01). Furthermore, AKAP4 was highly susceptible to adduction by 4-hydroxynonenal (4HNE), which resulted in a global reduction in the phosphorylation profiles following capacitation. In conclusion, the proteomic profiles of high- and low-quality stallion spermatozoa differ substantially, and proteins such as AKAP4 and HK1 could serve as biomarkers of ejaculate quality.

Functions and effects of reactive oxygen species in male fertility

Publisher: Elsevier BV

Date: 09-2020

DOI: 10.1016/J.ANIREPROSCI.2020.106456

The influence of medium composition, osmolarity and albumin content on the acrosome reaction and fertilizing capacity of human spermatozoa: development of an improved zona‐free hamster egg penetration

Publisher: Wiley

Date: 04-1983

DOI: 10.1111/J.1365-2605.1983.TB00337.X

Abstract: The influence of medium composition, osmolarity and albumin concentration on the ability of human spermatozoa to undergo the acrosome reaction and penetrate zona-free hamster ova has been investigated. Raising the osmolarity but not the albumin concentration of the media was found to significantly increase the proportion of spermatozoa exhibiting an acrosome reaction and penetrating hamster ova, without influencing motility. There was, however, no correlation between the size of the acrosome reacted population and penetration rates between s les, suggesting that the zona-free hamster egg penetration test is more than just a measure of the availability of acrosome reacted cells. As a result of this study, a revised protocol for the hamster egg assay is described which is shorter and considerably more sensitive than conventional procedures.

The correlates of fertilizing capacity in normal fertile men

Publisher: Elsevier BV

Date: 07-1982

DOI: 10.1016/S0015-0282(16)46398-3

Abstract: An investigation has been carried out in normal fertile men on the correlates of fertilizing capacity as defined by the zona-free hamster egg penetration test. The results demonstrate that the apparent fertility of these men is compatible with a wide range of intrinsic sperm quality as reflected by penetration rates ranging from 14% to 90% and differences in the minimum concentration of motile spermatozoa required to initiate penetration. These differences in sperm function could not be correlated with any of the conventional parameters of semen analysis or any of a large number of sperm movement characteristics analyzed by time-exposure photomicrography, with two exceptions: (1) the percentage of progressively (greater than 25 micron/sec) motile spermatozoa exhibiting an litude of lateral head displacement (Ah) of less than 10 micron, which was positively correlated with penetrating capacity (P less than 0.01), and (2) the percentage of progressively (less than 25 micron/sec) motile spermatozoa exhibiting an Ah of greater than 10 micron, which was negatively correlated with penetration rate (P less than 0.05). The information obtained in this study should provide a useful basis against which to compare the properties of spermatozoa in cases of suspected infertility.

Deficient Tryptophan Catabolism along the Kynurenine Pathway Reveals That the Epididymis Is in a Unique Tolerogenic State

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1074/JBC.M110.172114

Development of a model for studying the developmental consequences of oxidative sperm DNA damage by targeting redox-cycling naphthoquinones to the Sertoli cell population

Publisher: Elsevier BV

Date: 09-2023

DOI: 10.1016/J.FREERADBIOMED.2023.06.008

Glycogen synthase kinase 3 regulates acrosomal exocytosis in mouse spermatozoa via dynamin phosphorylation

Publisher: Wiley

Date: 25-03-2015

DOI: 10.1096/FJ.14-265553

Abstract: The dynamin family of GTPases has been implicated as novel regulators of the acrosome reaction, a unique exocytotic event that is essential for fertilization. Dynamin activity during the acrosome reaction is accompanied by phosphorylation of key serine residues. We now tested the hypothesis that glycogen synthase kinase 3 (GSK3) is the protein kinase responsible for dynamin phosphorylation at these phosphosites in mouse spermatozoa. Pharmacologic inhibition of GSK3 in mature mouse spermatozoa (CHIR99021: IC50 = 6.7 nM) led to a significant reduction in dynamin phosphorylation (10.3% vs. 27.3% P < 0.001), acrosomal exocytosis (9.7% vs. 25.7% P < 0.01), and in vitro fertilization (53% vs. 100% P < 0.01). GSK3 was shown to be present in developing germ cells where it colocalized with dynamin in the peri-acrosomal domain. However, additional GSK3 was acquired by maturing mouse spermatozoa within the male reproductive tract, via a novel mechanism involving direct interaction of sperm heads with extracellular structures known as epididymal dense bodies. These data reveal a novel mode for the cellular acquisition of a protein kinase and identify a key role for GSK3 in the regulation of sperm maturation and acrosomal exocytosis.

Sperm capacitation: a distant landscape glimpsed but unexplored

Publisher: Oxford University Press (OUP)

Date: 26-09-2013

DOI: 10.1093/MOLEHR/GAT067

Abstract: Capacitation is a remarkable process whereby spermatozoa prepare themselves for engagement with the oocyte. Although the existence of this process has been appreciated as a biological phenomenon for more than half a century, its molecular underpinnings still await clarification. We know that some of the major changes involve sterol oxidation and efflux from the plasma membrane, the anterior movement of lipid rafts, changes in the surface expression of a variety of proteins including hyaluronidase and receptors for the zona pellucida, an increase in intracellular cyclic adenosine monophosphate (cAMP), the induction of tyrosine phosphorylation and the expression of hyperactivated motility. These changes are dependent on the presence of bicarbonate, to facilitate cAMP generation, maintain an alkaline intracellular pH and support an optimal level of reactive oxygen species generation and are enhanced by the presence of albumin to provide antioxidant protection to the plasma membrane and promote cholesterol efflux. In vivo, the rate at which sperm cells capacitate is carefully controlled in order to ensure that the release of capacitated spermatozoa from a post-insemination reservoir in the isthmic region of the oviduct is synchronized with ovulation. The factors that control these critical events are now being resolved, aided by proteomic studies that are providing critical definitive information on the range of receptors that exist in the sperm plasma membrane and define the manner in which these exquisitely complex cells interact with their environment. Progress in this area has been enhanced by IVF technology pioneered by Bob Edwards and will ultimately facilitate the design of safe, effective culture conditions for optimization of this revolutionary therapy.

Contraceptive vaccines

Publisher: Informa Healthcare

Date: 08-2003

DOI: 10.1517/14712598.3.5.829

Abstract: The control of human fertility would be revolutionised by the development of a safe, effective, long-acting contraceptive vaccine. The pursuit of this objective has involved the selection of appropriate targets within the reproductive process that are amenable to interference with antibodies. To date, three major targets have been researched. The zona pellucida (ZP) plays key roles in folliculogenesis, fertilisation and early development, and is comprised of powerful cell-specific antigens. The induction of infertility requires high ZP antibody titres that are difficult to maintain without inducing ovarian pathology characterised by a premature loss of primordial follicles. As a premature menopause would be a high price to pay for long-term contraception, this approach to a vaccine cannot progress until the cause of the ovarian pathology has been resolved. Sperm surface antigens represent another promising approach to contraceptive vaccine development. While there is some clinical data to support the likely efficacy of this strategy, none of the gamete-specific molecules characterised to date have fulfilled this promise. Anti-human chorionic gonadotropin (hCG) vaccines terminate pregnancy by preventing the maternal recognition of pregnancy. This vaccine has reached the stage of clinical trials, and preliminary indications are that the approach is safe and potentially effective. However, reliability may be an issue, given the observed inter-in idual variability in antibody generation. The future of contraceptive vaccine development will clearly involve a continuation of the intense search for suitable targets and the development of improved immunisation procedures that exploit the latest innovations in vaccine technology.

Analysis of Human Sperm Function Following Exposure to the Ionophore A23187: Comparison of Normospermic and Oligozoospermic Men

Publisher: Wiley

Date: 10-09-1984

DOI: 10.1002/J.1939-4640.1984.TB00796.X

Abstract: Time exposure photomicrography and interspecific in vitro fertilization techniques have been used to compare the responses to the alent cation ionophore A23187 of spermatozoa from normal fertile and oligozoospermic men. The fertilizing capacity of spermatozoa from the fertile controls produced a bell-shaped dose response curve when assessed in the presence of ionophore. The optimal responses occurred in the presence of 50 and 100 microM A23187. At this concentration, a mean penetration rate of about 75%, in association with multiple polyspermy, was observed without significant changes in motility patterns. At higher doses of A23187, there was a decline in fertilization rates, an independent reduction in sperm motility, and a significant decrease in the litude of lateral sperm head displacement. In contrast to the fertile controls, spermatozoa recovered from patients with oligozoospermia failed to exhibit a significant change in their fertilizing potential following exposure to A23187. Calculations based on the Poisson distribution theory indicated that this lack of responsiveness was not related to any differences in the motility of the spermatozoa from the oligozoospermic patients compared to the controls. These results suggest that calcium ionophores may be of value in providing a rapid and sensitive indicator of the functional competence of human spermatozoa, which circumvents problems concerning the rate and efficiency of sperm capacitation encountered with conventional protocols.

Analysis of proteomic changes associated with sperm capacitation through the combined use of IPG-strip prefractionation followed by RP chromatography LC-MS/ MS analysis

Publisher: Wiley

Date: 02-2010

DOI: 10.1002/PMIC.200900574

Abstract: Following ejaculation, mammalian spermatozoa undergo an obligatory process known as capacitation, which enables these cells to bind to and fertilize an oocyte. Since spermatozoa are transcriptionally and translationally silent, the functional metamorphosis of these cells during capacitation is accomplished entirely by PTMs. Despite the importance of this process, very few studies have attempted to define the precise nature of the proteomic changes that allow spermatozoa to attain a capacitated state. Here we report the use of an IPG-strip pre-fractionation approach to isolate and purify tryptic peptides derived from mouse spermatozoa exhibiting varying degrees of capacitation. Following focusing, the strips were cut into 1 cm segments, the peptides extracted and run into a mass spectrometer. Label-free, quantitative analysis of proteomic changes associated with capacitation was then performed. In total, we found 210 significant peptide changes. Of these, we could conclusively interpret the tandem mass spectra of 71 peptides, corresponding to 52 protein changes during capacitation. Many proteins including VDAC2, Fascin-3 and sorbitol dehydrogenase (SORD) have not previously been implicated in this process. To validate our data, we were able to show significant upregulation of SORD activity during capacitation, suggesting that the polyol pathway is activated during this process.

Sperm Motility Is Lost In Vitro as a Consequence of Mitochondrial Free Radical Production and the Generation of Electrophilic Aldehydes but Can Be Significantly Rescued by the Presence of Nucleophilic

Publisher: Oxford University Press (OUP)

Date: 11-2012

DOI: 10.1095/BIOLREPROD.112.102020

Abstract: The prolonged incubation of human spermatozoa in vitro was found to induce a loss of motility associated with the activation of mitochondrial reactive oxygen species generation in the absence of any change in mitochondrial membrane potential. The increase in mitochondrial free radical production was paralleled by a loss of protein thiols and a concomitant rise in the formation of 4-hydroxynonenal, an electrophilic product of lipid peroxidation that was found to directly suppress sperm movement. These results prompted a search for nucleophiles that could counteract the action of such cytotoxic aldehydes, as a means of ensuring the long-term survival of spermatozoa in vitro. Four nucleophilic compounds were consequently assessed (penicillamine, homocysteine, N-acetylcysteine, and mercaptosuccinate) in three species (human, rat, and horse). The results of this analysis revealed drug and species specificity in the manner in which these compounds affected sperm function, with penicillamine conferring the most consistent, effective support. This prosurvival effect was achieved downstream of mitochondrial reactive oxygen species generation and was associated with the stabilization of 4-hydroxynonenal generation, the preservation of sperm thiols, and a reduction in 8-hydroxy-2'-deoxyguanosine formation. Theoretical calculations of Fe-S and Cu-S bond distances and corresponding binding energies suggested that the particular effectiveness of penicillamine may, in part, reflect the ability of this nucleophile to form stable complexes with transition metals that catalyze lipid peroxidation. The practical implications of these findings were indicated by the effective preservation of equine spermatozoa for 8 days at ambient temperature when the culture medium was supplemented with penicillamine.

On methods for the detection of reactive oxygen species generation by human spermatozoa: analysis of the cellular responses to catechol oestrogen, lipid aldehyde, menadione and arachidonic acid

Publisher: Wiley

Date: 13-01-2013

DOI: 10.1111/J.2047-2927.2012.00056.X

Abstract: Oxidative stress is known to have a major impact on human sperm function and, as a result, there is a need to develop sensitive methods for measuring reactive oxygen species (ROS) generation by these cells. A variety of techniques have been developed for this purpose including chemiluminescence (luminol and lucigenin), flow cytometry (MitoSOX Red, dihydroethidium, 4,5-diaminofluorescein diacetate and 2',7'-dichlorodihydrofluorescein diacetate) and spectrophotometry (nitroblue tetrazolium). The relative sensitivity of these assays and their comparative ability to detect ROS generated in different subcellular compartments of human spermatozoa, have not previously been investigated. To address this issue, we have compared the performance of these assays when ROS generation was triggered with a variety of reagents including 2-hydroxyestradiol, menadione, 4-hydroxynonenal and arachidonic acid. The results revealed that menadione predominantly induced release of ROS into the extracellular space where these metabolites could be readily detected by luminol-peroxidase and, to a lesser extent, 2',7'-dichlorodihydrofluorescein. However, such sensitivity to extracellular ROS meant that these assays were particularly vulnerable to interference by leucocytes. The remaining reagents predominantly elicited ROS generation by the sperm mitochondria and could be optimally detected by MitoSOX Red and DHE. Examination of spontaneous ROS generation by defective human spermatozoa revealed that MitoSOX Red was the most effective indicator of oxidative stress, thereby emphasizing the general importance of mitochondrial dysregulation in the aetiology of defective sperm function.

WHAT POTENTIAL OVUM DONORS THINK

Publisher: Elsevier BV

Date: 05-1984

DOI: 10.1016/S0140-6736(84)91493-4

Development of a technique for monitoring the contamination of human semen samples with leukocytes

Publisher: Elsevier BV

Date: 06-1992

DOI: 10.1016/S0015-0282(16)55094-8

Abstract: To determine whether receptors for the N-formyl chemotactic peptide, FMLP, exist on the surface of human spermatozoa and regulate reactive oxygen species generation by these cells. Chemiluminescent analysis of reactive oxygen species generation by suspensions of human spermatozoa before and after removal of leukocytes using a magnetic cell separation technique. Academic Research Institute. Unselected male volunteers. Human sperm suspensions responded to FMLP and phorbol ester (PMA) with a burst of reactive oxygen species production. Autoradiographic analyses employing 3H FMLP and chemiluminescence studies involving the selective removal of leucocytes with anti-CD 45-coated magnetic beads demonstrated that the FMLP responses were because of leukocyte contamination. In contrast, reactive oxygen species production in response to PMA appeared to reflect the oxidant-generating capacity of both leukocytes and spermatozoa. The only cells present in the human ejaculate possessing detectable receptors for FMLP and capable of generating reactive oxygen species in response to this reagent come from the leukocyte population. Luminol-dependent, FMLP-induced, chemiluminescence provides a rational basis for monitoring the presence of leukocytes in suspensions of human spermatozoa.

Cyclic variation in concentrations of protein and hexose in human uterine flushings collected by an improved technique

Publisher: Bioscientifica

Date: 03-1978

DOI: 10.1530/JRF.0.0520289

Abstract: Uterine flushings were obtained from fertile women at various stages of the menstrual cycle. A technique was used which excluded contamination with cervical mucus and significantly lowered contamination with blood in comparison with an established technique. Contamination of the uterine flushings with seminal plasma and tubal fluid was also prevented. The concentrations of protein and hexose in the uterine flushings, corrected for contamination with plasma, were significantly lower in the secretory stages than in the proliferative stages of the cycle. It is concluded that proteins and carbohydrates are present within the uterine lumen not only after ovulation but also during the preovulatory period.

Suppression of sperm function by depot medroxyprogesterone acetate and testosterone enanthate in steroid male contraception

Publisher: Elsevier BV

Date: 04-1989

DOI: 10.1016/S0015-0282(16)60623-4

Abstract: Ten normal men were given three monthly intramuscular injections of 200 mg of depot medroxyprogesterone acetate (MPA) and 250 mg of testosterone (T) enanthate. Six men became azoospermic, while four remained oligozoospermic, with a mean sperm density of 1.5 +/- 0.3 standard error of the mean million/ml. Zona-free hamster oocyte penetration was abolished in all oligozoospermic s les at the end of treatment. Twenty of the 21 oligozoospermic s les yielding at least 0.6 to 5.0 million motile spermatozoa showed a complete absence of oocyte penetration. Semen parameters returned to normal, although some took up to 12 months. These findings demonstrated an antifertility action of MPA and T enanthate on the functional capacity of residual spermatozoa and support the view that extreme oligozoospermia may be a tenable target for reversible steroid male contraception.

Damage to sperm DNA mediated by reactive oxygen species: Its impact on human reproduction and the health trajectory of offspring

Publisher: Springer International Publishing

Date: 2015

DOI: 10.1007/978-3-319-18881-2_2

Abstract: Disruptions to the genetic integrity of the mammalian spermatozoon play a major role in determining the subsequent developmental trajectory of the embryo. This chapter examines the causative links that connect DNA damage in human spermatozoa and the appearance of mutations in the progeny responsible for a variety of clinical conditions from autism to cancer. Integral to this discussion is an abundance of evidence indicating that human spermatozoa are vulnerable to free radical attack and the generation of oxidative DNA damage. The resolution of this damage appears to be initiated by the spermatozoa but is driven to completion by the oocyte in a round of DNA repair that follows fertilization. The persistence of unresolved oxidative DNA damage following zygote formation has the potential to create mutations/epimutations in the offspring that may have a profound impact on the health of the progeny. It is proposed that the creation of oxidative stress in the male germ line is a consequence of a wide variety of environmental/lifestyle factors that influence the health and well-being of the offspring as a consequence of mutational change induced by the aberrant repair of oxidative DNA damage in the zygote. Factors such as paternal age, subfertility, smoking, obesity, and exposure to a range of environmental influences, including radio-frequency electromagnetic radiation and xenobiotics, have all been implicated in this process. Identifying the contributors to oxidative stress in the germ line and resolving the mechanisms by which such stressors influence the mutational load carried by the progeny will be an important task for the future. This task is particularly pressing, given the extensive use of assisted reproductive technologies to achieve pregnancies in vitro that would have been prevented in vivo by the complex array of mechanisms that nature has put in place to ensure that only the fittest gametes participate in the generative process.

CABYR is essential for fibrous sheath integrity and progressive motility in mouse spermatozoa

Publisher: The Company of Biologists

Date: 2016

DOI: 10.1242/JCS.193151

Abstract: Calcium binding tyrosine phosphorylation regulated protein (CABYR) has been implicated in sperm physiological function in several in vitro studies. It has also been implicated as a potential cause and diagnostic tool in asthenozoospermic human males. CABYR is known to be localized to the fibrous sheath, an accessory structure in the flagellar principal piece. Utilizing the CRISPR/Cas9 technology, we have knocked out this gene in mice to understand its role in male fertility. Cabyr KO male mice showed severe subfertility with a defect in sperm motility as well as a significant disorganisation in the fibrous sheath. Further, abnormal configuration of doublet microtubules was observed in the Cabyr KO spermatozoa, suggesting that the fibrous sheath is important for the correct organization of the axoneme. Our results show that it is the role of CABYR in the formation of the fibrous sheath that is essential for male fertility.

DNA damage to spermatozoa has impacts on fertilization and pregnancy

Publisher: Springer Science and Business Media LLC

Date: 24-05-2005

DOI: 10.1007/S00441-005-1097-5

Abstract: DNA damage in the male germ line has been associated with poor semen quality, low fertilization rates, impaired preimplantation development, increased abortion and an elevated incidence of disease in the offspring, including childhood cancer. The causes of this DNA damage are still uncertain but the major candidates are oxidative stress and aberrant apoptosis. The weight of evidence currently favours the former and, in keeping with this conclusion, positive results have been reported for antioxidant therapy both in vivo and in vitro. Resolving the causes of DNA damage in the male germ line will be essential if we are to prevent the generation of genetically damaged human embryos, particularly in the context of assisted conception therapy.

Head and flagella subcompartmental proteomic analysis of human spermatozoa

Publisher: Wiley

Date: 2013

DOI: 10.1002/PMIC.201200350

Abstract: Subcellular proteomics not only deepens our knowledge of what proteins are present within cells, but also opens our understanding as to where those proteins reside. Given the highly differentiated, cross-linked state of spermatozoa, such studies have proven difficult to perform. In this study we have fractionated spermatozoa into two components, consisting of either the head or flagellar region. Following SDS-PAGE, 1 mm slices were digested and used for LC-MS/MS analysis. In total, 1429 proteins were identified with 721 proteins being exclusively found in the tail and 521 exclusively in the head. Not only is this the largest reported proteomic analysis of human spermatozoa, but also it has provided novel insights into the compartmentalization of proteins, particularly receptors, never previously reported to be present in this cell type.

Antioxidant trials—the need to test for stress

Publisher: Oxford University Press (OUP)

Date: 2021

DOI: 10.1093/HROPEN/HOAB007

Mechanistic Insight into the Regulation of Lipoxygenase-Driven Lipid Peroxidation Events in Human Spermatozoa and Their Impact on Male Fertility

Publisher: MDPI AG

Date: 31-12-2020

DOI: 10.3390/ANTIOX10010043

Abstract: A prevalent cause of sperm dysfunction in male infertility patients is the overproduction of reactive oxygen species, an attendant increase in lipid peroxidation and the production of cytotoxic reactive carbonyl species such as 4-hydroxynonenal. Our previous studies have implicated arachidonate 15-lipoxygenase (ALOX15) in the production of 4-hydroxynonenal in developing germ cells. Here, we have aimed to develop a further mechanistic understanding of the lipoxygenase-lipid peroxidation pathway in human spermatozoa. Through pharmacological inhibition studies, we identified a protective role for phospholipase enzymes in the liberation of peroxidised polyunsaturated fatty acids from the human sperm membrane. Our results also revealed that arachidonic acid, linoleic acid and docosahexanoic acid are key polyunsaturated fatty acid substrates for ALOX15. Upon examination of ALOX15 in the spermatozoa of infertile patients compared to their normozoospermic counterparts, we observed significantly elevated levels of ALOX15 protein abundance in the infertile population and an increase in 4-hydroxynonenal adducts. Collectively, these data confirm the involvement of ALOX15 in the oxidative stress cascade of human spermatozoa and support the notion that increased ALOX15 abundance in sperm cells may accentuate membrane lipid peroxidation and cellular dysfunction, ultimately contributing to male infertility.

Oxidative stress and the etiology of male infertility

Publisher: Springer Science and Business Media LLC

Date: 20-08-2016

DOI: 10.1007/S10815-016-0791-4

Analysis of lipid peroxidation mechanisms in human spermatozoa

Publisher: Wiley

Date: 07-1993

DOI: 10.1002/MRD.1080350313

Abstract: The mechanisms by which ferrous ion promoters induce malondialdehyde generation by human spermatozoa have been investigated in order to provide a rational basis for the quantification and interpretation of lipid peroxidation assays. Incubation of human spermatozoa with a ferrous ion promoter in the presence of thiobarbituric acid (TBA) led to the generation of the bone fide malondialdehyde-TBA adduct. The importance of iron in the stimulation of lipid peroxidation was emphasized by the ability of Desferal and EDTA to suppress malondialdehyde generation. Paradoxically, when the concentration of EDTA relative to iron was equimolar or greater, the suppression of malondialdehyde formation was accompanied by the generation of hydroxyl radicals. These results suggested that the addition of promoter did not effect the first-chain initiation of lipid peroxidation but favored an alternative mechanism involving the catalytic decomposition of pre-existing lipid peroxides. This conclusion was reinforced by the inability of reagents that would limit the formation (superoxide dismutase and/or catalase) or availability (mannitol, formate) of hydroxyl radicals, to influence malondialdehyde generation. While hydroxyl radicals were not directly involved in Fe(2+)-promoted malondialdehyde generation, the existence of significant correlations between reactive oxygen species production and the outcome of the TBA assay, suggested that Fenton chemistry might be important in the initiation of peroxidative damage. It is proposed that the impeded propagation of peroxidation initiated by Fenton or Haber Weiss reactions would lead to the accumulation of lipid peroxides in the spermatozoa and it is these peroxides that are induced to decompose during the Fe(2+)-promoted TBA assay, stimulating a lipoperoxidative chain reaction and malondialdehyde formation.

Oxidative stress, DNA damage and the Y chromosome

Publisher: Bioscientifica

Date: 10-2001

DOI: 10.1530/REP.0.1220497

Abstract: Recent advances in understanding of male infertility have implicated two major causative factors, oxidative stress and Y chromosome deletions. A major cause of oxidative stress appears to be the high rate of reactive oxygen species generation associated with the retention of excess residual cytoplasm in the sperm midpiece. Other possible causes include the redox cycling of xenobiotics, and antioxidant depletion or apoptosis. Oxidative stress induces peroxidative damage in the sperm plasma membrane and DNA damage in both the mitochondrial and nuclear genomes. Nuclear DNA damage in the germ line of the father may be associated with pathology in the offspring, including childhood cancer and infertility. Gene deletions on the non-recombining region of the Y chromosome account for the infertility observed in about 15% of patients with azoospermia and 5-10% of subjects with severe oligozoospermia. The Y chromosome is particularly susceptible to gene deletions because of the inability of the haploid genome to deploy recombination repair in retrieving lost genetic information. Aberrant recombination, defective chromatin packaging, abortive apoptosis and oxidative stress may all be involved in the aetiology of DNA damage in the germ line. The factors responsible for Y chromosome deletions in spermatozoa remain unresolved but may be one facet of a central reproductive problem: controlling the amount of oxidative stress experienced by germ cells during their differentiation and maturation in the male reproductive tract.

Just how safe is assisted reproductive technology for treating, male factor infertility?

Publisher: Informa UK Limited

Date: 05-2008

DOI: 10.1586/17474108.3.3.267

On the Use of Paramagnetic Beads and Ferrofluids to Assess and Eliminate the Leukocytic Contribution to Oxygen Radical Generation by Human Sperm Suspensions

Publisher: Wiley

Date: 06-1996

DOI: 10.1111/J.1600-0897.1996.TB00055.X

Abstract: To develop a methodology to determine a) the leukocytic contribution to reactive oxygen species generation by human sperm suspensions and b) the therapeutic value of removing these cellular contaminants. Leukocytes were removed with paramagnetic beads or colloidal ferrofluids coated with anti-CD45 antibody. The sperm suspensions were monitored for oxidant generation by chemiluminescence, leukocyte contamination by immunocytochemistry, and fertilizing potential using zona-free hamster oocytes. Percoll -prepared human sperm suspensions exhibited a competence for PMA-induced reactive oxygen species generation which was significantly correlated with leukocyte contamination. However, the purified spermatozoa remaining after paramagnetic bead treatment, also demonstrated an intrinsic capacity for PMA-responsive reactive oxygen species generation and, freed from the oxidative stress created by the leukocytes, exhibited a significantly enhanced capacity for sperm-oocyte fusion. Although human spermatozoa can generate reactive oxygen species, sperm function is inhibited by the additional oxidative stress created by contaminating leukocytes. Removal of these cells with paramagnetic beads enhances fertilizing potential.

Transgenerational inheritance: how impacts to the epigenetic and genetic information of parents affect offspring health

Publisher: Oxford University Press (OUP)

Date: 02-08-2019

DOI: 10.1093/HUMUPD/DMZ017

Abstract: A defining feature of sexual reproduction is the transmission of genomic information from both parents to the offspring. There is now compelling evidence that the inheritance of such genetic information is accompanied by additional epigenetic marks, or stable heritable information that is not accounted for by variations in DNA sequence. The reversible nature of epigenetic marks coupled with multiple rounds of epigenetic reprogramming that erase the majority of existing patterns have made the investigation of this phenomenon challenging. However, continual advances in molecular methods are allowing closer examination of the dynamic alterations to histone composition and DNA methylation patterns that accompany development and, in particular, how these modifications can occur in an in idual’s germline and be transmitted to the following generation. While the underlying mechanisms that permit this form of transgenerational inheritance remain unclear, it is increasingly apparent that a combination of genetic and epigenetic modifications plays major roles in determining the phenotypes of in iduals and their offspring. Information pertaining to transgenerational inheritance was systematically reviewed focusing primarily on mammalian cells to the exclusion of inheritance in plants, due to inherent differences in the means by which information is transmitted between generations. The effects of environmental factors and biological processes on both epigenetic and genetic information were reviewed to determine their contribution to modulating inheritable phenotypes. Articles indexed in PubMed were searched using keywords related to transgenerational inheritance, epigenetic modifications, paternal and maternal inheritable traits and environmental and biological factors influencing transgenerational modifications. We sought to clarify the role of epigenetic reprogramming events during the life cycle of mammals and provide a comprehensive review of how the genomic and epigenomic make-up of progenitors may determine the phenotype of its descendants. We found strong evidence supporting the role of DNA methylation patterns, histone modifications and even non-protein-coding RNA in altering the epigenetic composition of in iduals and producing stable epigenetic effects that were transmitted from parents to offspring, in both humans and rodent species. Multiple genomic domains and several histone modification sites were found to resist demethylation and endure genome-wide reprogramming events. Epigenetic modifications integrated into the genome of in iduals were shown to modulate gene expression and activity at enhancer and promoter domains, while genetic mutations were shown to alter sequence availability for methylation and histone binding. Fundamentally, alterations to the nuclear composition of the germline in response to environmental factors, ageing, diet and toxicant exposure have the potential to become hereditably transmitted. The environment influences the health and well-being of progeny by working through the germline to introduce spontaneous genetic mutations as well as a variety of epigenetic changes, including alterations in DNA methylation status and the post-translational modification of histones. In evolutionary terms, these changes create the phenotypic ersity that fuels the fires of natural selection. However, rather than being adaptive, such variation may also generate a plethora of pathological disease states ranging from dominant genetic disorders to neurological conditions, including spontaneous schizophrenia and autism.

As the world grows: contraception in the 21st century

Publisher: American Society for Clinical Investigation

Date: 04-2008

DOI: 10.1172/JCI33873

Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition

Publisher: The Company of Biologists

Date: 15-07-2004

DOI: 10.1242/JCS.01214

Abstract: Mammalian spermatozoa undergo a series of molecular and biochemical changes collectively termed capacitation prior to acquiring the ability to fertilise the oocyte. Although phosphorylation of sperm proteins on tyrosine residues has been recognised as an important component of this process, the precise relationship between the phosphorylation status of mammalian spermatozoa and their capacity for fertilisation has remained unclear. In this study we demonstrate a causal relationship between tyrosine phosphorylation in spermatozoa and sperm-zona interaction. The phosphotyrosine expression associated with sperm capacitation localised to internal flagellar structures in permeabilised cells but could also be detected on the exterior surface of the sperm head in live cells. Importantly, almost all spermatozoa bound to the zona pellucida demonstrated this pattern of phosphoprotein localisation, compared to fewer than 15% of the free-swimming population. These data suggest that tyrosine phosphorylation plays a significant role in remodelling the sperm surface, so that these cells are able to recognise the zona pellucida. Phosphoproteome analysis yielded the first evidence of molecular chaperones, endoplasmin (erp99) and heat shock protein 60 (hsp60), as targets for phosphorylation on the surface of mouse spermatozoa, whereas immunofluorescence localised these proteins to the precise region of the sperm head that participates in zona recognition. Based on these results, we propose a novel mechanism for mammalian gamete interaction whereby the activation of sperm-surface chaperones by tyrosine phosphorylation during capacitation may trigger conformational changes facilitating the formation of a functional zona pellucida receptor complex on the surface of mammalian spermatozoa.

Analysis of the biological properties of antibodies raised against intact and deglycosylated porcine zonae pellucidae

Publisher: Wiley

Date: 04-1987

DOI: 10.1002/MRD.1120160407

Abstract: Zonae pellucidae (ZP) were isolated from 1,500 porcine ovaries and heat solubilized to generate approximately 15 mg ZP glycoprotein. Analysis of this material by isoelectric focusing, one-dimensional electrophoresis, and gas chromatography indicated the presence of a major glycoprotein species that exhibited considerable microheterogeneity with respect to its charge (pI 7.5-3.5) and molecular mass (45-85 kDa) and that contained 39.6% carbohydrate, predominantly N-acetylglucosamine. Chemical deglycosylation of porcine ZP using trifluoromethanesulphonic acid (TFMS) resulted in the production of five discrete protein bands on one-dimensional sodium dodecyl sulphate olyacrylamide gel electrophoresis (SDS/PAGE) with molecular masses of 66, 52, 36, 32, and 16 kDa. Antisera raised in rabbits and marmosets to ZP and/or deglycosylated ZP (DGZP) were used in immunoblotting experiments to demonstrate the retention of immunogenicity by DGZP and the cross-reactivity of the antisera with their heterologous antigen. These studies indicated that antisera that were capable of inhibiting the fertility of primates in vivo and the penetration of the human ZP in vitro reacted preferentially with 3 of the 5 products of deglycosylation, with molecular masses of 66, 52, and 36 kDa. Anti-DGZP antibodies were also shown to interact with intact porcine and human ZP and, with the latter, to block the ability of human spermatozoa to both bind to and penetrate this structure.

Cloning, sequencing and site of origin of the rat sperm receptor protein, ZP3

Publisher: Cambridge University Press (CUP)

Date: 02-1999

DOI: 10.1017/S0967199499000362

Abstract: The ZP3 gene encodes for a zona glycoprotein that serves as both a cell-specific binding site for capacitated spermatozoa and an inducer of acrosomal exocytosis during fertilisation. In this study we have determined the nucleotide sequence of rat ZP3 (accession no. Y10823), predicted primary amino acid structure and determined the cellular origin of this molecule within the ovary. Rat ZP3 was found to have an open reading frame of 1272 nucleotides encoding a polypeptide chain of 424 amino acids that was expressed exclusively by the actively growing oocyte population. Rat ZP3 exhibited 91%, 78% and 66% identity with the mouse, hamster and human homologues, respectively. Key features of mouse ZP3, including the number and location of cysteine and proline residues and N-linked glycosylation sites, were also conserved in the rat homologue. The putative O-linked glycosylation sites, a series of serine residues at ZP3(329-334), were also conserved in rat and mouse ZP3, although immediately downstream of this site the amino acid sequences deviated over a short stretch of amino acids. The hydropathicity profile revealed two hydrophobic domains. The first was associated with a putative N-terminal signal sequence which is unusual in the rat in possessing a proline residue at the -1 position relative to the signal cleavage site, a feature it shares with human and marmoset ZP3 but not mouse. The second hydrophobic domain was observed at the C-terminus downstream of a TGF-beta type III receptor domain that appears to be common to all ZP3 sequences examined to date.

Proteomic changes in mammalian spermatozoa during epididymal maturation

Publisher: Medknow

Date: 07-2007

DOI: 10.1111/J.1745-7262.2007.00280.X

Abstract: Epididymal maturation is associated with the activation of a cAMP-induced tyrosine phosphorylation cascade, which is ultimately associated with the expression of capacitation-dependent sperm functions, such as hyperactivated movement and acrosomal exocytosis. As spermatozoa progress through the epididymis they first acquire the capacity to phosphorylate tyrosine on targets on the principal piece, followed by the midpiece. By the time these cells have reached the cauda epididymidis they can phosphorylate the entire tail from neck to endpiece. This particular pattern of phosphorylation is associated with the ontogeny of fully functional spermatozoa that are capable of fertilizing the oocyte. Proteomic analyses indicate that this change is associated with the phosphorylation of several mitochondrial proteins, creation of a mitochondrial membrane potential and activation of mitochondrial free radical generation. At least in rodent species, activation of sperm mitochondria appears to be a particularly important part of epididymal maturation.

Oxidation of sperm nucleus in mammals: A physiological necessity to some extent with adverse impacts on oocyte and offspring

Publisher: MDPI AG

Date: 23-01-2020

DOI: 10.3390/ANTIOX9020095

Abstract: Sperm cells have long been known to be good producers of reactive oxygen species, while they are also known to be particularly sensitive to oxidative damage affecting their structures and functions. As with all organic cellular components, sperm nuclear components and, in particular, nucleic acids undergo oxidative alterations that have recently been shown to be commonly encountered in clinical practice. This review will attempt to provide an overview of this situation. After a brief coverage of the biological reasons why the sperm nucleus and associated DNA are sensitive to oxidative damage, a summary of the most recent results concerning the oxidation of sperm DNA in animal and human models will be presented. The study will then attempt to cover the possible consequences of sperm nuclear oxidation on male fertility and beyond.

Identification of gene products present in Triton X-100 soluble and insoluble fractions of human spermatozoa lysates using LC-MS/MS analysis

Publisher: Wiley

Date: 05-2007

DOI: 10.1002/PRCA.200601013

Abstract: A comprehensive analysis of the proteins found in human spermatozoa is essential for understanding the events leading up to, and including, fertilization and development. Proteomics offers a platform for investigating this process, provided that the dynamic range is relatively low. In this report, spermatozoa from a number of human sperm ejaculates were isolated in a pure state using discontinuous Percoll gradient centrifugation. Triton X-100 soluble and insoluble proteins were recovered and separated by SDS-PAGE. The separation lanes were dissected into 96 fractions and analyzed in idually by LC-MS(n) . A comprehensive protocol, involving LC-MS/MS analysis eventually down to the ninth most intense peak found in the MS-survey scan, was performed. Analysis of purified human sperm populations resulted in the identification of 1056 gene products, of which approximately 8% have not previously been characterized. The data were supported by the large number of proteins represented by expressed sequence tags in the testis. Bioinformatic analysis demonstrated that 437 of the gene products were involved in various metabolic pathways including glycolysis and oxidative phosphorylation. The inventory of proteins present in the human sperm proteome includes a number of notable discoveries including the first description of a nicotinamide adenine dinucleotide phosphate oxidase, dual-oxidase 2, finally laying to rest any doubts about the presence of such enzymes in spermatozoa. Furthermore, a number of different classes of receptor have also been detected in these cells and are potential regulators of sperm function. This list includes at least six seven-pass transmembrane receptors, six tyrosine kinase receptors, a tyrosine phosphatase receptor, glutamate-gated ion channel receptors, transient receptor potential cation channels, and a non-genomic progesterone receptor. This is the first published list of identified proteins in human spermatozoa using LC-MS/MS analysis.

Male reproductive ageing: a radical road to ruin

Publisher: Oxford University Press (OUP)

Date: 11-08-2023

DOI: 10.1093/HUMREP/DEAD157

Abstract: In modern post-transition societies, we are reproducing later and living longer. While the impact of age on female reproductive function has been well studied, much less is known about the intersection of age and male reproduction. Our current understanding is that advancing age brings forth a progressive decline in male fertility accompanied by a reduction in circulating testosterone levels and the appearance of age-dependent reproductive pathologies including benign prostatic hypertrophy and erectile dysfunction. Paternal ageing is also associated with a profound increase in sperm DNA damage, the appearance of multiple epigenetic changes in the germ line and an elevated mutational load in the offspring. The net result of such changes is an increase in the disease burden carried by the progeny of ageing males, including dominant genetic diseases such as Apert syndrome and achondroplasia, as well as neuropsychiatric conditions including autism and spontaneous schizophrenia. The genetic basis of these age-related effects appears to involve two fundamental mechanisms. The first is a positive selection mechanism whereby stem cells containing mutations in a mitogen-activated protein kinase pathway gain a selective advantage over their non-mutant counterparts and exhibit significant clonal expansion with the passage of time. The second is dependent on an age-dependent increase in oxidative stress which impairs the steroidogenic capacity of the Leydig cells, disrupts the ability of Sertoli cells to support the normal differentiation of germ cells, and disrupts the functional and genetic integrity of spermatozoa. Given the central importance of oxidative stress in defining the impact of chronological age on male reproduction, there may be a role for antioxidants in the clinical management of this process. While animal studies are supportive of this strategy, carefully designed clinical trials are now needed if we are to realize the therapeutic potential of this approach in a clinical context.

Standards in semen examination: publishing reproducible and reliable data based on high-quality methodology

Publisher: Oxford University Press (OUP)

Date: 16-09-2022

DOI: 10.1093/HUMREP/DEAC189

Abstract: Biomedical science is rapidly developing in terms of more transparency, openness and reproducibility of scientific publications. This is even more important for all studies that are based on results from basic semen examination. Recently two concordant documents have been published: the 6th edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen, and the International Standard ISO 23162:2021. With these tools, we propose that authors should be instructed to follow these laboratory methods in order to publish studies in peer-reviewed journals, preferable by using a checklist as suggested in an Appendix to this article.

Remodeling of the plasma membrane in preparation for sperm-egg recognition: roles of acrosomal proteins

Publisher: Medknow

Date: 2015

DOI: 10.4103/1008-682X.152817

Causes and consequences of apoptosis in spermatozoa; contributions to infertility and impacts on development

Publisher: UPV/EHU Press

Date: 2013

DOI: 10.1387/IJDB.130146JA

Abstract: During early development, apoptosis plays a major role in the ontogeny of the germ line as a means of regulating the germ cell:Sertoli cell ratio. In the adult, apoptosis fulfils another function in removing damaged germ cells from the seminiferous epithelium in response to a wide range of physiological and environmental triggers. These include various forms of electromagnetic radiation, chemotherapeutic agents and commonly encountered toxicants such as phthalate esters, bisphenol A and cadmium. This form of apoptosis can lead to spermatogenic arrest and is predominantly mediated by the Fas/FasL system. In addition, senescent mature spermatozoa can undergo a truncated form of apoptosis in order to ensure their efficient phagocytosis within the male and female reproductive tracts. This apoptotic cascade appears to be triggered by oxidative stress and lipid peroxidation, which leads to activation of mitochondrial reactive oxygen species (ROS) generation in a self-perpetuating redox cycle. The electrophilic aldehydes generated as a result of lipid peroxidation also lead to a rapid loss of sperm motility followed some hours later by caspase activation and phosphatidylserine exposure on the sperm surface. The nuclear DNA suffers oxidative damage during this process but there is no immediate DNA cleavage by endonucleases as there is in somatic cells. The reasons for this deviation from the normal pattern of apoptosis involve the unusual physical architecture of spermatozoa and the limited capacity these cells possess for base-excision repair. These findings have practical implications for the approaches that might be used to detect and prevent DNA damage in spermatozoa.

Do sperm find eggs attractive?

Publisher: Springer Science and Business Media LLC

Date: 05-1991

DOI: 10.1038/351019B0

Biosynthesis of the Canine Zona Pellucida Requires the Integrated Participation of Both Oocytes and Granulosa Cells1

Publisher: Oxford University Press (OUP)

Date: 08-2004

DOI: 10.1095/BIOLREPROD.104.028779

Endogenous redox activity in mouse spermatozoa and its role in regulating the tyrosine phosphorylation events associated with sperm capacitation

Publisher: Oxford University Press (OUP)

Date: 07-2003

DOI: 10.1095/BIOLREPROD.102.012716

Influence of caffeine on movement characteristics, fertilizing capacity and ability to penetrate cervical mucus of human spermatozoa

Publisher: Bioscientifica

Date: 1983

DOI: 10.1530/JRF.0.0670019

Abstract: When added to frozen-thawed human semen, the 3 doses of caffeine tested (2, 5 and 10 mM) induced a significant increase in the percentage of motile spermatozoa but did not influence the quality of movement. Considerable variability was noted between s les in their responsiveness to caffeine which, at the 5 and 10 mM doses, was significantly correlated with the degree of motility lost during cryostorage. Caffeine treatment of frozen-thawed human spermatozoa also increased the number of spermatozoa penetrating cervical mucus in unit time, by increasing the frequency rather than the success of collisions between spermatozoa and the cervical mucus interface. When caffeine-stimulated spermatozoa were washed free of seminal plasma containing this compound they were no longer at an advantage with respect to their motility or fertilizing ability. When 2 mM-caffeine was added to washed suspensions of capacitated spermatozoa it failed to stimulate motility but did significantly enhance the fertilizing ability of the spermatozoa, indicating a possible clinical role for this compound in in-vitro fertilization therapy.

The Impact of Sperm Metabolism duringIn VitroStorage: The Stallion as a Model

Publisher: Hindawi Limited

Date: 2016

DOI: 10.1155/2016/9380609

Abstract: In vitro sperm storage is a necessary part of many artificial insemination or in vitro fertilization regimes for many species, including the human and the horse. In many situations spermatozoa are chilled to temperatures between 4 and 10°C for the purpose of restricting the metabolic rate during storage, in turn, reducing the depletion of ATP and the production of detrimental by-products such as reactive oxygen species (ROS). Another result of lowering the temperature is that spermatozoa may be “cold shocked” due to lipid membrane phase separation, resulting in reduced fertility. To overcome this, a method of sperm storage must be developed that will preclude the need to chill spermatozoa. If a thermally induced restriction-of-metabolic-rate strategy is not employed, ATP production must be supported while ameliorating the deleterious effects of ROS. To achieve this end, an understanding of the nature of energy production by the spermatozoa of the species of interest is essential. Human spermatozoa depend predominantly on glycolytic ATP production, producing significantly less ROS than oxidative phosphorylation, with the more efficient pathway predominantly employed by stallion spermatozoa. This review provides an overview of the implications of sperm metabolism for in vitro sperm storage, with a focus on ambient temperature storage in the stallion.

Causes and consequences of oxidative stress in spermatozoa

Publisher: CSIRO Publishing

Date: 2016

DOI: 10.1071/RD15325

Abstract: Spermatozoa are highly vulnerable to oxidative attack because they lack significant antioxidant protection due to the limited volume and restricted distribution of cytoplasmic space in which to house an appropriate armoury of defensive enzymes. In particular, sperm membrane lipids are susceptible to oxidative stress because they abound in significant amounts of polyunsaturated fatty acids. Susceptibility to oxidative attack is further exacerbated by the fact that these cells actively generate reactive oxygen species (ROS) in order to drive the increase in tyrosine phosphorylation associated with sperm capacitation. However, this positive role for ROS is reversed when spermatozoa are stressed. Under these conditions, they default to an intrinsic apoptotic pathway characterised by mitochondrial ROS generation, loss of mitochondrial membrane potential, caspase activation, phosphatidylserine exposure and oxidative DNA damage. In responding to oxidative stress, spermatozoa only possess the first enzyme in the base excision repair pathway, 8-oxoguanine DNA glycosylase. This enzyme catalyses the formation of abasic sites, thereby destabilising the DNA backbone and generating strand breaks. Because oxidative damage to sperm DNA is associated with both miscarriage and developmental abnormalities in the offspring, strategies for the amelioration of such stress, including the development of effective antioxidant formulations, are becoming increasingly urgent.

Chromosome abnormalities in human embryos after in vitro fertilization

Publisher: Springer Science and Business Media LLC

Date: 05-1983

DOI: 10.1038/303336A0

Abstract: In vitro fertilization and embryo transfer is now an established treatment for certain forms of human infertility. High success rates have been reported following oocyte recovery and fertilization in vitro, the only major remaining difficulty being the high failure rate (80%) of implantation after replacement of the embryo into the mother. One possible reason for this failure of implantation is that some embryos may carry a lethal chromosome abnormality which does not allow development beyond the preimplantation stage. The need for information on the chromosomal status of early embryos fertilized and developed in vitro is recognized but two reported attempts to examine them have met with technical problems. Here we describe a method for examining chromosomes in 8-cell human embryos. Although we have made complete chromosome analyses for only three oocytes, our findings are surprising in that two of the three embryos were chromosomally abnormal. Data on the DNA content of the nuclei in a further eight cases showed that approximately 20% overall may be haploid.

Tyrosine Phosphorylation of HSP-90 during Mammalian Sperm Capacitation

Publisher: Oxford University Press (OUP)

Date: 12-2003

DOI: 10.1095/BIOLREPROD.103.017350

Selection of Peptides Targeting the Human Sperm Surface Using Random Peptide Phage Display Identify Ligands Homologous to ZP3

Publisher: Oxford University Press (OUP)

Date: 11-2000

DOI: 10.1095/BIOLREPROD63.5.1396

Abstract: Analysis of the surface architecture of human spermatozoa is a necessary step in the development of new approaches to contraception and resolving the causes of human infertility. In this study we have utilized phage display technology to identify peptides that bind with high affinity to the surface of human spermatozoa. Fifteen- and twelve-mer random peptide phage display libraries were screened against paraformaldehyde-fixed spermatozoa and a number of sperm-binding peptides were identified. One peptide, M6, displayed a high level of affinity for the sperm surface and showed sequence homology with a dominant human ZP3 epitope (hZP 25-33). This peptide bound preferentially to the equatorial and post acrosomal domains of the sperm head and exhibited contraceptive activity by virtue of its capacity to impair the fusion of acrosome-reacted spermatozoa with the vitelline membrane of the oocyte. A similar form of contraceptive activity was also observed within an unrelated peptide, K6, derived from screening the 12-mer library. These results indicate that phage display technology is a powerful tool for developing reagents capable of targeting the human sperm surface, providing insights into the composition of this structure and the identity of targets susceptible to contraceptive attack and pathological disruption.

Characterization of structure and expression of the Dzip1 gene in the rat and mouse

Publisher: Elsevier BV

Date: 02-2006

DOI: 10.1016/J.YGENO.2005.10.007

Abstract: A transcript encoding a rat homologue of DZIP1 (DAZ-interacting protein) was isolated from testis RNA. Like human DZIP1, it contains a C(2)H(2) zinc finger domain. A predicted mouse homologue of DZIP1 was found in the GenBank database. Genome analysis indicated that while DZIP1 and mouse Dzip1 contain 22 and 20 exons, respectively, the rat sequence was intronless, confirmed by PCR on genomic DNA. This rat Dzip1 sequence is homologous to mouse Dzip1 exons 1-6 and DZIP1 exons 5-9. As this rat sequence was shorter than DZIP1 it was designated rat Dzip1S. The rat genome also contained a further predicted homologue of DZIP1 displaying conserved linkage homology with mouse Dzip1 and DZIP1. This sequence, if expressed, is the true rat homologue of DZIP1, designated rat Dzip1. Rat Dzip1S mRNA was present in all tissues examined by qualitative RT-RCR, and in situ hybridization of rat testis confirmed that expression of rat Dzip1S mRNA was confined to the spermatogenic lineage, specifically premeiotic spermatogonia.

Proteomic insights into spermatozoa: Critiques, comments and concerns

Publisher: Informa UK Limited

Date: 12-2009

DOI: 10.1586/EPR.09.76

Abstract: Understanding the cellular mechanisms that regulate mammalian sperm function is strategically important for both the management of male infertility and the development of novel approaches to male contraception. The spermatozoon is a transcriptionally and translationally suppressed cell that is released from the testes in a functionally inert state. Functional activation occurs in the epididymis and female tract via mechanisms that are entirely dependent on post-translational modifications. Proteomics is, therefore, the ideal technology to investigate this cell type. Herein, we comment on the proteomic analyses that have been applied to mammalian spermatozoa, including some concerns relating to data interpretation. Three comprehensive liquid chromatography-mass spectrometry lists of human, mouse and rat spermatozoa are then compared, insights into the molecular regulation of sperm function discussed and future directions speculated upon.

Mass spectrometry, proteomics, and the study of sperm cell biology

Publisher: Springer New York

Date: 2013

DOI: 10.1007/978-1-4614-6651-2_18

Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding

Publisher: Wiley

Date: 28-10-2014

DOI: 10.1002/JCP.24728

Abstract: The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

Adjuncts in the IVF laboratory: where is the evidence for ‘add-on’ interventions?

Publisher: Oxford University Press (OUP)

Date: 31-01-2017

DOI: 10.1093/HUMREP/DEX004

Abstract: Globally, IVF patients are routinely offered and charged for a selection of adjunct treatments and tests or 'add-ons' that they are told may improve their chance of a live birth, despite there being no clinical evidence supporting the efficacy of the add-on. Any new IVF technology claiming to improve live birth rates (LBR) should, in most cases, first be tested in an appropriate animal model, then in clinical trials, to ensure safety, and finally in a randomized controlled trial (RCT) to provide high-quality evidence that the procedure is safe and effective. Only then should the technique be considered as 'routine' and only when applied to the similar patient population as those studied in the RCT. Even then, further pediatric and long-term follow-up studies will need to be undertaken to examine the long-term safety of the procedure. Alarmingly, there are currently numerous ex les where adjunct treatments are used in the absence of evidence-based medicine and often at an additional fee. In some cases, when RCTs have shown the technique to be ineffective, it is eventually withdrawn from the clinic. In this paper, we discuss some of the adjunct treatments currently being offered globally in IVF laboratories, including embryo glue and adherence compounds, sperm DNA fragmentation, time-lapse imaging, preimplantation genetic screening, mitochondria DNA load measurement and assisted hatching. We examine the evidence for their safety and efficacy in increasing LBRs. We conclude that robust studies are needed to confirm the safety and efficacy of any adjunct treatment or test before they are offered routinely to IVF patients.

The John Hughes Memorial Lecture: Aspects of Sperm Physiology—Oxidative Stress and the Functionality of Stallion Spermatozoa

Publisher: Elsevier BV

Date: 2014

DOI: 10.1016/J.JEVS.2013.10.120

Men’s Preconception Health: A Primary Health-Care Viewpoint

Publisher: SAGE Publications

Date: 18-05-2018

DOI: 10.1177/1557988318776513

Abstract: The purpose of this article is to theoretically explore men’s preconception health as a mechanism to enhance fertility, as well as the health and well-being of the subject and his descendants. Premorbid risk factors and behaviors associated with stress, environmental toxins, excessive alcohol consumption, smoking, lack of exercise/obesity, and the use of illicit drugs are all known to affect fecundity. While there are many health clinics available to women, where advice in areas such as postnatal care of the newborn, family planning, and couples fertility is provided, there are few, if any, equivalent health clinics available to men. Additionally, getting men to attend primary health-care services has also been continuously problematic, even in the context of there being a clearly discernible need for treatment. It is argued in this article that an impetus is required to encourage men to focus on and improve their preconception health and to utilize primary health-care services to take action. An assertive men’s preconception health outlook can positively influence the conjugal relationship, fathering, male self-esteem, and continued good health. Using the sometimes complex concept of preconception health as a motivating factor for healthy lifestyle adaptation has the potential to improve male fertility outcomes and general health and well-being, as well as the health of future generations.

Prospective controlled trial of an electrophoretic method of sperm preparation for assisted reproduction: comparison with density gradient centrifugation

Publisher: Oxford University Press (OUP)

Date: 09-2008

DOI: 10.1093/HUMREP/DEN330

Abstract: A membrane-based electrophoretic filtration system, known as the Cell Sorter-10 (CS-10), that preferentially isolates spermatozoa with very low levels of DNA damage has recently been developed. However, it remains to be proven whether spermatozoa prepared in this way are capable of achieving fertilization in assisted conception. Therefore, this clinical trial was designed to answer this question. A split-s le split-cohort study design was employed to control for differences in semen and oocyte quality between 28 couples undergoing either intracytoplasmic sperm injection (ICSI) or IVF in this clinical trial. Each semen s le was split between preparation using the CS-10 and preparation by standard density gradient centrifugation (DGC) and each cohort of oocytes was split for insemination using either CS-10 (n = 197) or DGC (n = 195) prepared spermatozoa. Both methods of sperm preparation yielded comparable rates of sperm recovery, motility and DNA fragmentation. There was no significant difference between the ability of CS-10 and DGC prepared spermatozoa to produce fertilization (62.4% versus 63.6%), cleavage (99.0% versus 88.5%) and high-quality embryos (27.4% versus 26.1%). This pilot study demonstrates that membrane-based electrophoresis is as effective as DGC in preparing sperm for IVF and ICSI, although it takes only a fraction of the time.

Electrophilic Aldehydes Generated by Sperm Metabolism Activate Mitochondrial Reactive Oxygen Species Generation and Apoptosis by Targeting Succinate Dehydrogenase

Publisher: Elsevier BV

Date: 09-2012

DOI: 10.1074/JBC.M112.366690

Conception and early pregnancy in the mare: lipidomics the unexplored frontier

Publisher: Bioscientifica

Date: 03-2022

DOI: 10.1530/RAF-21-0104

Abstract: Lipids are dynamic biological molecules that play key roles in metabolism, inflammation, cell signalling and structure. They are biologically significant in the physiology of conception and reproduction. Many of the mechanisms surrounding equine conception and the early feto-maternal dialogue are yet to be understood at a biochemical level. Recently, lipidomic technologies have advanced considerably and analytical strategies have been enhanced and ersified. Consequently, in-depth lipidomic exploration now has the potential to reveal new lipid biomarkers and biochemical relationships that improve our understanding of the processes leading to efficient and successful reproduction. This review considers the role of lipids in conception and establishment of pregnancy, providing new insights into the enigmatic pathways governing early reproductive physiology of the mare. This paper discusses the role that lipids play in the very early stages of pregnancy in the mare. Lipids are microscopic non-soluble molecules that are important components of living cells. The manuscript discusses how lipids influence the reproductive cycle of mares, including ovulation and the detailed biological process of becoming pregnant. It explains how lipids are identified in a laboratory setting with a newly developing technology known as ‘lipodomics’. The technology may lead to a more detailed understanding of how mares become pregnant. The focus of the paper is on mare reproduction, but it also draws on similarities with reproduction in other mammals. Remarkably there are gaps in much of our knowledge about the finer details of pregnancy in the horse, and the paper summarises what we already know about lipids, highlighting areas for further research.

Application of a Poisson-gamma model to study the influence of gamete concentration on sperm-oocyte fusion in the zona-free hamster egg penetration test

Publisher: Bioscientifica

Date: 11-1986

DOI: 10.1530/JRF.0.0780733

Abstract: A Poisson-gamma distribution model has been developed for studying sperm-oocyte interaction in the zona-free hamster egg penetration test. With the aid of this model we have analysed the relationship between the penetration results achieved with semen of normal fertile men and the concentration of spermatozoa and oocytes used in the assay. From this analysis we have drawn up reference tables estimating the number of spermatozoa and oocytes which must be present in the assay for the result to represent a significant (P less than 0.05) decline in sperm function relative to the normal fertile population. Since there is, as yet, no fixed protocol for the penetration assay, these tables have been constructed to fit a range of methodologies varying with respect to the nature of the preincubation phase and including (a) a 7-h preincubation in Medium BWW, (b) a 3-h preincubation in hyperosmotic Medium BWW or (c) a 3-h preincubation in the presence of A23187.

Definition of the optimal criteria for identifying hyperactivated human spermatozoa at 25 hz using in-vitro fertilization as a functional end-point

Publisher: Oxford University Press (OUP)

Date: 11-1995

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A135822

Abstract: Spermatozoa from fertile donors were used to select and validate criteria for the automated classification of hyperactivated (HA) motility using a computer-aided sperm analysis (CASA) system operating at 25 Hz. In the first phase of this analysis, 710 sperm trajectories were analysed and classified as forward progressive (FP), transition phase (TP) or HA. These tracks were then subjected to a CASA analysis and the various attributes of sperm movement defined for each category of motion. From this analysis, criteria were identified and subsequently validated that permitted the automated selection of FP, TP+HA and HA cells, with > 90% accuracy. To determine which attributes of movement best predicted the fertilizing potential of human spermatozoa, a detailed analysis of sperm motion was undertaken in patients undergoing in-vitro fertilization therapy. This analysis indicated that the single most important aspect of sperm movement was the incidence of HA motility (defined as curvilinear velocity > 90 microns/s, linearity 45.8 microns) after 3 h of incubation. Using the latter criterion as the first incorporated variable, multiple regression equations were created that explained up to 50% of the variance in fertilization rates. None of the other patterns of motion (FP, TP, TP+HA) was correlated with fertilization rates, and none of the other published algorithms for identifying HA cells were as efficient as those described here.

Cis-Unsaturated Fatty Acids Stimulate Reactive Oxygen Species Generation and Lipid Peroxidation in Human Spermatozoa

Publisher: The Endocrine Society

Date: 10-2006

DOI: 10.1210/JC.2006-1309

Abstract: Defective sperm function is the largest defined cause of human infertility however, the etiology of this condition is poorly understood. Although oxidative stress is acknowledged as a key contributor to this pathology, there are also data indicating that defective human spermatozoa contain abnormally high amounts of cis-unsaturated fatty acids. This study investigated whether a causative relationship exists between these two attributes of impaired semen quality. The objective of this study was to determine whether polyunsaturated fatty acids can induce oxidative stress in human spermatozoa. Dihydroethidium and SYTOX Green were used in conjunction with flow cytometry and HPLC to investigate reactive oxygen species (ROS) generation by human spermatozoa after fatty acid exposure. Arachidonic acid (AA) induced a time- and dose-dependent increase in ROS generation by human spermatozoa that led to the promotion of peroxidative damage and a loss of sperm motility. This effect could not be blocked with inhibitors of the cyclooxygenase or lipoxygenase pathways of AA metabolism, rotenone, protein kinase C antagonists, or known inhibitors of plasma membrane redox systems. However, ROS generation could be triggered with other cis-unsaturated fatty acids including linoleic and docosahexaenoic acids. Saturated fatty acids, methyl esters of unsaturated fatty acids, or other hiphiles were all ineffective. However in a cell-free system, AA could trigger a redox signal via mechanisms that were profoundly disrupted by diphenylene iodonium, a flavoprotein inhibitor. The presence of excess unsaturated fatty acids in defective human spermatozoa may precipitate the oxidative stress encountered in male infertility.

Quantitative analysis of gene-specific DNA damage in human spermatozoa

Publisher: Elsevier BV

Date: 08-2003

DOI: 10.1016/S0027-5107(03)00101-5

Abstract: Recent studies have suggested that human spermatozoa are highly susceptible to DNA damage induced by oxidative stress. However, a detailed analysis of the precise nature of this damage and the extent to which it affects the mitochondrial and nuclear genomes has not been reported. To induce DNA damage, human spermatozoa were treated in vitro with hydrogen peroxide (H2O2 0-5 mM) or iron (as Fe(II)SO4, 0-500 microM). Quantitative PCR (QPCR) was used to measure DNA damage in in idual nuclear genes (hprt, beta-pol and beta-globin) and mitochondrial DNA. Single strand breaks were also assessed by alkaline gel electrophoresis. H2O2 was found to be genotoxic toward spermatozoa at concentrations as high as 1.25 mM, but DNA damage was not detected in these cells with lower concentrations of H2O2. The mitochondrial genome of human spermatozoa was significantly (P<0.001) more susceptible to H2O2-induced DNA damage than the nuclear genome. However, both nDNA and mtDNA in human spermatozoa were significantly (P<0.001) more resistant to damage than DNA from a variety of cell lines of germ cell and myoblastoid origin. Interestingly, significant DNA damage was also not detected in human spermatozoa treated with iron. These studies report, for the first time, quantitative measurements of DNA damage in specific genes of male germ cells, and challenge the commonly held belief that human spermatozoa are particularly vulnerable to DNA damage.

Advantages of using the CRISPR/Cas9 system of genome editing to investigate male reproductive mechanisms using mouse models

Publisher: Medknow

Date: 2015

DOI: 10.4103/1008-682X.153851

Human spermatozoa: The future of sex

Publisher: Springer Science and Business Media LLC

Date: 02-2002

DOI: 10.1038/415963A

The extragenomic action of progesterone on human spermatozoa: evidence for a ubiquitous response that is rapidly down-regulated.

Publisher: The Endocrine Society

Date: 09-1996

DOI: 10.1210/ENDO.137.9.8756577

Abstract: Image analysis techniques have been used to demonstrate that progesterone induces a rapid calcium transient in the acrosomal domain of greater than 90% of human spermatozoa (n = 2354). These results are at variance with previous reports, suggesting that progesterone receptors are only expressed on a small subpopulation of these cells, by virtue of their ability to bind fluorescent probes incorporating progesterone 3- (O-carboxymethyl) oxime conjugated to BSA. In the present study, we could confirm that such probes only bound to a small proportion of human spermatozoa (3.01 +/- 0.29% n = 7557) although 91.79 +/- 1.8% of the same sperm populations exhibited a calcium transient in response to progesterone. These results indicate that the binding of labeled progesterone conjugates to human spermatozoa does not reflect the size of the progesterone responsive population the response elicited by this steroid is essentially ubiquitous. Progesterone action was shown to involve an influx of extracellular calcium via mechanisms that did not involve voltage sensitive- or second messenger operated-channels, phospholipase C, or G proteins. Despite previous evidence suggesting that progesterone action might involve a GABAA receptor/chloride channel, neither GABA nor the GABA agonist muscimol had any effect on intracellular calcium concentrations in human spermatozoa or influenced their functional competence. The only factor that disrupted the responses of human spermatozoa to progesterone was this steroid itself. Progesterone exposure induced a prolonged period of refractoriness to further stimulation that influenced the capacity of these cells to generate calcium transients, and their ability to exhibit a biological response to changes in intracellular calcium. There are implications in these results for our understanding of the extragenomic action of progesterone on human spermatozoa and the clinical manipulation of this system for the assessment and suppression of human sperm function.

Are sperm capacitation and apoptosis the opposite ends of a continuum driven by oxidative stress?

Publisher: Medknow

Date: 2015

DOI: 10.4103/1008-682X.153850

Residual sperm function in oligozoospermia induced by testosterone enanthate administered as a potential steroid male contraceptive

Publisher: Wiley

Date: 10-1992

DOI: 10.1111/J.1365-2605.1992.TB01356.X

Abstract: To investigate the fertility of men who remain oligozoospermic despite sex steroid suppression, the in-vitro fertilizing capacity of residual spermatozoa was assessed in 30 men receiving intramuscular testosterone enanthate (TE). Spermatozoa were prepared by either Percoll or repetitive centrifugation/washing. Although the mean (+/- SEM) pretreatment zona-free hamster oocyte penetration (HOP) rates were similar (59.4 +/- 10.1 and 63.8 +/- 10.8%), following the induction of oligozoospermia the Percoll-prepared spermatozoa exhibited a penetration rate (26.9 +/- 10.2%) which was markedly greater than that obtained for sperm prepared by repetitive washing (0 +/- 0%). In addition, the partners of two men exhibiting a HOP test with Percoll-prepared spermatozoa, conceived despite a sperm concentration of 3 x 10(6) ml-1 and a negative HOP test with spermatozoa prepared by repetitive washing. These results suggest that Percoll preparation optimizes the assessment of in-vitro sperm function and that the fertility of men with TE-induced severe oligozoospermia is suppressed but not abolished.

The 12 ^th International Symposium on Spermatology

Publisher: Medknow

Date: 2015

DOI: 10.4103/1008-682X.153852

Novel characterization of the HSPA2-stabilizing protein BAG6 in human spermatozoa

Publisher: Oxford University Press (OUP)

Date: 07-07-2015

DOI: 10.1093/MOLEHR/GAV041

Abstract: While a large cohort of sperm surface receptors underpin sperm-oocyte adhesion processes, our recent work has revealed that the molecular chaperone Heat Shock Protein A2 (HSPA2) is a key regulator of zona pellucida-receptor complex assembly in our own species. Indeed, in the infertile population, spermatozoa that fail to interact with the zona pellucida of the oocyte consistently lack HSPA2 protein expression. While the mechanisms behind this protein deficiency are under consideration, BCL2-associated athanogene 6 (BAG6) has been identified as a key regulator of HSPA2 stability in mouse germ cells. However, in the human, the presence of BAG family proteins remains completely uncharacterized. Consequently, this study aimed to determine the presence of BAG6 in human sperm cells and to characterize its putative interaction with HSPA2 throughout sperm cell development. BAG6 was shown to co-localize with HSPA2 in human testicular germ cells and epididymal spermatozoa. Similarly, BAG6 was identified in the equatorial region of non-capacitated spermatozoa but underwent a marked relocation to the anterior region of the head upon the induction of capacitation in these cells. Protein-protein interaction assays revealed the stable interaction of BAG6 and HSPA2 proteins in mature spermatozoa. Furthermore, examination of the spermatozoa of infertile men with zona pellucida binding defects, related to a lack of HSPA2, revealed a concomitant deficiency in BAG6 protein expression. In view of the findings described in this study, we propose that BAG6 is likely a key regulator of HSPA2 stability/function in human germ cells. Moreover, its under-representation in spermatozoa with zona pellucida binding deficiency suggests that BAG6 may be an important candidate to study for a further understanding of male idiopathic infertility.

Melatonin Prevents Postovulatory Oocyte Aging in the Mouse and Extends the Window for Optimal Fertilization In Vitro1

Publisher: Oxford University Press (OUP)

Date: 03-2013

DOI: 10.1095/BIOLREPROD.112.106450

Abstract: The quality of metaphase II oocytes deteriorates rapidly following ovulation as the result of an aging process associated with impaired fertilizing potential, disrupted developmental competence, and increased likelihood of embryonic resorption. Because oxidative stress accelerates the onset of apoptosis in oocytes and influences their capacity for fertilization, this study aimed to characterize the significance of such stress in the postovulatory aging of mouse oocytes in vitro. We investigated the ability of the potent antioxidant melatonin to arrest the aging process when used to supplement oocyte culture medium. This study demonstrated that oxidative stress may occur in oocytes after as little as 8 h in culture and coincides with the appearance of early apoptotic markers such as phosphatidylserine externalization, followed 16 h later by caspase activation (P < 0.05) and morphological evidence of oocyte senescence. Importantly, supplementation of oocyte culture medium with 1 mM melatonin was able to significantly relieve the time-dependent appearance of oxidative stress in oocytes (P < 0.05) and, as a result, significantly delay the onset of apoptosis (P < 0.05). Furthermore, melatonin supplementation extended the optimal window for fertilization of oocytes aged for 8 and 16 h in vitro (P < 0.05) and significantly improved the quality of the resulting embryos (P < 0.01). We conclude that melatonin may be a useful tool in a clinical setting to prevent the time-dependent deterioration of oocyte quality following prolonged culture in vitro.

DNA damage and repair in the female germline: contributions to ART.

Publisher: Oxford University Press (OUP)

Date: 12-12-2019

DOI: 10.1093/HUMUPD/DMY040

Abstract: DNA integrity and stability are critical determinants of cell viability. This is especially true in the female germline, wherein DNA integrity underpins successful conception, embryonic development, pregnancy and the production of healthy offspring. However, DNA is not inert rather, it is subject to assault from various environment factors resulting in chemical modification and/or strand breakage. If structural alterations result and are left unrepaired, they have the potential to cause mutations and propagate disease. In this regard, reduced genetic integrity of the female germline ranks among the leading causes of subfertility in humans. With an estimated 10% of couples in developed countries taking recourse to ART to achieve pregnancy, the need for ongoing research into the capacity of the oocyte to detect DNA damage and thereafter initiate cell cycle arrest, apoptosis or DNA repair is increasingly more pressing. This review documents our current knowledge of the quality control mechanisms utilised by the female germline to prevent and remediate DNA damage during their development from primordial follicles through to the formation of preimplantation embryos. The PubMed database was searched using the keywords: primordial follicle, primary follicle, secondary follicle, tertiary follicle, germinal vesical, MI, MII oocyte, zygote, preimplantation embryo, DNA repair, double-strand break and DNA damage. These keywords were combined with other phrases relevant to the topic. Literature was restricted to peer-reviewed original articles in the English language (published 1979-2018) and references within these articles were also searched. In this review, we explore the quality control mechanisms utilised by the female germline to prevent, detect and remediate DNA damage. We follow the trajectory of development from the primordial follicle stage through to the preimplantation embryo, highlighting findings likely to have important implications for fertility management, age-related subfertility and premature ovarian failure. In addition, we survey the latest discoveries regarding DNA repair within the metaphase II (MII) oocyte and implicate maternal stores of endogenous DNA repair proteins and mRNA transcripts as a primary means by which they defend their genomic integrity. The collective evidence reviewed herein demonstrates that the MII oocyte can engage in the activation of major DNA damage repair pathway(s), therefore encouraging a reappraisal of the long-held paradigm that oocytes are largely refractory to DNA repair upon reaching this late stage of their development. It is also demonstrated that the zygote can exploit a number of protective strategies to mitigate the risk and/or effect the repair, of DNA damage sustained to either parental germline affirming that DNA protection is largely a maternally driven trait but that some aspects of repair may rely on a collaborative effort between the male and female germlines. The present review highlights the vulnerability of the oocyte to DNA damage and presents a number of opportunities for research to bolster the stringency of the oocyte's endogenous defences, with implications extending to improved diagnostics and novel therapeutic applications to alleviate the burden of infertility.

Evaluation of a spectrophotometric assay for the measurement of malondialdehyde and 4‐hydroxyalkenals in human spermatozoa: relationships with semen quality and sperm function

Publisher: Wiley

Date: 05-1998

DOI: 10.1046/J.1365-2605.1998.00106.X

Abstract: A spectrophotometric assay for the measurement of malondialdehyde and 4 hydroxyalkenals (MA + 4HA) has been evaluated for the detection of sperm pathologies involving oxidative stress. In order to make sensitive measurements of MA + 4HA on human spermatozoa, the stimulation of a lipid peroxidation cascade with a ferrous ion promoter was found to be necessary. The optimal configuration for the promoter was defined (0.64 mM FeSO4 + 20 mM ascorbate for 2 h in Ca2+ and Mg2 free Hanks' balanced salt solution) and the assay used in a series of studies to elucidate the functional significance of MA + 4HA determinations. Such measurements were found to give highly significant correlations (p < 0.001) with the loss of motility induced by oxidative stress created either with a xanthine oxidase, free radical generating system or by prolonged incubation under aerobic conditions. Experiments involving the stimulation and suppression of lipid peroxide release from human sperm suspensions, in concert with a bioassay for cytotoxicity, confirmed the strength and causative nature of these associations. Measurements of lipid peroxidation potential in highly purified, leucocyte-free sperm suspensions revealed the presence of inverse correlations with the motility of the spermatozoa, their viability, their competence for sperm-oocyte fusion and, most significantly, the quality of sperm movement in the original semen s les. Similar negative correlations were observed between sperm function and phorbol ester-stimulated reactive oxygen species generation but, unlike the MA + 4HA determinations, these relationships were obfuscated by the presence of leucocytes. We conclude that the measurement of MA + 4HA in human spermatozoa provides important information on the underlying quality of spermatogenesis and should be of value in the clinical diagnosis of infertility involving oxidative stress and the selection of patients for antioxidant therapy.

From Peptide Masses to Pregnancy Maintenance: A Comprehensive Proteomic Analysis of The Early Equine Embryo Secretome, Blastocoel Fluid, and Capsule

Publisher: Wiley

Date: 09-2017

DOI: 10.1002/PMIC.201600433

Abstract: Early pregnancy in the mare is a poorly understood, high risk period during which the embryo communicates its presence to the maternal endometrium. Remarkably, the maternal recognition of pregnancy signal is unknown in the horse. This study aimed to profile the proteins secreted by equine blastocysts into their immediate environment, along with proteins contained in the blastocoel and within the acellular embryo capsule. Embryos were recovered on day 8 after ovulation and cultured for 48 hours. Secretomes of day 9 and day 10 embryos were analyzed by LC-MS/MS and supported by analysis of blastocoel fluid and embryo capsule. Analyses revealed 72 (24 h) and 97 (48 h) unique protein IDs in the embryo secretome, 732 protein IDs in blastocoel fluid, and 11 proteins IDs in the embryo capsule. Novel findings of interest include secretion of a pregnancy specific proteinase (PAG) by the equine embryo at day 10, along with detection of a prostaglandin receptor inhibiting protein (PTGFRN) and a progesterone potentiating factor (FKBP4) in blastocoel fluid. This is the first comprehensive proteomic analysis of the equine embryo secretome, and provides new insights into the unique physiology of early pregnancy in this species.

COVID‐19 and male infertility: An update

Publisher: Wiley

Date: 31-08-2021

DOI: 10.1111/ANDR.13098

The extragenomic action of progesterone on human spermatozoa is influenced by redox regulated changes in tyrosine phosphorylation during capacitation

Publisher: Elsevier BV

Date: 03-1996

DOI: 10.1016/0303-7207(95)03733-0

Abstract: Capacitation had no effect on the ability of progesterone to elicit a rapid calcium transient in the acrosomal domain of human spermatozoa but had a marked influence of the ability of this steroid to induce a biological response. The development of this responsiveness to progesterone appeared to be redox regulated in that it was promoted by the stimulation of reactive oxygen species generation and inhibited by the presence of antioxidants, including catalase and membrane permeant thiols. The ability of redox conditions to influence the biological responsiveness of human spermatozoa did not involve changes in the dynamics of the calcium transients induced by progesterone but was causally linked with clear differences in tyrosine phosphorylation. We conclude that the ability of human spermatozoa to respond to the calcium transients induced by progesterone depends on a background of phosphotyrosine expression that can be profoundly influenced by the redox status of the spermatozoa during capacitation.

Oxidative stress and human spermatozoa: diagnostic and functional significance of aldehydes generated as a result of lipid peroxidation

Publisher: Oxford University Press (OUP)

Date: 02-04-2015

DOI: 10.1093/MOLEHR/GAV014

Abstract: Oxidative stress is known to compromise human sperm function and to activate the intrinsic apoptotic cascade in these cells. One of the key features of oxidatively stressed spermatozoa is the induction of a lipid peroxidation process that results in the formation of aldehydes potentially capable of disrupting sperm function through the formation of adducts with DNA and key proteins. In this study, we have examined the impact of a range of small molecular mass aldehydes generated as a consequence of lipid peroxidation on human sperm function and also compared the two most commonly formed compounds, 4-hydroxynonenal (4HNE) and malondialdehyde (MDA), for their relative ability to reflect a state of oxidative stress in these cells. Dramatic differences in the bioactivity of in idual aldehydes were observed, that generally correlated with the second order rate constants describing their interaction with the model nucleophile, glutathione. Our results demonstrate that acrolein and 4HNE were the most reactive lipid aldehydes, inhibiting sperm motility while augmenting reactive oxygen species production, lipid peroxidation, oxidative DNA damage and caspase activation, in a dose-dependent manner (P < 0.001). In contrast, a variety of saturated aldehydes and the well-known marker of oxidative stress, MDA, were without effect on this cell type. While MDA was not cytotoxic per se, its generation did reflect the induction of oxidative stress in vivo and in vitro in a manner that was highly correlated with the bioactive lipid aldehyde, 4HNE. Despite such overall correlations, in idual patient s les were observed in which either MDA or 4HNE predominated. Given the relative cytotoxicity of 4HNE, we propose that this aldehyde should be the preferred criterion for diagnosing oxidative stress in the male germ line.

Protein Kinase C- and Ca2+ Ionophore-Stimulated Production of Reactive Oxygen Species in Mechanically Dispersed Isolated Bovine Luteal Cells1

Publisher: Oxford University Press (OUP)

Date: 08-1997

DOI: 10.1095/BIOLREPROD57.2.428

Abstract: We measured the production of reactive oxygen species (ROS) using luminol-horseradish peroxidase-induced chemiluminescence in mechanically dispersed cell suspensions from bovine corpus luteum (CL). Since other cell types besides luteal cells were present in crude cell suspensions from CL, cell preparations were purified by centrifugation on Percoll. Only cell suspensions that gave no significant response when stimulated with formyl-methionyl-leucyl-phenylalanine, a potent stimulator of ROS production by phagocytes, were used routinely. Basal ROS production by purified bovine luteal cell preparations was low but could be stimulated rapidly and in a dose-dependent manner by nanomolar concentrations of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA), though only in cells purified by fractionation on Percoll. Luteal ROS responses to PMA were quenched by returning bovine erythrocytes to purified luteal cells, or by exogenous catalase or superoxide dismutase. The magnitude of the response to PMA varied markedly from one luteal cell preparation to another but appeared to be unrelated to the stage of the luteal phase of the CL from which the cells were prepared. The luteal ROS response to PMA was blocked by staurosporine, an inhibitor of PKC. Although the inactive phorbol ester (4alpha-phorbol didecanoate 4alphaPDD) alone had little or no effect on luteal ROS production, 4alphaPDD significantly potentiated the effects of submaximal concentrations of PMA in a dose-dependent manner. ROS production could also be stimulated by the Ca2+ ionophore A23187. This response was rapidly abolished by treatment with EDTA or EGTA. A23187 also augmented the response to submaximal PMA levels: however, pretreatment with 4alphaPDD did not significantly enhance the ROS response to A23187. In conclusion, we have shown that isolated bovine luteal cell suspensions are capable of generating a marked acute ROS response triggered by activation of PKC and/or elevation of cytosolic calcium.

Mechanism of sperm‐binding inhibition by anti‐zona antisera

Publisher: Wiley

Date: 1981

DOI: 10.1002/MRD.1120040107

Clinical evaluation of the zona‐free hamster egg penetration test in the management of the infertile couple. Prospective and retrospective studies

Publisher: Wiley

Date: 10-1986

DOI: 10.1111/J.1365-2605.1986.TB00948.X

Paradoxical stimulation of human sperm motility by 2-deoxyadenosine

Publisher: Bioscientifica

Date: 11-1986

DOI: 10.1530/JRF.0.0780515

Abstract: Exposure of cryostored human spermatozoa to 2-deoxyadenosine resulted in significant increases in percentage motility, the linear velocity of progression and the frequency of sperm head rotation, which were maximal at a dose of 2.5 mM. At the same dose both adenosine and caffeine significantly increased percentage motility, although neither compound influenced the quality of sperm movement as assessed by time-exposure photomicrography. 2-Deoxyadenosine was also significantly more effective than caffeine in sustaining the motility of cryostored spermatozoa as well as in enhancing the motility of fresh and washed preparations of human spermatozoa. The ability of caffeine and 2-deoxyadenosine to influence sperm motility was counteracted by the presence of calcium in the external medium although the latter was less susceptible to such inhibition and still enhanced motility in the presence of calcium levels (1.7 mM) typical of media used for in-vitro fertilization. The mechanism of action of 2-deoxyadenosine was associated with an increase of intracellular cAMP levels, which were sustained over a time course lasting from 5 to 180 min and exhibited significant dose dependency over the range 1-10 mM. The response to 2-deoxyadenosine did not involve any changes in the steady state levels of ATP and was augmented by the presence of the phosphodiesterase inhibitors, IBMX and caffeine. We conclude that 2-deoxyadenosine is a powerful stimulator of human sperm motility and that this effect involves an increase of intracellular cAMP levels via mechanisms which do not involve the classical 'R'-site receptor mediated pathway.

Seeds of concern

Publisher: Springer Science and Business Media LLC

Date: 11-2004

DOI: 10.1038/432048A

A free radical theory of male infertility

Publisher: CSIRO Publishing

Date: 1994

DOI: 10.1071/RD9940019

Abstract: There is a growing body of evidence to indicate that a significant factor in the aetiology of male infertility involves a loss of sperm function as a consequence of oxidative stress. This stress originates from the excessive generation of reactive oxygen species by the spermatozoa and results in the peroxidation of unsaturated fatty acids in the sperm plasma membrane. It is possible that reactive oxygen species originating from infiltrating leucocytes could also stress the spermatozoa although the protective properties of seminal plasma would render this unlikely in vivo. Whatever the source of the reactive oxygen species, the lipid peroxides thereby generated exhibit powerful negative correlations with the movement characteristics of the spermatozoa and their capacity for sperm-oocyte fusion. These findings should have important implications for the development of rational techniques for the diagnosis and treatment of male infertility.

Investigation of the stallion sperm proteome by mass spectrometry

Publisher: Bioscientifica

Date: 03-2015

DOI: 10.1530/REP-14-0500

Abstract: Stallion spermatozoa continue to present scientific and clinical challenges with regard to the biological mechanisms responsible for their survival and function. In particular, deeper understanding of sperm energy metabolism, defence against oxidative damage and cell–cell interactions should improve fertility assessment and the application of advanced reproductive technologies in the equine species. In this study, we used highly sensitive LC–MS/MS technology and sequence database analysis to identify and characterise the proteome of Percoll-isolated ejaculated equine spermatozoa, with the aim of furthering our understanding of this cell's complex biological machinery. We were able to identify 9883 peptides comprising 1030 proteins, which were subsequently attributed to 975 gene products. Gene ontology analysis for molecular and cellular processes revealed new information about the metabolism, antioxidant defences and receptors of stallion spermatozoa. Mitochondrial proteins and those involved in catabolic processes constituted dominant categories. Several enzymes specific to β-oxidation of fatty acids were identified, and further experiments were carried out to ascertain their functional significance. Inhibition of carnitine palmitoyl transferase 1, a rate-limiting enzyme of β-oxidation, reduced motility parameters, indicating that β-oxidation contributes to maintenance of motility in stallion spermatozoa.

Human spermatozoa possess an IL4I1 l-amino acid oxidase with a potential role in sperm function.

Publisher: Bioscientifica

Date: 06-2015

DOI: 10.1530/REP-14-0621

Abstract: Reactive oxygen species (ROS) are known to play an important role in the regulation of human sperm function. In this study, we demonstrate for the first time that human spermatozoa possess interleukin-induced gene 1 (IL4I1), an l -amino acid oxidase (LAAO) which is capable of generating ROS on exposure to aromatic amino acids in the presence of oxygen. The preferred substrates were found to be phenylalanine and tryptophan while the enzyme was located in the acrosomal region and midpiece of these cells. In contrast to equine and bovine spermatozoa, enzyme activity was lost as soon as the spermatozoa became non-viable. On a cell-to-cell basis human spermatozoa were also shown to generate lower levels of hydrogen peroxide than their equine counterparts on exposure to phenylalanine. Stimulation of LAAO activity resulted in the induction of several hallmarks of capacitation including tyrosine phosphorylation of the sperm flagellum and concomitant activation of phospho-SRC expression. In addition, stimulation of LAAO resulted in an increase in the levels of acrosomal exocytosis in both the presence and absence of progesterone stimulation, via mechanisms that could be significantly reversed by the presence of catalase. As is often the case with free radical-mediated phenomena, prolonged exposure of human spermatozoa to phenylalanine resulted in the stimulation of apoptosis as indicated by significant increases in mitochondrial superoxide generation and the activation of intracellular caspases. These results confirm the existence of an LAAO in human spermatozoa with a potential role in driving the redox regulation of sperm capacitation and acrosomal exocytosis.

On the nature of semen quality and infertility

Publisher: Oxford University Press (OUP)

Date: 02-1995

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A135922

Stimulation of mitochondrial reactive oxygen species production by unesterified, unsaturated fatty acids in defective human spermatozoa

Publisher: Elsevier BV

Date: 2010

DOI: 10.1016/J.FREERADBIOMED.2009.10.033

Abstract: Male infertility is a relatively common condition affecting 1 in 20 men of reproductive age. The etiology of this condition is thought to involve the excessive generation of reactive oxygen species by human spermatozoa however, the cause of this aberrant activity is unknown. In this study we demonstrate that defective human sperm populations are characterized by high cellular contents of both esterified and unesterified fatty acids and a decrease in the proportion of the total fatty acid pool made up by docosahexaenoic acid. The free unsaturated fatty acid content of these cells was positively correlated with the induction of mitochondrial superoxide generation (P<0.001). This relationship was causal and mediated by the range of unesterified, unsaturated fatty acids that are present in human spermatozoa. Thus direct exposure of these cells to free unsaturated fatty acids stimulated mitochondrial superoxide generation and precipitated a loss of motility and an increase in oxidative DNA damage, two key attributes of male infertility. We conclude that defective human spermatozoa are characterized by an abnormally high content of fatty acids that, in their unesterified, unsaturated form, promote ROS generation by sperm mitochondria, creating a state of oxidative stress and a concomitant loss of functional competence.

Introduction

Publisher: Elsevier

Date: 2018

DOI: 10.1016/B978-0-12-812501-4.02001-0

An analysis of the direct effects of gossypol on human spermatozoa

Publisher: Wiley

Date: 04-1983

DOI: 10.1111/J.1365-2605.1983.TB00334.X

Abstract: The direct effects of gossypol and its acetic acid adduct, on the movement and functional competence of human spermatozoa were investigated employing exposure times of 1, 5 or 15 min and concentrations of 50 microM, 500 microM and 1000 microM. These compounds markedly reduced the motility, velocity, frequency of sperm head rotation and linearity of sperm progression, the most significant effects being observed with gossypol acetic acid on populations of 'capacitated' spermatozoa. Significant direct effects of gossypol on the ability of human spermatozoa to penetrate both cervical mucus and zona-free hamster ova were also observed, which were independent of any effects on motility. These results reinforce the notion that gossypol may serve a contraceptive role in the female as a 'spermicidal' agent, and suggest that this compound may also be of scientific value as a probe for identifying and isolating functionally important components of the human spermatozoon.

Role of Oxidative Stress in the Etiology of Male Infertility and the Potential Therapeutic Value of Antioxidants

Publisher: Elsevier

Date: 2019

DOI: 10.1016/B978-0-12-812501-4.00010-9

Studies on Men's Health and Fertility

Publisher: Humana Press

Date: 2012

DOI: 10.1007/978-1-61779-776-7

Chemokine receptor-dependent control of skin tissue-resident memory T Cell formation

Publisher: The American Association of Immunologists

Date: 10-2017

DOI: 10.4049/JIMMUNOL.1700571

Abstract: Infection or inflammation of the skin recruits effector CD8+ T cells that enter the epidermis and form populations of long-lived tissue-resident memory T (TRM) cells. These skin TRM cells migrate within the constrained epidermal environment by extending multiple dynamic dendritic projections and squeezing between keratinocytes to survey the tissue for pathogens. In this study, we examined the signals required for this distinctive mode of T cell migration by inhibiting key cytoskeletal components and performing intravital two-photon microscopy to visualize TRM cell behavior. We found that TRM cell motility and dendrite formation required an intact actomyosin cytoskeleton and the Rho-associated coiled-coil containing kinases. We also identified an essential role for microtubules for maintaining skin TRM cell shape and cellular integrity. We reveal a role for pertussis toxin–sensitive signaling for TRM cell dendritic morphology and migration that is independent of CXCR3 or CXCR6, or the skin-selective chemokine receptors CCR10 and CCR8. However, we found that CXCR6 and CCR10 expression by CD8+ T cells was required for the optimal formation of memory T cell populations, in particular TRM cell populations in the skin.

Stallion fertility: A focus on the spermatozoon

Publisher: Wiley

Date: 18-08-2014

DOI: 10.1111/EVJ.12308

Abstract: Stallion fertility is a vast subject, with a wide array of permutations that can impact reproductive performance in either positive or negative ways. This review is intended to address a mere segment of the male fertility issue, but the very essence of the male contribution to fertilisation, that of the spermatozoon. Spermatozoal ultrastructure and form-to-function are detailed and spermatozoal metabolism is discussed, with specific reference to distinctive characteristics of stallion spermatozoa. Lastly, methods for assessment of spermatozoal function are considered, with emphasis on spermatozoal motility, the acrosome reaction and spermatozoon-oocyte interactions. Closing comments address the need for development and standardisation of molecular-based assays for use with spermatozoa of stallions whose subfertility cannot be explained with conventional tests.

Life and death in the germ line: Apoptosis and the origins of dna damage in human spermatozoa

Publisher: Cambridge University Press

Date: 03-02-2011

DOI: 10.1017/CBO9780511921896.012

The contraceptive potential of ZP3 and ZP3 peptides in a primate model

Publisher: Elsevier BV

Date: 2002

DOI: 10.1016/S0165-0378(01)00105-X

Abstract: It has been known for some time that antibodies raised against ZP3, the major component of the glycoprotein shell that surrounds all mammalian oocytes, can successfully inhibit sperm-egg interaction in vitro. In our own studies using the non-human primate Callithrix jacchus, active immunisation was successfully achieved when homologous or heterologous ZP3 was used as an immunogen. However this long-term suppression of fertility was at the expense of ovarian function. An ovarian pathology was observed which was characterised by a disruption of folliculogenesis and depletion of the primordial follicle pool. Adverse auto-immune reactions have also been observed in mice following induction of immunity to mouse ZP3. Following careful selection of B-cell epitopes on mouse ZP3, peptide vaccines were formulated which could circumvent these adverse side effects and induce reversible infertility in actively immunised mice. To identify similar epitopes on primate ZP3, epitope mapping studies were performed and several candidate regions of the molecule were identified. These were incorporated into chimeric peptide vaccines and administered as single or triple peptide vaccines. Active immunisation successfully induced antibodies that bound exclusively to the zona pellucida of marmoset and human ovarian sections. These antibodies were able to suppress human sperm-egg binding by up to 60% in vitro. Encouragingly, no adverse side effects on ovarian function were observed following long-term immunisation however, no loss of fertility was consistently observed in vivo. Thus considerable research is still required to identify a combination of ZP epitopes that will induce reversible infertility in the absence of any ovarian dysfunction.

Molecular basis of sperm-egg interaction and the prospects for immunocontraception

Publisher: Elsevier BV

Date: 11-1998

DOI: 10.1016/S0165-0378(98)00039-4

Abstract: As the molecular basis of sperm-egg interaction is resolved, so new opportunities are created for the development of immunological approaches to disrupt the process of conception. Thus, realisation that the zona glycoprotein, ZP3, serves as a specific receptor for spermatozoa, has prompted a detailed examination of its contraceptive potential. In primate models, recombinant ZP3 has been shown to suppress fertility very efficiently, however this efficacy is tempered by the appearance of adverse side-effects involving accelerated primordial follicle depletion and a lymphocytic infiltration of the ovarian stroma. Synthetic peptides encoding B-cell epitopes have been found to circumvent the lymphocyte response although the effectiveness of such reagents in preventing the loss of primordial follicles has not yet been determined. The induction of active immunity against sperm-specific antigens has also been shown to generate long term infertility in both males and females. Molecular and immunological techniques are now being used to produce a rapidly expanding list of unique sperm antigens which are currently being evaluated to determine their potential contribution to the development of safe, effective, contraceptive vaccines.

Identification of RARhoGAP, a novel putative RhoGAP gene expressed in male germ cells☆

Publisher: Elsevier BV

Date: 08-2004

DOI: 10.1016/J.YGENO.2004.03.004

Urinary phthalate metabolites and semen quality: a review of a potential biomarker of susceptibility

Publisher: Wiley

Date: 06-12-2007

DOI: 10.1111/J.1365-2605.2007.00844.X

Abstract: Phthalates are a class of chemicals with widespread general population exposure. Some phthalates are reproductive and developmental toxicants in laboratory animals. Advances in the field of phthalate research in humans are dependent on the development and implementation of biomarkers to assess exposure and outcome, as well as potential markers that may be indicative of increased susceptibility. Recently, we incorporated a novel biomarker of potential 'susceptibility' into our study on the relationship of phthalates with semen quality and sperm DNA damage among men recruited from an infertility clinic. We measured urinary concentrations of three di(2-ethylhexyl) phthalate (DEHP) metabolites, mono(2-ethylhexyl) phthalate (MEHP) and two oxidative metabolites, mono-(2-ethyl-5-hydroxylhexyl) phthalate (MEHHP) and mono-(2-ethyl-5-oxohexyl) phthalate (MEOHP). We calculated the percent of DEHP excreted as the hydrolytic monoester (i.e., MEHP). We referred to this as %MEHP and considered it a phenotypic marker of the proportion of DEHP excreted in the urine as MEHP. In our sperm DNA study, we found novel results for the DEHP metabolites. Although MEHP was positively correlated with the oxidative metabolites, the association of sperm DNA damage with MEHP, as compared to MEHHP and MEOHP, were in opposite directions. We hypothesized that MEHP is the bioactive toxicant and further metabolism to MEHHP/MEOHP may lower internal burden of MEHP and thus be protective from sperm DNA damage. An alternative explanation may include that the relative percentage of DEHP excreted as MEHP was a surrogate for the function of phase I enzymes. Men with high %MEHP may have higher levels of sperm DNA damage because of poor metabolism (detoxification) of other genotoxic chemicals. Our hypothesis that %MEHP may represent a phenotypic marker of metabolism is novel but requires further exploration to confirm.

Importance of sperm-to-epithelial cell contact for the capacitation of human spermatozoa in fallopian tube epithelial cell cocultures

Publisher: Elsevier BV

Date: 10-2000

DOI: 10.1016/S0015-0282(00)01514-4

Abstract: To investigate the mechanisms involved in the stimulatory effect of fallopian tube epithelial cell coculture on sperm movement characteristics. Human spermatozoa were cultured with human fallopian tube epithelial cell monolayers. A microporous membrane was used to prevent sperm-to-epithelial cell contact. Sperm movement characteristics were measured at 4 and 24 hours. University hospital and fertility center. Voluntary donors. None. Movement characteristics of human spermatozoa. Fallopian tube epithelial cell coculture increased sperm motility, curvilinear velocity, litude of lateral head displacement, and hyperactivated motility, mainly at 24 hours, compared with controls. These stimulatory effects were inhibited when a microporous membrane prevented cell-to-cell contact between sperm and fallopian tube epithelial cells. Physical contact between sperm and epithelial cells in coculture systems seems to be the main factor in stimulating sperm movement characteristics, and this could be the main mechanism of in vivo sperm capacitation.

Male infertility and somatic health — insights into lipid damage as a mechanistic link

Publisher: Springer Science and Business Media LLC

Date: 13-09-2022

DOI: 10.1038/S41585-022-00640-Y

Abstract: Over the past decade, mounting evidence has shown an alarming association between male subfertility and poor somatic health, with substantial evidence supporting the increased incidence of oncological disease, cardiovascular disease, metabolic disorders and autoimmune diseases in men who have previously received a subfertility diagnosis. This paradigm is concerning, but might also provide a novel window for a crucial health reform in which the infertile phenotype could serve as an indication of potential pathological conditions. One of the major limiting factors in this association is the poor understanding of the molecular features that link infertility with comorbidities across the life course. Enzymes involved in the lipid oxidation process might provide novel clues to reconcile the mechanistic basis of infertility with incident pathological conditions. Building research capacity in this area is essential to enhance the early detection of disease states and provide crucial information about the disease risk of offspring conceived through assisted reproduction.

Analysis of lipid peroxidation in human spermatozoa using BODIPY C11

Publisher: Oxford University Press (OUP)

Date: 27-02-2007

DOI: 10.1093/MOLEHR/GAL119

Abstract: Lipid peroxidation is known to be a major factor in the aetiology of defective sperm function. Although biochemical assays for this process exist, they are relatively insensitive and require large numbers of spermatozoa a condition that cannot be met with many infertility specimens. Recently, a new approach for monitoring peroxidative damage has been introduced, involving the probe BODIPY (581/591) C(11), which readily incorporates into cells and undergoes a spectral emission shift when attacked by reactive oxygen metabolites. We have examined the applicability of this probe as an indicator of oxidative stress in human sperm populations using flow cytometry as an end point. The measurement of peroxidation with BODIPY C(11) demonstrated significant dependence on the presence of a ferrous ion promoter (P < 0.001), which was significantly enhanced in sperm recovered from low-density Percoll fractions (P < 0.05) and was particularly damaging to the sperm midpiece. Iron-induced radical formation was suppressed by ascorbate in a dose-dependent manner (P < 0.001) and could only be promoted by Fe(II) and Cu(II) nickel, zinc and Fe(III) were ineffective. The Fe(II)-promoted BODIPY C(11) signal was significantly correlated with the measurement of reactive oxygen species generation with dihydroethidium. We conclude that BODIPY C(11) is an extremely useful probe for indexing peroxidative damage in human spermatozoa.

Development of vaccines targeting the zona pellucida

Publisher: Elsevier BV

Date: 1990

DOI: 10.1016/0952-7915(90)90043-G

Abstract: Three-dimensional (3-D) cardiac activation imaging (3-DCAI) is a recently developed technique that aims at imaging the activation sequence throughout the the ventricular myocardium. 3-DCAI entails the modeling and estimation of the cardiac equivalent current density (ECD) distribution from which the activation time at any myocardial site is determined as the time point with the peak litude of local ECD estimates. In this paper, we report, for the first time, an in vivo validation study assessing the feasibility of 3-DCAI in comparison with the 3-D intracardiac mapping, for a group of four healthy rabbits undergoing the ventricular pacing from various locations. During the experiments, the body surface potentials and the intramural bipolar electrical recordings were simultaneously measured in a closed-chest condition. The ventricular activation sequence noninvasively imaged from the body surface measurements by using 3-DCAI was generally in agreement with that obtained from the invasive intramural recordings. The quantitative comparison between them showed a root mean square (rms) error of 7.42 +/-0.61 ms, a relative error (RE) of 0.24 +/-0.03, and a localization error (LE) of 5.47 +/-1.57 mm. The experimental results were also consistent with our computer simulations conducted in well-controlled and realistic conditions. The present study suggest that 3-DCAI can noninvasively capture some important features of ventricular excitation (e.g., the activation origin and the activation sequence), and has the potential of becoming a useful imaging tool aiding cardiovascular research and clinical diagnosis of cardiac diseases.

The role of free oxygen radicals and sperm function

Publisher: Wiley

Date: 04-1989

DOI: 10.1111/J.1365-2605.1989.TB01290.X

Field trial of a diluent for the transportation of human semen at ambient temperatures

Publisher: Elsevier BV

Date: 02-1997

DOI: 10.1016/S0015-0282(97)81922-X

Abstract: As the US healthcare system moves towards value-based care, hospitals have increased efforts to improve quality and reduce unnecessary resource use. Surgery is one of the most resource-intensive areas of healthcare and we aim to compare health resource utilization between open and minimally invasive cancer procedures. We retrospectively analyzed cancer patients who underwent colon resection, rectal resection, lobectomy, or radical nephrectomy within the Premier hospital database between 2014 and 2019. Study outcomes included length of stay (LOS), discharge status, reoperation, and 30-day readmission. The open surgical approach was compared to minimally invasive approach (MIS), with subgroup analysis of laparoscopic/video-assisted thoracoscopic surgery (LAP/VATS) and robotic (RS) approaches, using inverse probability of treatment weighting. MIS patients had shorter LOS compared to open approach: - 1.87 days for lobectomy, - 1.34 days for colon resection, - 0.47 days for rectal resection, and - 1.21 days for radical nephrectomy (all p .001). All MIS procedures except for rectal resection are associated with higher discharge to home rates and lower reoperation and readmission rates. Within MIS, robotic approach was further associated with shorter LOS than LAP/VATS: - 0.13 days for lobectomy, - 0.28 days for colon resection, - 0.67 days for rectal resection, and - 0.33 days for radical nephrectomy (all p .05) and with equivalent readmission rates. Our data demonstrate a significant shorter LOS, higher discharge to home rate, and lower rates of reoperation and readmission for MIS as compared to open procedures in patients with lung, kidney, and colorectal cancer. Patients who underwent robotic procedures had further reductions in LOS compare to laparoscopic/video-assisted thoracoscopic approach, while the reductions in LOS did not lead to increased rates of readmission.

The electrophoretic separation of spermatozoa: an analysis of genotype, surface carbohydrate composition and potential for capacitation

Publisher: Wiley

Date: 12-05-2011

DOI: 10.1111/J.1365-2605.2011.01164.X

Abstract: This study examines the properties of an electrophoretic device designed to effect the rapid isolation of spermatozoa for assisted conception purposes. In light of previous reports suggesting that X- and Y-bearing spermatozoa can be separated in an electric field, the first characteristic examined was the sex chromosome status of electrophoretically isolated spermatozoa. Exploiting sex chromosome-specific differences in the structure of the amelogenin gene, a quantitative PCR protocol was designed that allowed the rapid genotyping of isolated sperm suspensions. Reassuringly, application of this procedure demonstrated that the electrophoretic method did not result in a significant skewing of the ratio of X- and Y-bearing spermatozoa. Analysis of the molecular basis for electrophoretic sperm isolation demonstrated that sperm suspensions prepared in this manner were enriched in surface sialic acid residues that bound the Sambucus nigra agglutinin (SNA) lectin. Western blot analyses demonstrated the presence of four major SNA binding proteins, three of which were identified by MALDI-Tof mass spectrometry as aminopeptidase B, fucosyltransferase and prostatic acid phosphatase. The ability of neuraminidase to significantly suppress the electrophoretic isolation of spermatozoa emphasized the causative nature of this association between cell surface sialation and sperm behaviour in an electric field. Finally, seminal plasma proteins possessing decapacitation properties were shown to co-migrate with spermatozoa during their electrophoresis, necessitating their removal prior to in vitro fertilization. In terms of function, electrophoretically isolated cells were found to capacitate normally, exhibiting high levels of tyrosine phosphorylation and a capacity for extensive binding to homologous zonae pellucidae. We conclude that the electrophoretic procedure rapidly isolates functional spermatozoa via mechanisms that are independent of their genotype but reliant upon a net electronegative charge that is largely conferred by sperm surface glycoproteins.

Composition and significance of detergent resistant membranes in mouse spermatozoa

Publisher: Wiley

Date: 2009

DOI: 10.1002/JCP.21575

Abstract: Mammalian spermatozoa acquire the ability to fertilize an oocyte as they ascend the female reproductive tract. This process is characterized by a complex cascade of biophysical and biochemical changes collectively know as "capacitation." The attainment of a capacitated state is accompanied by a dramatic reorganization of the surface architecture to render spermatozoa competent to recognize the oocyte and initiate fertilization. Emerging evidence indicates that this process is facilitated by molecular chaperone-mediated assembly of a multimeric receptor complex on the sperm surface. However, the mechanisms responsible for gathering key recognition molecules within this putative complex have yet to be defined. In this study, we provide the first evidence that chaperones partition into detergent resistant membrane fractions (DRMs) within capacitated mouse spermatozoa and co-localize in membrane microdomains enriched with the lipid raft marker, G(M1) ganglioside. During capacitation, these microdomains coalesce within the apical region of the sperm head, a location compatible with a role in sperm-zona pellucida interaction. Significantly, DRMs isolated from spermatozoa possessed the ability to selectively bind to the zona pellucida of unfertilized, but not fertilized, mouse oocytes. A comprehensive proteomic analysis of the DRM fractions identified a total of 100 proteins, a number of which have previously been implicated in sperm-oocyte interaction. Collectively, these data provide compelling evidence that mouse spermatozoa possess membrane microdomains that provide a platform for the assembly of key recognition molecules on the sperm surface and thus present an important mechanistic insight into the fundamental cell biological process of sperm-oocyte interaction.

B cells flying solo

Publisher: Wiley

Date: 2008

DOI: 10.1038/SJ.ICB.7100142

Abstract: Systemic autoimmunity such as systemic lupus erythematosus (SLE) is associated with the loss of B-cell tolerance, B-cell dysregulation and autoantibody production. While some autoantibodies may contribute to the pathology seen with SLE, numerous studies have shown that dysregulation of T-cell function is another critical aspect driving disease. The positive results obtained in clinical trials using T-cell- or B-cell-specific treatments have suggested that cooperation between T and B cells probably underlies disease progression in many patients. A similar cooperative mechanism seemed to explain SLE developing in mice overexpressing the B-cell-activating factor from the tumor necrosis factor family (BAFF). However, surprisingly, T-cell-deficient BAFF transgenic (Tg) mice develop SLE similar to T-cell-sufficient BAFF Tg mice, and the disease was linked to innate activation of B cells and production of proinflammatory autoantibody isotypes. In conclusion, dysregulated innate activation of B cells alone can drive disease independently of T cells, and as such this aspect represents a new pathogenic mechanism in autoimmunity.

Stimulation of oxidant generation by human sperm suspensions using phorbol esters and formyl peptides: Relationships with motility and fertilization in vitro

Publisher: Elsevier BV

Date: 09-1994

DOI: 10.1016/S0015-0282(16)56952-0

Abstract: To investigate the influence of reactive oxygen species generated by human spermatozoa and contaminating leukocytes on sperm movement and fertilization in vitro. A chemiluminescence technique, using luminol and peroxidase, was used to monitor the generation of reactive oxygen species by human sperm suspensions and the results were correlated with sperm movement and the fertilization of human ova in vitro. Diagnostic Andrology Laboratory and IVF Clinic. Infertile couples undergoing IVF therapy. An N-formyl-methionyl-leucyl-phenylalanine (FMLP) provocation test was used to demonstrate that the presence of leukocytes in 28.5% of the sperm preparations was associated with elevated levels of spontaneous reactive oxygen species production, impaired movement, and a reduced capacity for fertilization in vitro. In the absence of leukocytes, exposure to phorbol ester stimulated a burst of reactive oxygen species generation by human spermatozoa, the magnitude of which was correlated highly with a loss of sperm motility but not with fertilization rates observed in the concurrent IVF cycle. Leukocyte contamination of human sperm preparations can be detected readily by FMLP-induced, luminol-dependent chemiluminescence and the results have an important bearing on the fertilizing capacity of the spermatozoa in vitro.

Identification of a key role for permeability glycoprotein in enhancing the cellular defense mechanisms of fertilized oocytes

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.YDBIO.2016.06.035

Abstract: Double strand breaks (DSBs) are highly damaging DNA lesions that can destabilize the genome and generate a suite of adverse physiological outcomes in the oocyte and early embryo. While it is therefore likely that these cells possess a sophisticated suite of protective mechanisms to ameliorate such damage, the precise nature of these defense systems are yet to be fully elucidated. This study characterizes the sensitivity of the oocyte to etoposide, a chemotherapeutic agent with the ability to elicit DSBs. We demonstrate significant developmental changes in etoposide vulnerability, with fertilization of the oocyte leading to an enhancement of its cellular defense machinery. Using a parthenogenic model we show that this response is mediated, at least in part, by permeability glycoprotein (PGP), an endogenous multidrug efflux transporter that is up-regulated, translocated to the oolemma and phosphorylated upon oocyte activation. Moreover, evidence from dye exclusion assays in the presence of a specific PGP pharmacological inhibitor (PSC833), illustrates that these events effectively increase oocyte efflux activity, thereby enhancing the ability of these cells to exclude genotoxicants capable of eliciting DSB formation.

Development of the signalling pathways associated with sperm capacitation during epididymal maturation

Publisher: Wiley

Date: 13-02-2003

DOI: 10.1002/MRD.10255

Abstract: As spermatozoa mature within the epididymis they acquire the potential for capacitation and ultimately fertilization. In biochemical terms, the former is reflected in the progressive activation of a signal transduction pathway characterized by cAMP-mediated induction of phosphotyrosine expression on the sperm tail. In this study, we have examined the cellular mechanisms controlling this maturational event. Caput epididymal spermatozoa exhibited tyrosine phosphorylation on the sperm head that was largely unresponsive to cAMP and not significantly impaired by removal of extracellular HCO(3) (-). In contrast, caudal epididymal spermatozoa exhibited low levels of phosphorylation on the sperm head, yet responded dramatically to cAMP by phosphorylating a new set of proteins on the sperm tail via mechanisms that were highly dependent on extracellular HCO(3) (-). The impact of extracellular HCO(3) (-) depletion on caudal cells was not associated with a significant change in the redox regulation of cAMP but could be fully reversed by buffering the intracellular pH with N-Tris[Hydroxymethyl]methyl-3-amino-propanesulfonic acid (TAPS). The pattern of tyrosine phosphorylation was also profoundly influenced by the presence or absence of added extracellular calcium. In the presence of this cation, only caudal spermatozoa could respond to increased extracellular cAMP with tyrosine phosphorylation of the sperm tail. However, in calcium-depleted medium, this difference completely disappeared. Under these conditions, caput and caudal spermatozoa were equally competent to exhibit phosphotyrosine expression on the sperm tail in response to cAMP. These results emphasize the pivotal role played by calcium and HCO(3) (-) in modulating the changes in tyrosine phosphorylation observed during epididymal maturation.

Plasma Progesterone Levels throughout the Ovarian Cycle of the Common Marmoset (Callithrix jacchus)

Publisher: S. Karger AG

Date: 1982

DOI: 10.1159/000460023

Abstract: Radioimmunoassay of progesterone in marmoset plasma has been used to determine ovarian cycle length. Total cycle length was 30.1 +/- 3.8 days (mean +/- SD, n = 30, range 24-41 days, median 29.5 days). The pre-ovulatory (follicular) phase, during which progesterone levels were below 10 ng/ml, lasted for 8.8 +/- 3.7 days (mean +/- SD, n = 30, range 3-20 days, median 8.5 days). The post-ovulatory (luteal) phase, during which progesterone levels were greater than 10 ng/ml, lasted for 21.5 +/- 2.2 days (mean +/- SD, n = 30, range 14-29 days, median 21.5 days). Total cycle length was almost twice that recorded in an earlier study. The reasons for this difference are discussed.

OXIDATIVE STRESS and REPRODUCTIVE FUNCTION Oxidative stress and in vitro ageing of the post-ovulatory oocyte: an update on recent advances in the field

Publisher: Bioscientifica

Date: 12-2022

DOI: 10.1530/REP-22-0206

Abstract: Post-ovulatory ageing of oocytes leads to poor oocyte and embryo quality as well as abnormalities in offspring. This review provides an update on the contributions of oxidative stress to this process and discusses the current literature surrounding the use of antioxidant media to delay post-ovulatory oocyte ageing. Following ovulation, the metaphase II stage oocyte has a limited functional lifespan before succumbing to a process known as post-ovulatory oocyte ageing. This progressive demise occurs both in vivo and in vitro and is accompanied by a deterioration in oocyte quality, leading to a well-defined sequelae of reduced fertilisation rates, poor embryo quality, post-implantation errors, and abnormalities in the offspring. Although the physiological consequences of post-ovulatory oocyte ageing have largely been characterised, less is known regarding the molecular mechanisms that drive this process. This review presents an update on the established relationships between the biochemical changes exhibited by the ageing oocyte and the myriad of symptoms associated with the ageing phenotype. In doing so, we consider the molecular events that are potentially involved in orchestrating post-ovulatory ageing with a particular focus on the role of oxidative stress. We highlight the mounting evidence that oxidative stress acts as an initiator for a cascade of events that create the aged oocyte phenotype. Specifically, oxidative stress has the capacity to disrupt mitochondrial function and directly damage multiple intracellular components of the oocyte such as lipids, proteins, and DNA. Finally, this review addresses emerging strategies for delaying post-ovulatory oocyte ageing with emphasis placed on the promise afforded by the use of selected antioxidants to guide the development of media tailored for the preservation of oocyte integrity during in vitro fertilisation procedures.

Blockade of ROS production inhibits oncogenic signaling in acute myeloid leukemia and amplifies response to precision therapies

Publisher: American Association for the Advancement of Science (AAAS)

Date: 28-03-2023

DOI: 10.1126/SCISIGNAL.ABP9586

Abstract: Mutations in the type III receptor tyrosine kinase FLT3 are frequent in patients with acute myeloid leukemia (AML) and are associated with a poor prognosis. AML is characterized by the overproduction of reactive oxygen species (ROS), which can induce cysteine oxidation in redox-sensitive signaling proteins. Here, we sought to characterize the specific pathways affected by ROS in AML by assessing oncogenic signaling in primary AML s les. The oxidation or phosphorylation of signaling proteins that mediate growth and proliferation was increased in s les from patient subtypes with FLT3 mutations. These s les also showed increases in the oxidation of proteins in the ROS-producing Rac/NADPH oxidase-2 (NOX2) complex. Inhibition of NOX2 increased the apoptosis of FLT3-mutant AML cells in response to FLT3 inhibitors. NOX2 inhibition also reduced the phosphorylation and cysteine oxidation of FLT3 in patient-derived xenograft mouse models, suggesting that decreased oxidative stress reduces the oncogenic signaling of FLT3. In mice grafted with FLT3 mutant AML cells, treatment with a NOX2 inhibitor reduced the number of circulating cancer cells, and combining FLT3 and NOX2 inhibitors increased survival to a greater extent than either treatment alone. Together, these data raise the possibility that combining NOX2 and FLT3 inhibitors could improve the treatment of FLT3 mutant AML.

Reactive oxygen species and human spermatozoa: Analysis of the cellular mechanisms involved in luminol‐ and lucigenin‐dependent chemiluminescence

Publisher: Wiley

Date: 06-1992

DOI: 10.1002/JCP.1041510305

Abstract: We have shown that human spermatozoa generate and release reactive oxygen species that can be detected by chemiluminescence techniques. Analysis of the cellular mechanisms responsible for this activity suggests that the probe, luminol, undergoes an intracellular dioxygenation reaction mediated by hydrogen peroxide and a sperm peroxidase located within the acrosome. Support for this model included the following observations: (1) the luminol-dependent signal could be suppressed with peroxidase inhibitors, phenylhydrazine and sodium azide (2) this suppression could be reversed by the addition of an azide-insensitive peroxidase, horse radish peroxidase (HRP) (3) inhibition of intracellular superoxide dismutase (SOD) with potassium cyanide (KCN) suppressed the luminol signal (4) peroxidase activity could be detected in purified populations of human spermatozoa with 3,3',5,5' tetramethylbenzidine (TMB) (5) this peroxidase was active at the pH prevailing within the acrosomal vesicle and (6) peroxidase activity and luminol-dependent chemiluminescence were minimal in spermatozoa exhibiting a congenital absence of acrosomes. Human spermatozoa could also generate lucigenin-dependent chemiluminescent signals that could neither be suppressed with peroxidase inhibitors nor enhanced by the addition of peroxidase. However, these signals could be enhanced by suppression of intracellular SOD with KCN or inhibited by exogenous SOD, suggesting that lucigenin was responding to superoxide anion released into the extracellular space. The ability of chemiluminescent techniques to detect and discriminate the production of superoxide and hydrogen peroxide by spermatozoa should facilitate the further analysis of reactive oxygen species as mediators of normal and abnormal human sperm function.

Phosphorylation and consequent stimulation of the tyrosine kinase c-Abl by PKA in mouse spermatozoa; its implications during capacitation

Publisher: Elsevier BV

Date: 09-2009

DOI: 10.1016/J.YDBIO.2009.06.022

Abstract: Upon ejaculation, spermatozoa undergo a series of post-translational modifications in a process known as capacitation in order to prepare for fertilization. In the absence of capacitation, fertilization cannot occur. Spermatozoa are unusual in that one of the hallmarks of capacitation is a global up-regulation in phosphotyrosine expression, which is known to be mediated upstream by PKA. Little is known about the signaling events downstream of PKA apart from the involvement of SRC, as a key mediator of PKA-induced tyrosine phosphorylation in the sperm tail. Here we describe the presence of c-Abl in mouse spermatozoa. In vitro analysis confirmed that PKA can up-regulate c-Abl kinase activity. In vivo, this tyrosine kinase was found to associate, and become threonine phosphorylated by PKA in the sperm flagellum. By treating spermatozoa with hemolysin we could demonstrate that a significant proportion of the tyrosine phosphorylation associated with capacitation could be suppressed by the c-Abl inhibitor, Gleevac. This is the first report of c-Abl being up-regulated by PKA for any cell type. We present a model, whereby these kinases may operate together with SRC to ensure optimal levels of tyrosine phosphorylation in the sperm flagellum during the attainment of a capacitated state.

Sperm DNA integrity: a special issue exploring the causes, consequences, and treatment of DNA damage in human spermatozoa

Publisher: Wiley

Date: 19-10-2023

DOI: 10.1111/ANDR.13503

Evidence that extrapancreatic insulin production is involved in the mediation of sperm survival

Publisher: Elsevier BV

Date: 04-2021

DOI: 10.1016/J.MCE.2021.111193

Workshop report: clinical CASA--the quest for consensus

Publisher: CSIRO Publishing

Date: 1995

DOI: 10.1071/RD9950951

Abstract: The goals of the Workshop were to establish the current state-of-the-art in the clinical use of computer-aided sperm analysis (CASA), to identify areas of contention or confusion in the use of CASA technology, and to reach consensus on these matters to facilitate the wider use, and usefulness, of these instruments in clinical and research applications. CASA assessments of sperm morphology were not considered as they were discussed in a separate Workshop (Advanced Techniques in Sperm Preparation and Imaging) on analytical techniques. Four topics were considered: (a) CASA and semen analysis (b) the diagnostic value of sperm kinematics in semen (c) human sperm hyperactivation and (d) CASA and fertility prediction. In all, 17 specific consensus points were identified.

Meiosis-inducing substances in human preovulatory follicular fluid related to time of follicle aspiration and to the potential of the oocyte to fertilize and cleave in vitro

Publisher: Elsevier BV

Date: 11-1985

DOI: 10.1016/S0015-0282(16)48984-3

Abstract: Meiosis-inducing substance (MIS) and steroid and gonadotropic hormones were investigated in 41 preovulatory follicular fluids (FFs) aspirated at either 0, 12, or 36 hours after human chorionic gonadotropin (hCG) administration in 25 women with clomiphene citrate-stimulated cycles. Twenty-one oocytes were recovered from these FFs and subjected to in vitro fertilization. MIS activity was present in 25 (61%) of the FFs. The frequency of MIS-active FFs increased from 11% (1 of 9) at 0 hours and 40% (2 of 5) at 12 hours to 81% (22 of 27) at 36 hours after hCG administration (P less than 0.001). The concentration of hormones in MIS-active FFs was not significantly different from that of MIS-inactive FFs. Twelve (86%) of 14 oocytes that fertilized and cleaved in vitro were recovered from MIS-active FFs. By contrast, all seven oocytes that remained unfertilized in vitro were recovered from MIS-inactive FFs. These findings support the notion that resumption of meiosis in the preovulatory oocyte is triggered by MIS in FF and suggest that follicular MIS production may be one of the factors that determines the success of in vitro fertilization and early embryonic development.

A Prospective Study of the Relationship Between Semen Quality and Fertility in Cases of Unexplained Infertility

Publisher: Wiley

Date: 08-07-1984

DOI: 10.1002/J.1939-4640.1984.TB00792.X

Abstract: The male partners of 68 couples exhibiting 5.1 +/- 0.3 (SEM) years of unexplained infertility were assessed using the conventional criteria of semen quality, the movement characteristics of the spermatozoa and the outcome of the zona-free hamster egg penetration test. After a follow-up period of 2.3 +/- 0.06 (SEM) years, 25 (37%) of these patients were found to have initiated a pregnancy, thereby permitting an analysis of those aspects of semen quality which most accurately predicted their subsequent fertility. A multivariate discriminant analysis revealed that the conventional semen profile, per se, was not of significant value in discriminating the incidence of pregnancy. However, significant discrimination (P = 0.0173) was obtained when the postcapacitation movement characteristics of the spermatozoa were incorporated into the analysis. The accuracy of this prognosis was further increased if either the duration of infertility or the outcome of the zona-free hamster egg penetration test was taken into consideration. Overall classifications of fertility were then 76.3% and 76.5% accurate, respectively. These results suggest that in vitro assessments of human sperm function are of significant value in evaluating male fertility.

Rosiglitazone improves stallion sperm motility, ATP content, and mitochondrial function

Publisher: Oxford University Press (OUP)

Date: 28-09-2016

DOI: 10.1095/BIOLREPROD.116.142687

Abstract: Media used for equine sperm storage often contain relatively high concentrations of glucose, even though stallion spermatozoa preferentially utilize oxidative phosphorylation (OXPHOS) over glycolysis to generate ATP and support motility. Rosiglitazone is an antidiabetic compound that enhances metabolic flexibility and glucose utilization in various cell types, but its effects on sperm metabolism are unknown. This study investigated the effects of rosiglitazone on stallion sperm function in vitro, along with the possible role of AMP-activated protein kinase (AMPK) in mediating these effects. Spermatozoa were incubated with or without rosiglitazone, GW9662 (an antagonist of peroxisome proliferator-activating receptor-gamma), and compound C (CC an AMPK inhibitor). Sperm motility, viability, reactive oxygen species production, mitochondrial membrane potential (mMP), ATP content, and glucose uptake capacity were measured. S les incubated with rosiglitazone displayed significantly higher motility, percentage of cells with normal mMP, ATP content, and glucose uptake capacity, while sperm viability was unaffected. The percentage of spermatozoa positive for mitochondrial ROS was also significantly lower in rosiglitazone-treated s les. AMPK localized to the sperm midpiece, and its phosphorylation, was increased in rosiglitazone-treated spermatozoa. CC decreased sperm AMPK phosphorylation and reduced sperm motility, and successfully inhibited the effects of rosiglitazone. Inclusion of rosiglitazone in a room temperature sperm storage medium maintained sperm motility above 60% for 6 days, attaining significantly higher motility than sperm stored in control media. The ability of rosiglitazone to substantially alleviate the time-dependent deterioration of stallion spermatozoa by erting metabolism away from OXPHOS and toward glycolysis has novel implications for the long-term, functional preservation of these cells.

Inhibition of arachidonate 15-lipoxygenase (ALOX15) prevents 4HNE-induced protein damage in male germ cells

Publisher: Oxford University Press (OUP)

Date: 02-02-2017

DOI: 10.1093/BIOLRE/IOX005

Abstract: Lipid peroxidation products, such as 4-hydroxynonenal (4HNE), are causative agents responsible for extensive protein damage within the male and female germlines. Recently, we have demonstrated that 4HNE production can initiate the proteolytic degradation of the molecular chaperone Heat Shock Protein A2 (HSPA2) in male germ cells. These events may be partially responsible for HSPA2 deficiency in the spermatozoa of patients that repeatedly fail in vitro fertilization. Given this, mechanisms that limit the production of 4HNE will be highly advantageous for the preservation of male fertility. The propagation of 4HNE in somatic cells has been linked to the enzymatic actions of arachidonate 15-lipoxygenase (ALOX15), a member of the lipoxygenase family of proteins. In view of this association, this study sought to explore ALOX15 as a physiological target to manipulate the levels of 4HNE produced in the male germline. Herein, we have demonstrated that ALOX15 is markedly upregulated in response to oxidative stress in round spermatids and the GC-2 cell line. Pharmacological inhibition of ALOX15 in GC-2 cells resulted in a significant reduction in both mitochondrial and cytoplasmic reactive oxygen species, as well as a dramatic reduction in 4HNE. Importantly, the reduced bioavailability of this aldehyde appears to confer positive downstream effects to its target proteins such that HSPA2 could be protected from damage by 4HNE. Taken together, these results suggest that the actions of ALOX15 are intimately tied to the production of 4HNE. Thus, the ALOX15 protein may be a promising new target for the mitigation of germline oxidative stress.

Dynamin regulates specific membrane fusion events necessary for acrosomal exocytosis in mouse spermatozoa

Publisher: Elsevier BV

Date: 11-2012

DOI: 10.1074/JBC.M112.392803

A novel pathway for the induction of DNA damage in human spermatozoa involving extracellular cell-free DNA

Publisher: Elsevier BV

Date: 05-2020

DOI: 10.1016/J.MRFMMM.2020.111722

Apoptosis and DNA damage in human spermatozoa

Publisher: Medknow

Date: 30-08-2010

DOI: 10.1038/AJA.2010.68

Measurement of intracellular calcium in human spermatozoa

Publisher: Wiley

Date: 09-1986

DOI: 10.1002/MRD.1120150107

The Male Gamete Is Not a Somatic Cell—the Possible Meaning of Varying Sperm RNA Levels

Publisher: Mary Ann Liebert Inc

Date: 10-01-2013

DOI: 10.1089/ARS.2012.4715

The effect of iron and iron chelators on the in-vitro block to development of the mouse preimplantation embryo: BAT6 a new medium for improved culture of mouse embryos in vitro

Publisher: Oxford University Press (OUP)

Date: 11-1990

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A137235

Abstract: The effect of iron and iron chelators on the development of the mouse embryo in vitro from the 1-cell stage to the blastocyst has been investigated. An adverse effect of iron was found. The high affinity iron chelator, desferal, also blocked development, whilst transferrin (whether as apoprotein or saturated with iron), DETAPAC and EDTA promoted development. The addition of transferrin permitted development to the blastocyst stage of embryos from stains normally exhibiting the 2-cell block. Under such circumstances both the rate of embryonic development and the proportion of embryos reaching the blastocyst stage approached levels found in vivo. Based on these results, a new medium, BAT6, is described for the optimal in-vitro culture of mouse embryos.

A comparative study of oxidative DNA damage in mammalian spermatozoa

Publisher: Wiley

Date: 2005

DOI: 10.1002/MRD.20285

Abstract: Alkaline gel electrophoresis, pulsed field gel electrophoresis, and quantitative PCR analyses (QPCR) of the nuclear (nDNA) and mitochondrial (mtDNA) genomes were used to assess DNA integrity in the spermatozoa of three species exposed to oxidative stress. In human and murine spermatozoa, the mtDNA was significantly more susceptible to H2O2-mediated damage than nDNA. In both eutherian species, exposure to 250 microM H2O2 induced around 0.6 lesions/10 kb of mtDNA. The mtDNA of human spermatozoa was particularly vulnerable to oxidative stress 0.25, 1, and 5 mM H2O2 inducing DNA damage equivalent to 0.62, 1.34, and 1.42 lesions/10 kb, respectively. Such results emphasize the diagnostic significance of mtDNA as a biomarker of oxidative stress in the male germ line. In contrast, no damage could be detected by QPCR in the nDNA of either eutherian species, on exposure to H2O2 at doses as high as 5 mM. However, electrophoretic analysis indicated that severe oxidative stress could induce detectable nDNA fragmentation in human, but not murine spermatozoa. The mtDNA of tammar wallaby spermatozoa was relatively resistant to oxidative stress, only exhibiting damage (0.6 lesions/10 kb DNA) on exposure to 5 mM H2O2. By contrast, the nDNA of wallaby spermatozoa was significantly more susceptible to this oxidant than the other species. Such vulnerability is consistent with the lack of disulfide cross-linking in marsupial sperm chromatin and suggests that chromatin condensation during epididymal maturation may be important in establishing the resistance of these cells to the genotoxic effects of reactive oxygen species.

Identification and characterization of a novel Mt-retrotransposon highly represented in the female mouse germline

Publisher: Elsevier BV

Date: 04-2006

DOI: 10.1016/J.YGENO.2005.08.015

Abstract: The control of primordial follicle recruitment into the growing follicle population is a major limiting process in female reproduction. In order to gain insight into the molecular processes occurring at the time of primordial follicle activation, a subtractive hybridization analysis was performed between cDNAs prepared from temporally distinct mouse neonatal ovarian tissues that differed according to the state of primordial follicle activation. One highly represented clone associated with activation was an Mt retrotransposon-like sequence designated Mtfull, which was subsequently cloned and determined to be novel and restricted in expression to the ovary. The polyadenylated 1684-bp sequence has long terminal repeats, is predicted to be noncoding, and is the predominant Mti-related sequence present in the mouse ovary. In situ hybridization further localized Mtfull expression to the oocyte and confirmed that expression is concomitant with follicle activation. Together with in silico data, we predict Mtfull plays an essential role in folliculogenesis through regulation of gene expression.

Heat exposure induces oxidative stress and DNA damage in the male germ line

Publisher: Oxford University Press (OUP)

Date: 17-01-2018

DOI: 10.1093/BIOLRE/IOY009

Abstract: The reproductive consequences of global warming are not currently understood. In order to address this issue, we have examined the reproductive consequences of exposing male mice to a mild heat stress. For this purpose, adult male mice were exposed to an elevated ambient temperature of 35°C under two exposure models. The first involved acute exposure for 24 h, followed by recovery periods between 1 day and 6 weeks. The alternative heating regimen involved a daily exposure of 8 h for periods of 1 or 2 weeks. In our acute model, we identified elevated sperm mitochondrial ROS generation (P < 0.05), increased sperm membrane fluidity (P < 0.05), DNA damage in the form of single-strand breaks (P < 0.001), and oxidative DNA damage (P < 0.05), characteristic of an oxidative stress cascade. This DNA damage was detected in pachytene spermatocytes (P < 0.001) and round spermatids (P 0.05). Collectively, our acute heat stress model supports the existence of heat susceptible stages of germ cell development, with the round spermatids being most perturbed and spermatogonial stem cells exhibiting resistance to this insult. Such findings were complemented by our chronic heat stress model, which further supported the vulnerability of the round spermatid population.

Oxidative stress, sperm survival and fertility control

Publisher: Elsevier BV

Date: 05-2006

DOI: 10.1016/J.MCE.2005.12.026

Abstract: The human spermatozoon is highly susceptible to oxidative stress. This process induces peroxidative damage in the sperm plasma membrane and DNA fragmentation in both the nuclear and mitochondrial genomes. Such stress may arise from a variety of sources including a lack of antioxidant protection, the presence of redox cycling xenobiotics, infiltrating leukocytes and excess reactive oxygen species production by the spermatozoa. Whenever the levels of oxidative stress in the male germ line are high, the peroxidation of unsaturated fatty acids in the sperm plasma membrane ensures that normal fertilization cannot occur. However, at lower levels of oxidative stress, spermatozoa may retain their capacity for fertilization while carrying significant levels of oxidative damage in their DNA. Epidemiological evidence suggests that subsequent aberrant repair of such damage in the zygote may result in the creation of mutations associated with pre-term pregnancy loss and a variety of pathologies in the offspring, including childhood cancer. Thus, while the induction of oxidative stress in spermatozoa is causally involved in the aetiology of male infertility, the prospects of using such a strategy for male contraception is fraught with potential problems, should the suppression of fertility be incomplete and DNA-damaged spermatozoa gain access to the oocyte.

Bridging the gap between male and female fertility control; contraception-on-demand

Publisher: Elsevier BV

Date: 10-2008

DOI: 10.1016/J.CONTRACEPTION.2008.03.020

Abstract: Contraception-on-demand refers to contraceptive methods that are only employed when needed, such as barrier or postcoital methods, as opposed to technologies, such as the IUD or pill, where the exposure is continuous irrespective of the risk of pregnancy. The development of women-centered approaches to contraception-on-demand is a high priority in current contraceptive research, with emphasis on the 15- to 25-year-old demographic. Since this cohort of potential users is also at high risk of contracting sexually transmitted disease, topical methods that would provide simultaneous protection against both fertility and infection are of particular interest. This review examines the current strategies that are being pursued to achieve this objective.

The Chaperonin Containing TCP1 Complex (CCT/TRiC) Is Involved in Mediating Sperm-Oocyte Interaction

Publisher: Elsevier BV

Date: 10-2011

DOI: 10.1074/JBC.M110.188888

Accuracy of human sperm DNA oxidation quantification and threshold determination using an 8-OHdG immuno-detection assay

Publisher: Oxford University Press (OUP)

Date: 23-02-2018

DOI: 10.1093/HUMREP/DEY038

Abstract: Can a discriminant threshold be determined for human sperm DNA oxidation? A discriminant threshold was found with 65.8% of 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive sperm cells and a mean intensity of fluorescence (MIF) of 552 arbitrary units. Oxidative stress is known to interfere with sperm quality and fertilizing capacity. However, current practice does not include the routine determination of oxidative DNA damage in spermatozoa optimized consensus protocols are lacking and no thresholds of normality have been established. Intra- and inter-method comparisons between four protocols (I-IV) were conducted to determine the most relevant and efficient means of assessing human sperm 8-OHdG content. Tests of assay repeatability, specificity, sensitivity and stability were performed to validate an optimized methodology for routine diagnostic use. This prospective study compared three immuno-detection methods including immunocytochemistry, fluorescence microscopy and flow cytometry. Sperm DNA oxidation for 80 patients was determined relative to semen parameters and clinical conditions, using the selected immuno-detection protocol in comparison with a commercial kit. These patients (age 35 ± 1 years: mean ± SEM) presented with normozoospermic (n = 40) or altered parameters (necro- or/and astheno- or/and teratozoospermia or/and leukocytospermia). Significant positive Pearson and Spearman correlations were determined for 8-OHdG values and sperm parameters using protocol III. A notable high and positive correlation was revealed for MIF with BMI and leukocyte concentration. Protocol III was the most discriminating method regarding assay repeatability, specificity, sensitivity, stability and reliability for sperm parameter alterations, in particular leukocytospermia according to parametric or non-parametric tests, effect-size determinations and factorial analysis such as principal component analysis and factor discriminant analysis. Of interest is that 39% of the subjects with 'pathological' sperm DNA oxidation values were normozoospermic. The oligozoospermic population was not evaluated in this study because insufficient material was available to carry out the comparisons. However, spermatozoa concentration was taken into account in the statistical analysis. Our study is the first validation of a protocol to determine a discriminant threshold for human sperm DNA oxidation. The protocol's detection accuracy for 8-OHdG human sperm DNA residues, stability over time, and relationship to human sperm quality were demonstrated. The assay should find application in the diagnosis of male factor infertility associated with oxidative stress. This work was funded by institutional grants from the CNRS, INSERM and Université Clermont Auvergne (to J.R.D.) and by Clermont-Ferrand Hospital-CECOS research funds (to L.J. and F.B.). P.G., A.M., R.J.A. and J.D. are, respectively, CEO, scientific director and scientific advisors of a US-based biotech company (Celloxess, Princeton, NJ, USA) involved in preventative medicine with a focus on the generation of antioxidant oral supplements.

Physiological and Pathological Aspects of Sperm Metabolism

Publisher: Cambridge University Press

Date: 25-05-2017

DOI: 10.1017/9781316411124.009

Sperm DNA: organization, protection and vulnerability: from basic science to clinical applications--a position report

Publisher: Oxford University Press (OUP)

Date: 06-02-2010

DOI: 10.1093/HUMREP/DEP465

Abstract: This article reports the results of the most recent in a series of EHSRE workshops designed to synthesize the current state of the field in Andrology and provide recommendations for future work (for details see Appendix). Its focus is on methods for detecting sperm DNA damage and potential application of new knowledge about sperm chromatin organization, vulnerability and repair to improve the diagnosis and treatment of clinical infertility associated with that damage. Equally important is the use and reliability of these tests to identify the extent to which environmental contaminants or pharmaceutical agents may contribute to the incidence of sperm DNA damage and male fertility problems. A working group (for workshop details, see Appendix) under the auspices of ESHRE met in May 2009 to assess the current knowledgebase and suggest future basic and clinical research directions. This document presents a synthesis of the working group's understanding of the recent literature and collective discussions on the current state of knowledge of sperm chromatin structure and function during fertilization. It highlights the biological, assay and clinical uncertainties that require further research and ends with a series of 5 key recommendations.

Analysis of the direct effects of prostaglandins on human sperm function

Publisher: Bioscientifica

Date: 1985

DOI: 10.1530/JRF.0.0730139

Abstract: Time-exposure photomicrography and interspecies in-vitro fertilization procedures have been used to examine the influence of prostaglandins on human sperm function. An analysis of variance indicated that the presence of PGs in the incubation media was associated with a significant increase in sperm velocity and the frequency of sperm head rotation, although there were no differences between in idual PGs in the degree of stimulation observed. Changes in the penetrating ability of human spermatozoa were detected after exposure to PGs, particularly PGE-1 and PGE-2. PGE-2 induced a sustained increase in penetration rates at all doses of greater than 8.4 micrograms/ml, while exposure to PGE-1 gave a bell-shaped dose-response curve which exhibited a peak between 8.4 and 33.3 micrograms/ml and progressively fell to reach control levels at the maximum concentration tested of 270 micrograms/ml. A combination of PGE-2 and PGE-1 produced a dose-response curve similar to that for PGE-1 alone, while exposure to PGF-2 alpha was without effect. Seminal extracts containing predominantly 19-hydroxy PGE-1, or equal amounts of 19-hydroxy PGE-1 + 2 induced a slight, but significant, rise in penetration rates while a combination of PGs representing the major components of human seminal plasma was without significant effect. We conclude that certain prostaglandins may have a direct action on the functional competence of human spermatozoa.

Redox regulation of human sperm function: From the physiological control of sperm capacitation to the etiology of infertility and DNA damage in the germ line

Publisher: Mary Ann Liebert Inc

Date: 02-2011

DOI: 10.1089/ARS.2010.3186

Abstract: Defective sperm function is the largest single defined cause of human infertility and one of the major reasons we are witnessing an exponential increase in the uptake of assisted conception therapy in the developed world. A major characteristic of defective human spermatozoa is the presence of large amounts of DNA damage, which is, in turn, associated with reduced fertility, increased rates of miscarriage, and an enhanced risk of disease in the offspring. This DNA damage is largely oxidative and is closely associated with defects in spermiogenesis. To explain the origins of this DNA damage, we postulate that spermiogenesis is disrupted by oxidative stress, leading to the creation of defective gametes with poorly remodeled chromatin that are particularly susceptible to free radical attack. To compound the problem, these defective cells have a tendency to undergo an unusual truncated form of apoptosis associated with high amounts of superoxide generation by the sperm mitochondria. This leads to significant oxidative DNA damage that eventually culminates in the DNA fragmentation we see in infertile patients. In light of the significance of oxidative stress in the etiology of defective sperm function, a variety of antioxidant therapies are now being assessed for their therapeutic potential.

Proteomics of Human Spermatozoa

Publisher: Springer Berlin Heidelberg

Date: 2009

DOI: 10.1007/978-3-642-01379-9_1

Oxidative stress in the male germ line and its role in the aetiology of male infertility and genetic disease

Publisher: Elsevier BV

Date: 2003

DOI: 10.1016/S1472-6483(10)61730-0

Abstract: The human male is characterized by extremely poor semen quality as reflected in the number, morphology and motility of the spermatozoa and a high incidence of nuclear and mitochondrial DNA damage. As a consequence of these factors, defective sperm function is thought to be a major contributor to the aetiology of human infertility, as well as childhood diseases including dominant genetic mutations such as achondroplasia and cancer. Factors associated with the origin of poor semen quality include: (i) a lack of selection pressure for high fecundity genes in developed countries, (ii) an evolutionary lineage associated with the deterioration of several male fertility genes in humans and their close ancestors, (iii) genetic factors including, but not limited to, Y-chromosome deletions (iv) paternal age and (v) environmental factors. A model is proposed whereby factors such as ageing or environmental toxicants initiate DNA strand breakage in the spermatozoa of affected males, eventually leading to a mutation in the embryo. This hypothesis stresses the importance of discovering the identity of those environmental factors that are capable of damaging DNA integrity in the male germ line. Such information could make an important contribution to understanding of the origins of both male infertility and a variety of pathological conditions that affect humans, including cancer and dominant genetic disease.

Immunocontraception with Zona pellucida Proteins

Publisher: S. Karger AG

Date: 2000

DOI: 10.1159/000016735

Abstract: The prospect of an immunological approach to contraception that would disrupt the process of fertilisation itself has resulted in a considerable interest into research in this area. It has been known for some time that antibodies raised against the zona pellucida (ZP) can suppress fertility very effectively. However, the initial optimism of this approach has been marred by the appearance of an ovarian pathology characterised by disruption of folliculogenesis and depletion of the primordial follicle pool. Adverse auto-immune reactions have been observed in the ovaries of mice after the induction of immunity with mouse ZP3 epitopes. However, this was associated with lymphocytic infiltration of the ovarian stroma, which could be circumvented by careful selection of B-cell epitopes to induce reversible infertility. In order to identify similar epitopes on primate ZP3, epitope-mapping studies were performed and incorporated into chimeric vaccines that included a promiscuous T-helper cell epitope. Both single and triple peptide vaccines have been evaluated in vivo and no detrimental effects on ovarian function were observed. The resulting high titre antibodies bound exclusively to the ZP of marmoset and human ovarian sections and could suppress in vitro human sperm-egg binding by approximately 60%, but did not prevent pregnancy in actively immunised female marmosets. Thus, considerable research is still required to identify a combination of ZP3 epitopes that will induce infertility free of any unwanted side effects.

Identification of ZP binding sites on human spermatozoa

Publisher: Elsevier BV

Date: 08-1997

DOI: 10.1016/S0165-0378(97)90392-2

New horizons in contraception

Publisher: Springer Science and Business Media LLC

Date: 10-1988

DOI: 10.1038/335492A0

Abstract: The microbiota of the mammalian intestinal tract represents a formidable barrier to colonization by pathogens. To overcome this resistance to colonization, bacterial pathogens use virulence factors to induce intestinal inflammation, which liberates nutrients for selective use by the infecting microbe. Studies of Salmonella enterica serovar Typhimurium (S. Typhimurium) infection in a streptomycin-treated mouse colitis model show how virulence factor-induced inflammation can produce nutrients used selectively by the pathogen. Type III secreted effectors of invading S. Typhimurium induce inflammation in the intestine (epithelial cells and lamina propria macrophages) that causes changes in the composition of the lumen. For ex le, neutrophils entering the intestine produce superoxide, resulting in production of tetrathionate, which S. Typhimurium in the lumen uses as an electron acceptor for anaerobic respiration. In their recent study, Lopez et al. demonstrate that S. Typhimurium strains that are lysogenized with a phage encoding type III effector SopE induce the host to produce nitric oxide synthetase (iNOS) in the intestine (C. A. Lopez et al., mBio 3:e00143-12, 2012). Nitric oxide is converted to a highly favorable electron acceptor, nitrate. As a result, growth of sopE(+) S. Typhimurium in the intestine lumen is boosted by nitrate respiration. This is a striking ex le of how acquisition of a virulence factor by horizontal gene transfer can increase the metabolic fitness of a pathogen. Interestingly, survival of the invading bacteria is probably decreased as a result of the SopE-induced immune response, and yet the S. Typhimurium bacteria that multiply in the lumen of the intestine can efficiently disseminate to another host, ensuring success for the pathogen.

Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: a possible role for GSH regulation in spermatozoa†

Publisher: Oxford University Press (OUP)

Date: 10-11-2018

DOI: 10.1093/BIOLRE/IOY241

Abstract: We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001). Furthermore, the deleterious effects of GSH depletion using menadione and 1,3 dimethoxy 1,4, naphtoquinone (DMNQ) were able to be prevented by the addition of cysteine, but no other antioxidant. Pre-incubation with cysteine prevented menadione and DMNQ induced damage to sperm membranes after 1 h (P < 0.001 P < 0.05) and after 3 h of incubation (P < 0.001, P < 0.05). Pre-incubation with cysteine ameliorated both the menadione- and DMNQ-induced increase in 4-hydroxynonenal (P < 0.001). As cysteine is a precursor of GSH, we hypothesized that stallion spermatozoa are able to synthesize this tripeptide using exogenous cysteine. To test this hypothesis, we investigated the presence of two enzymes required to synthesize GSH (GSH and GCLC) and using western blotting and immunocytochemistry we detected both enzymes in stallion spermatozoa. The inhibition of GCLC reduced the recovery of GSH by addition of cysteine after depletion, suggesting that stallion spermatozoa may use exogenous cysteine to regulate GSH. Other findings supporting this hypothesis were changes in sperm functionality after BSO treatment and changes in GSH and GSSG validated using HPLC-MS, showing that BSO prevented the increase in GSH in the presence of cysteine, although important stallion to stallion variability occurred and suggested differences in expression of glutamate cysteine ligase. Mean concentration of GSH in stallion spermatozoa was 8.2 ± 2.1 μM/109 spermatozoa, well above the nanomolar ranges per billion spermatozoa reported for other mammals.

Reactive oxygen species in spermatozoa: methods for monitoring and significance for the origins of genetic disease and infertility

Publisher: Springer Science and Business Media LLC

Date: 29-11-2005

DOI: 10.1186/1477-7827-3-67

Abstract: Human spermatozoa generate low levels of reactive oxygen species in order to stimulate key events, such as tyrosine phosphorylation, associated with sperm capacitation. However, if the generation of these potentially pernicious oxygen metabolites becomes elevated for any reason, spermatozoa possess a limited capacity to protect themselves from oxidative stress. As a consequence, exposure of human spermatozoa to intrinsically- or extrinsically- generated reactive oxygen intermediates can result in a state of oxidative stress characterized by peroxidative damage to the sperm plasma membrane and DNA damage to the mitochondrial and nuclear genomes. Oxidative stress in the male germ line is associated with poor fertilization rates, impaired embryonic development, high levels of abortion and increased morbidity in the offspring, including childhood cancer. In this review, we consider the possible origins of oxidative damage to human spermatozoa and reflect on the important contribution such stress might make to the origins of genetic disease in our species.

Proteomic and functional analysis of human sperm detergent resistant membranes

Publisher: Wiley

Date: 22-07-2007

DOI: 10.1002/JCP.22615

Abstract: Mammalian spermatozoa attain the ability to fertilize an oocyte as they negotiate the female reproductive tract. This acquisition of functional competence is preceded by an intricate cascade of biochemical and functional changes collectively known as "capacitation." Among the universal correlates of the capacitation process is a remarkable remodeling of the lipid and protein architecture of the sperm plasma membrane. While the mechanisms that underpin this dynamic reorganization remain enigmatic, emerging evidence has raised the prospect that it may be coordinated, in part, by specialized membrane microdomains, or rafts. In the present study we have demonstrated that human spermatozoa express recognized markers of membrane rafts. Further, upon depletion of membrane cholesterol through either physiological (capacitation) or pharmacological (methyl-β-cyclodextrin) intervention, these membrane rafts appear to undergo a polarized redistribution to the peri-acrosomal region of the sperm head. This finding encourages speculation that membrane rafts represent platforms for the organization of proteins involved in sperm-oocyte interactions. Support for this notion rests with the demonstration that membrane rafts isolated on the basis of their biochemical composition in the form of detergent resistant membranes (DRMs), possess the ability to adhere to homologous zona pellucidae. Furthermore a comprehensive proteomic analysis of the DRMs identified a number of proteins known for their affinity for the zona pellucida in addition to other candidates putatively involved in the mediation of downstream binding and/or fusion with the oolemma. Collectively these data afford novel insights into the subcellular localization and potential functions of membrane rafts in human spermatozoa.

Adenylyl cyclase isoforms in rat testis and spermatozoa from the cauda epididymidis

Publisher: Springer Science and Business Media LLC

Date: 12-2003

DOI: 10.1007/S00441-003-0801-6

Abstract: Expression of adenylyl cyclase genes in rat testis and spermatozoa from the cauda epididymidis was investigated using RT-PCR analysis. Genes encoding the transmembrane adenylyl cyclases (tmAC) II, III, IV, V, VI, VII, and VIII were expressed in the testis, whereas only the gene for tmAC III was expressed in caudal spermatozoa. Immunocytochemistry was used to investigate which tmAC were translated into putative, functional proteins in spermatozoa. Indirect immunofluorescence localized the tmAC II enzyme to a region on the head occupied by the acrosome. The tmAC III enzyme was localized to the posterior margin of the head and to the flagellum, whereas tmAC V and/or VI was localized to the region where the ventral surface of the acrosomal equatorial segment is located. The tmAC VII and VIII enzymes were localized to the convex margin of the head, covering the dorsal region of the acrosomal crescent. To our knowledge, this is the first demonstration that five apparently different tmAC enzymes are localized to discrete subcellular regions of mammalian spermatozoa. These findings provide a fundamental basis for future studies, to determine the physiological roles of tmAC in testis and mature spermatozoa.

Changes in the protein content of mouse uterine flushings during normal pregnancy and delayed implantation, and after ovariectomy and oestradiol administration

Publisher: Bioscientifica

Date: 05-1977

DOI: 10.1530/JRF.0.0500029

Abstract: The protein content of the mouse uterine lumen increased significantly (P less than 0-001) on Day 4 of pregnancy, the day of implantation. This increase was associated with the presence of 14 serum and 22 non-serum proteins in the lumen the major serum proteins were classed as high molecular weight slow alpha-globulins, while the dominant non-serum components consisted of slow and fast alpha-globulins, 6 prealbumins and a large quantity of proteinaceous material migrating near the origin of the gels. During experimental and lactational delayed implantation the protein levels were constantly low, transferrin, haemoglobin and albumin dominating the protein pattern. After administration of oestradiol-17beta, however, a biphasic uterine response was detected, significant increases in luminal protein concentration being observed within 12 h and again at 40-48 h after injection. The first phase of this response involved an influx of serum and non-serum proteins into the uterine lumen, most proteins migrating as high molecular weight slow alpha-globulins. The second phase involved an increase in the intensity of many non-serum components, the major proteins having Ra values of 0-06, 0-10 and 0-32. The qualitative, but not the quantitative, aspects of this response to oestradiol were identical in the absence of blastocysts.

Role of poor semen quality for current infertility and future fertility rates – lessons from the clinic and current population studies

Publisher: Wiley

Date: 02-2006

DOI: 10.1111/J.1365-2605.2005.00678.X

DNA Damage in Human Spermatozoa Is Highly Correlated with the Efficiency of Chromatin Remodeling and the Formation of 8-Hydroxy-2′-Deoxyguanosine, a Marker of Oxidative Stress1

Publisher: Oxford University Press (OUP)

Date: 09-2009

DOI: 10.1095/BIOLREPROD.109.076836

Abstract: DNA damage in human spermatozoa has been associated with a range of adverse clinical outcomes, including infertility, abortion, and disease in the offspring. We have advanced a two-step hypothesis to explain this damage involving impaired chromatin remodeling during spermiogenesis followed by a free radical attack to induce DNA strand breakage. The objective of the present study was to test this hypothesis by determining whether impaired chromatin protamination is correlated with oxidative base damage and DNA fragmentation in human spermatozoa. DNA fragmentation, chromatin protamination, mitochondrial membrane potential, and formation of the oxidative base adduct, 8-hydroxy-2'-deoxyguanosine (8OHdG), were monitored by flow cytometry/fluorescence microscopy. Impairment of DNA protamination during late spermatogenesis was highly correlated (P < 0.001) with DNA damage in human spermatozoa. The disruption of chromatin remodeling also was associated with a significant elevation in the levels of 8OHdG (P < 0.001), and the latter was itself highly correlated with DNA fragmentation (P < 0.001). The significance of oxidative stress in 8OHdG formation was demonstrated experimentally using H2O2/Fe2+ and by the correlation observed between this base adduct and superoxide generation (P < 0.001). That 8OHdG formation was inversely associated with mitochondrial membrane potential (P < 0.001) suggested a possible role for these organelles in the creation of oxidative stress. These results clearly highlight the importance of oxidative stress in the induction of sperm DNA damage and carry significant implications for the clinical management of this condition.

The importance of redox regulated pathways in sperm cell biology

Publisher: Elsevier BV

Date: 03-2004

DOI: 10.1016/J.MCE.2003.10.068

Lineage tracing reveals B cell antibody class switching is stochastic, cell-autonomous, and tuneable

Publisher: Elsevier BV

Date: 10-2022

DOI: 10.1016/J.IMMUNI.2022.08.004

Abstract: To optimize immunity to pathogens, B lymphocytes generate plasma cells with functionally erse antibody isotypes. By lineage tracing single cells within differentiating B cell clones, we identified the heritability of discrete fate controlling mechanisms to inform a general mathematical model of B cell fate regulation. Founder cells highly influenced clonal plasma-cell fate, whereas class switch recombination (CSR) was variegated within clones. In turn, these CSR patterns resulted from independent all-or-none expression of both activation-induced cytidine deaminase (AID) and IgH germline transcription (GLT), with the latter being randomly re-expressed after each cell ision. A stochastic model premised on these molecular transition rules accurately predicted antibody switching outcomes under varied conditions in vitro and during an immune response in vivo. Thus, the generation of functionally erse antibody types follows rules of autonomous cellular programming that can be adapted and modeled for the rational control of antibody classes for potential therapeutic benefit.

CALCIUM AND ZINC IN THE ENDOMETRIUM AND UTERINE FLUSHINGS OF THE ROE DEER (CAPREOLUS CAPREOLUS) DURING DELAYED IMPLANTATION

Publisher: Bioscientifica

Date: 10-1974

DOI: 10.1530/JRF.0.0400333

Abstract: The US Environmental Protection Agency (EPA) has designated a handful of instruments as Federal Reference or Federal Equivalency Methods (FRM and FEM, respectively) for the monitoring of fine particulate matter (PM

Label-Free Quantitation of Phosphopeptide Changes During Rat Sperm Capacitation

Publisher: American Chemical Society (ACS)

Date: 08-01-2010

DOI: 10.1021/PR900513D

Abstract: Before fertilization can occur, ejaculated mammalian spermatozoa must undergo a maturation process known as capacitation, which is dominated by post-translational modifications, particularly phosphorylation. Despite its biological importance, characterization of those proteins targeted for phosphorylation during capacitation remains ill-defined. Here, we report the isolation and purification of 288 phosphorylated peptides from rat spermatozoa using titanium dioxide columns in combination with nanoflow mass spectrometry. This equated to 120 identified phosphorylated proteins present in pure populations of spermatozoa. The MS survey scans of replicate titanium dioxide eluates, derived from both noncapacitated and capacitated sperm lysates, were then compared in silico using a virtual 2D PAGE format and DeCyderMS software. This analysis found 15 differentially phosphorylated proteins during capacitation. Included in this list were sperm qualifiers such as Izumo, a known sperm-oocyte fusion protein. To demonstrate that this label-free quantitative approach to phosphoprotein analysis was viable, we measured the enzymatic activity of 5'-nucleotidase, the phosphorylation status of which changed during capacitation. The results revealed, for the first time, that 5'-nucleotidase activity is up-regulated as sperm capacitate. This change, together with the other protein identifications reported in this study, constitute important new leads in elucidating the biochemical mechanisms by which spermatozoa attain a capacitated state.

Cryopreservation-induced human sperm DNA damage is predominantly mediated by oxidative stress rather than apoptosis

Publisher: Oxford University Press (OUP)

Date: 12-06-2009

DOI: 10.1093/HUMREP/DEP214

Abstract: Whereas studies have revealed that the cryopreservation of human semen increases sperm DNA fragmentation, the mechanisms involved in this type of cryo-injury are largely unknown. Elucidation of these mechanisms may provide insight into preventing such injury. We obtained 60 semen s les from 60 men and conducted experiments to determine the cause of cryopreservation-induced DNA fragmentation using 8-oxo-7,8-dihydro-2'deoxyguanosine (8OHdG) as a biomarker of oxidative stress, percentage caspase positive cells as an indicator of apoptosis, the potential antioxidant genistein and the caspase inhibitor Z-VAD(OMe)-FMK. Cryopreservation led to a significant increase in percentage DNA fragmentation, percentage 8OHdG and percentage caspase positive cells (P < 0.001). Percentage DNA fragmentation was positively correlated with percentage 8OHdG before (r = 0.756, P < 0.001) and after cryopreservation (r = 0.528, P = 0.017). The addition of 50 and 100 microM genistein to the cryoprotectant had a significant protective effect on sperm DNA (P < 0.001) although the caspase inhibitor demonstrated no difference to the control. Human sperm DNA fragmentation is associated with an increase in oxidative stress during cryopreservation, rather than the activation of caspases and apoptosis. The estrogenic compound genistein may be useful in reducing this effect but larger trials are needed to confirm this.

Prolactin Exerts a Prosurvival Effect on Human Spermatozoa via Mechanisms that Involve the Stimulation of Akt Phosphorylation and Suppression of Caspase Activation and Capacitation

Publisher: The Endocrine Society

Date: 23-12-2009

DOI: 10.1210/EN.2009-0964

Abstract: The purpose of this study was to examine the impact of prolactin (PRL) on human sperm function, in light of a recent proteomic analysis indicating that these cells express the PRL receptor (PRLR). Immunocytochemical analyses confirmed the presence of PRLR in human spermatozoa and localized this receptor to the postacrosomal region of the sperm head as well as the neck, midpiece, and principal piece of the sperm tail. Nested PCR analysis indicated that these cells possess four splice variants of the PRLR: the long form and three short isoforms, one of which is reported for the first time. A combination of Western blot analyses and immunocytochemistry demonstrated that PRL inhibited sperm capacitation in a dose-dependent manner, suppressing SRC kinase activation and phosphotyrosine expression, two hallmarks of this process. The suppression of sperm capacitation was accompanied by a powerful prosurvival effect, supporting the prolonged motility of these cells and preventing the formation of spontaneous DNA strand breaks via mechanisms that involved the concomitant suppression of caspase activation. Western blot analyses indicated that the prosurvival effect of PRL on human spermatozoa involved the stimulation of Akt phosphorylation, whereas inhibitors of phosphatidylinositol-3-OH kinase and Akt negated this effect, as did the direct induction of sperm capacitation with cAMP analogues. We conclude that PRL is a prosurvival factor for human spermatozoa that prevents these cells from defaulting to an intrinsic apoptotic pathway associated with cell senescence. These findings have implications for preservation of sperm integrity in vivo and in vitro.

Correction: Mobile Phone Radiation Induces Reactive Oxygen Species Production and DNA Damage in Human Spermatozoa In Vitro

Publisher: Public Library of Science (PLoS)

Date: 11-03-2013

DOI: 10.1371/ANNOTATION/9A8A0172-3850-4059-B852-72C330769C1B

Motility activation and second messenger signalling in spermatozoa from rat cauda epididymidis

Publisher: Bioscientifica

Date: 02-2003

DOI: 10.1530/REP.0.1250175

Abstract: This study examined molecular mechanisms involved in the activation of motility in spermatozoa from the cauda epididymidis of rats. A 1.05-fold dilution of semen from the cauda epididymidis with 300 mmol sucrose l(-1) did not activate motility in spermatozoa. Addition of dibutyryl cAMP, pentoxifylline or Ca(2+) to the sucrose activated motility in the short term ( -60 min). A fivefold dilution of semen from the cauda epididymidis with a modified Tyrode's medium (BWW) activated and sustained vigorous motility that could not be attenuated with kinase inhibitors. This motility was associated with a transient increase in intracellular cAMP during the first 60 s of activation. Lower motility was activated in Ca(2+)-deficient media but this was not associated with an increase in cAMP. A fivefold dilution with plasma from the cauda epididymidis did not activate motility. The addition of Ca(2+) to the sucrose induced an increase in cAMP of similar duration but lower magnitude to that associated with dilution in BWW. The results from this study indicate that the cAMP and Ca(2+) signal transduction pathways are involved in activation of sperm motility, and that the increase in intracellular cAMP in rat spermatozoa from the cauda epididymidis undergoing motility activation is Ca(2+)-dependent. This is the first study to report a Ca(2+)-dependent increase in cAMP associated with motility activation in immotile mammalian spermatozoa. In light of these data, a model is proposed whereby cAMP and Ca(2+) act as synarchic messengers, initiating a signal transduction cascade, which is independent of protein kinase A-mediated phosphorylation of flagella proteins in immotile spermatozoa from the cauda epididymidis.

Biochemical entities involved in reactive oxygen species generation by human spermatozoa

Publisher: Springer Science and Business Media LLC

Date: 05-2003

DOI: 10.1007/S00709-002-0057-0

Abstract: Spermatozoa were the first cell type suggested to generate reactive oxygen species. However, a lack of standardization in sperm preparation techniques and the obfuscating impact of contaminating cell types in human ejaculates have made it difficult to confirm that mammalian germ cells do, in fact, make such reactive metabolites. By identifying, on a molecular level, those entities involved in reactive oxygen species generation and demonstrating their presence in spermatozoa, the role of redox chemistry in the control of sperm function can be elucidated. Two major proteins have apparently been identified in this context, namely, NOX5, a calcium-activated NADPH oxidase, and nitric oxide synthase. Understanding the involvement of these enzymes in sperm physiology is essential if we are to understand the causes of oxidative stress in the male germ line.

The Role of Genetics and Oxidative Stress in the Etiology of Male Infertility—A Unifying Hypothesis?

Publisher: Frontiers Media SA

Date: 30-09-2020

DOI: 10.3389/FENDO.2020.581838

Cloning, sequencing and oocyte-specific expression of the marmoset sperm receptor protein, ZP3

Publisher: Cambridge University Press (CUP)

Date: 05-1993

DOI: 10.1017/S0967199400001350

Abstract: The zona pellucida surrounding the mammalian oocyte contains a major glycoprotein species, ZP3, that serves as a cell- and species-specific receptor for spermatozoa. In this study we have determined the primary amino acid structure of marmoset ZP3 (marZP3) and examined the expression of marZP3 mRNA within the ovary. The marZP3 gene possesses an open reading frame of 1272 nucleotides which is expressed specifically by the oocyte and encodes a polypeptide chain of 424 amino acids that exhibits 91% homology with the human ZP3 sequence. The disparity between these molecules was confined to a short domain spanning residues 322–352 otherwise the molecules were very similar, showing conservation of many structural features including the N –linked glycosylation sites, location and number of cysteine and proline residues and hydrophobicity profile. The results of this study have important implications for the use of the marmoset monkey as an animal model for the development of contraceptive vaccines targeting ZP3.

Reactive Oxygen Species and Their Consequences on the Structure and Function of Mammalian Spermatozoa

Publisher: Mary Ann Liebert Inc

Date: 09-2022

DOI: 10.1089/ARS.2021.0235

Cysteine-Rich Secretory Protein 2 Binds to Mitogen-Activated Protein Kinase Kinase Kinase 11 in Mouse Sperm1

Publisher: Oxford University Press (OUP)

Date: 07-2007

DOI: 10.1095/BIOLREPROD.106.057166

Abstract: Cysteine-rich secretory protein (CRISP) 2 (previously TPX1) is a testis-enriched member of the CRISP family, and has been localized to both the sperm acrosome and tail. Like all members of the mammalian CRISP family, its expression pattern is strongly suggestive of a role in male fertility, but functional support for this hypothesis remains limited. In order to determine the biochemical pathways within which CRISP2 is a component, the putative mature form of CRISP2 was used as bait in a yeast two-hybrid screen of a mouse testis expression library. One of the most frequently identified interacting partners was mitogen-activated protein kinase kinase kinase 11 (MAP3K11). Sequencing and deletion experiments showed that the carboxyl-most 20 amino acids of MAP3K11 interacted with the CRISP domain of CRISP2. This interaction was confirmed using pull-down experiments and the cellular context was supported by the localization of CRISP2 and MAP3K11 to the acrosome of the developing spermatids and epididymal spermatozoa. Interestingly, mouse epididymal sperm contained an approximately 60-kDa variant of MAP3K11, which may have been a result of proteolytic cleavage of the longer 93-kDa form seen in many tissues. These data raise the possibility that CRISP2 is a MAP3K11-modifying protein or, alternatively, that MAP3K11 acts to phosphorylate CRISP2 during acrosome development.

Evidence that fetal death is associated with placental aging

Publisher: Elsevier BV

Date: 10-2017

DOI: 10.1016/J.AJOG.2017.06.015

Abstract: The risk of unexplained fetal death or stillbirth increases late in pregnancy, suggesting that placental aging is an etiological factor. Aging is associated with oxidative damage to DNA, RNA, and lipids. We hypothesized that placentas at >41 completed weeks of gestation (late-term) would show changes consistent with aging that would also be present in placentas associated with stillbirths. We sought to determine whether placentas from late-term pregnancies and unexplained stillbirth show oxidative damage and other biochemical signs of aging. We also aimed to develop an in vitro term placental explant culture model to test the aging pathways. We collected placentas from women at 37-39 weeks' gestation (early-term and term), late-term, and with unexplained stillbirth. We used immunohistochemistry to compare the 3 groups for: DNA/RNA oxidation (8-hydroxy-deoxyguanosine), lysosomal distribution (lysosome-associated membrane protein 2), lipid oxidation (4-hydroxynonenal), and autophagosome size (microtubule-associated proteins 1A/1B light chain 3B, LC3B). The expression of aldehyde oxidase 1 was measured by real-time polymerase chain reaction. Using a placental explant culture model, we tested the hypothesis that aldehyde oxidase 1 mediates oxidative damage to lipids in the placenta. Placentas from late-term pregnancies show increased aldehyde oxidase 1 expression, oxidation of DNA/RNA and lipid, perinuclear location of lysosomes, and larger autophagosomes compared to placentas from women delivered at 37-39 weeks. Stillbirth-associated placentas showed similar changes in oxidation of DNA/RNA and lipid, lysosomal location, and autophagosome size to placentas from late-term. Placental explants from term deliveries cultured in serum-free medium also showed evidence of oxidation of lipid, perinuclear lysosomes, and larger autophagosomes, changes that were blocked by the G-protein-coupled estrogen receptor 1 agonist G1, while the oxidation of lipid was blocked by the aldehyde oxidase 1 inhibitor raloxifene. Our data are consistent with a role for aldehyde oxidase 1 and G-protein-coupled estrogen receptor 1 in mediating aging of the placenta that may contribute to stillbirth. The placenta is a tractable model of aging in human tissue.

Redox activity associated with the maturation and capacitation of mammalian spermatozoa

Publisher: Elsevier BV

Date: 04-2004

DOI: 10.1016/J.FREERADBIOMED.2004.01.017

The Simmet Lecture: New Horizons on an Old Landscape - Oxidative Stress, DNA Damage and Apoptosis in the Male Germ Line

Publisher: Wiley

Date: 25-07-2012

DOI: 10.1111/J.1439-0531.2012.02049.X

Abstract: Our ability to diagnose and treat male infertility is gradually improving in concert with advances in our understanding of the molecular mechanisms underpinning defective sperm function. In this context, one of the factors to emerge as a major causative agent in male infertility is oxidative stress. Spermatozoa are particularly susceptible to such stress because they are exceptionally rich in vulnerable substrates such as polyunsaturated fatty acids, proteins and DNA. The lack of sperm cytoplasm also provides these cells with little capacity to protect themselves from oxidative attack or to effect any repair, should damage occur. Similarly, sperm chromatin is in a quasi-crystalline state and has very little capacity to respond to any DNA damage induced by oxidative attack. When the latter does occur, it appears to be initiated by reactive oxygen species (ROS) generated by the sperm mitochondria. These free radicals attack the lipids present in the sperm mitochondria generating electrophilic aldehydes, which bind to components of the mitochondrial electron transport chain stimulating yet more ROS production. The oxidative stress created via this self-propagating mechanism initiates an apoptotic cascade as a result of which the spermatozoa loose their capacity for fertilization and suffer damage to their DNA. Phosphatidylserine externalization is a late event in sperm apoptosis and may facilitate the silent phagocytosis of moribund cells in the female reproductive tract, that is, the phagocytosis of senescent spermatozoa without the accompanying generation of an inflammatory response. Encouragingly, the involvement of oxidative stress in the aetiology of male infertility has opened up new opportunities for therapeutic interventions involving the judicious administration of nucleophiles and other forms of antioxidants.

Significance of Poisson distribution theory in analysing the interaction between human spermatozoa and zona-free hamster oocytes

Publisher: Bioscientifica

Date: 11-1984

DOI: 10.1530/JRF.0.0720311

Abstract: The value of Poisson distribution theory in describing and predicting the nature of sperm-egg interaction in vitro has been investigated using an interspecific in-vitro fertilization system, incorporating zona-free hamster oocytes and human spermatozoa. The frequency distribution of polyspermic oocyte penetrations in 72 experiments exhibited good agreement with the Poisson distribution at all levels of fertilization indicating that each oocyte must be of equal penetrability and that there can be no block to polyspermy in this interspecific system. Poisson distribution theory also accurately described the relationship between oocyte penetration and sperm motility in 50 out of 54 separate experiments spread across 10 serial dilution curves. For each dilution series the shape of the fitted curve was fixed but its location along the x-axis varied from donor to donor. The fixed nature of the relationship between sperm motility and egg penetration enables the results of such in-vitro fertilization experiments to be corrected for the number of motile spermatozoa in the incubation media. On the basis of these findings a protocol is described for assessing the results of the zona-free hamster oocyte penetration assay, which involves analysis of the degree of polyspermy followed by the application of Poisson distribution theory to correct the results to a standard concentration of motile spermatozoa. Changes in the penetrating ability of human spermatozoa after vasectomy and characterization of the degree of inter-ejaculate variation in penetrating potential are two clinical ex les of such analyses given in the text. The statistical methods described in this paper should also be of general relevance to the study of fertilization mechanisms, in providing a rationale by which to analyse the quantitative nature of sperm-egg interaction in vitro.

Ontogeny of Tyrosine Phosphorylation-Signaling Pathways During Spermatogenesis and Epididymal Maturation in the Mouse1

Publisher: Oxford University Press (OUP)

Date: 10-2006

DOI: 10.1095/BIOLREPROD.106.052712

Abstract: The objectives of this study were to map the ontogeny of tyrosine phosphorylation signal transduction pathways during germ cell development and to determine their association with the differentiation of a functional gamete. Until testicular germ cells differentiate into spermatozoa, cAMP-induced tyrosine phosphorylation is not detectable. Entry of these cells into the epididymis is accompanied by sudden activation of the tyrosine phosphorylation pathway, initially in the principal piece of the cell and subsequently in the midpiece. In the caput and corpus epididymides, the potential to express this pathway is inhibited by the presence of calcium in the extracellular medium. However, calcium has no effect on the expression of this pathway in caudal epididymal sperm. The competence of these cells to phosphorylate the entire sperm tail, from the neck to the tail-end piece, is accompanied by a capacity to exhibit hyperactivated motility on stimulation with cAMP. A distinctly different pattern of tyrosine phosphorylation, involving the acrosomal domain of the sperm head, is invoked as spermatozoa enter the caput epididymis, and phosphorylation remains high until these cells enter the distal corpus and cauda. The proportion of cells exhibiting this form of tyrosine phosphorylation is not affected by extracellular calcium or cAMP but is negatively correlated (R2 = 0.99) with their ability to acrosome-react. However, this relationship is not causative. Our findings indicate that the development of functional spermatozoa is accompanied by carefully orchestrated changes in tyrosine phosphorylation, controlled by independent regulatory mechanisms in distinct subcellular compartments of these highly specialized cells.

Uterine secretion of fructose in the roe deer

Publisher: Bioscientifica

Date: 03-1976

DOI: 10.1530/JRF.0.0460439

Abstract: Rabies virus from the brain of a striped skunk (Mephitis mephitis) from Ontario was inoculated into murine neuroblastoma (NA-C1300) cell cultures. These cultures were incubated and the cells were subcultured every three to four days. The presence of viral antigen in the cell cultures was monitored by direct immunofluorescent staining and in the culture fluids by titration in either baby hamster kidney (BHK/C13) or NA cells or in experimental mice. The virus-infected NA cultures evolved from an initial high viral concentration in supernatant fluid through a period of decreasing titers of infectious virus in the supernatant fluids to a final phase where no infectious virus has been found following cell culture and animal inoculation methods attempted although the persistently infected cells remained 95-100% viral nucleocapsid antigen-positive. Possible mechanisms involved in the perpetuation of this infection are discussed. This is the first report of a persistent infection of cell cultures by a field strain of rabies virus.

Relationship between calcium, cyclic AMP, ATP, and intracellular pH and the capacity of hamster spermatozoa to express hyperactivated motility

Publisher: Wiley

Date: 02-1989

DOI: 10.1002/MRD.1120220205

Abstract: Capacitation of hamster caudal spermatozoa at a density of 1 x 10(6)/ml is associated with a progressive rise in cAMP levels that precedes the onset of hyperactivated motility. This increase is not expressed by caput spermatozoa incubated under identical conditions. Both the incidence of hyperactivation and the rise in cAMP levels are severely attenuated in the absence of exogenous calcium. Neither factor is restored to control levels by the addition of the phosphodiesterase inhibitor IBMX, although in the presence of exogenous calcium, this reagent increased cAMP levels, stimulated percentage motility and advanced the appearance of hyperactivation. Treatment of spermatozoa at a density of 1 x 10(6)/ml with the calmodulin antagonist, calmidazolium (CZ), caused severe disruption of sperm motility and abolished hyperactivation, while causing only a slight reduction in cAMP content. Addition of IBMX in the presence of CZ elevated cAMP content to levels higher than normally observed during capacitation but did not restore either coordinated or hyperactivated motility. To determine both the mechanisms responsible for this elevation of cAMP content and the changes that occur during epididymal maturation to facilitate the expression of this increase, the free cytosolic calcium concentration, ATP levels, and intracellular pH of caput and caudal cells were compared. The calcium content of caudal spermatozoa rose significantly at a time when cAMP levels were increasing, while ATP content and intracellular pH fell. However, the inability of caput spermatozoa to express a rise in cAMP content was not due to deficiencies in any of these factors. These results indicate a positive role for the cAMP rise in the expression of hyperactivated motility and that the fundamental control mechanism governing both these events may be the influx of calcium that accompanies capacitation in this species.

Oxidative stress and reproductive function

Publisher: Bioscientifica

Date: 12-2022

DOI: 10.1530/REP-22-0368

OXIDATIVE STRESS AND REPRODUCTIVE FUNCTION The impact of oxidative stress on reproduction: a focus on gametogenesis and fertilization

Publisher: Bioscientifica

Date: 12-2022

DOI: 10.1530/REP-22-0126

Abstract: Many aspects of the reproductive process are impacted by oxidative stress. This article summarizes the chemical nature of reactive oxygen species and their role in both the physiological regulation of reproductive processes and the pathophysiology of infertility. This article lays out the fundamental principles of oxidative stress. It describes the nature of reactive oxygen species (ROS), the way in which these potentially toxic metabolites interact with cells and how they impact both cellular function and genetic integrity. The mechanisms by which ROS generation is enhanced to the point that the cells’ antioxidant defence mechanisms are overwhelmed are also reviewed taking ex les from both the male and female reproductive system, with a focus on gametogenesis and fertilization. The important role of external factors in exacerbating oxidative stress and impairing reproductive competence is also examined in terms of their ability to disrupt the physiological redox regulation of reproductive processes. Developing diagnostic and therapeutic strategies to cope with oxidative stress within the reproductive system will depend on the development of a deeper understanding of the nature, source, magnitude, and location of such stress in order to fashion personalized treatments that meet a given patient’s clinical needs.

Analysis of the relationships between oxidative stress, DNA damage and sperm vitality in a patient population: development of diagnostic criteria

Publisher: Oxford University Press (OUP)

Date: 17-08-2010

DOI: 10.1093/HUMREP/DEQ214

Abstract: DNA damage in human spermatozoa is known to be associated with a variety of adverse clinical outcomes affecting both reproductive efficiency and the health and wellbeing of the offspring. However, the origin of this damage, its biochemical nature and strategies for its amelioration, still await resolution. Using novel methods to simultaneously assess DNA fragmentation (modified TUNEL assay), DNA-base adduct formation (8-hydroxy-2'-deoxyguanosine [8OHdG]) and cell vitality, spermatozoa from a cohort of 50 assisted conception patients were examined and compared with a group of donors. Receiver operating characteristic (ROC) curve analysis was then used to examine the frequency distribution of the data and to determine optimized thresholds for identifying patients exhibiting abnormally high levels of DNA damage. 8OHdG formation and DNA fragmentation were highly correlated with each other and frequently associated with cell death. Percoll centrifugation improved sperm quality but, unexpectedly, increased 8OHdG formation in live cells, as did sperm fractionation using Puresperm gradients. ROC analysis indicated that the frequency distribution of 8OHdG and DNA fragmentation data were significantly different between patients and donors (P < 0.001), permitting the development of thresholds that would allow the accurate diagnosis of DNA damage in the male germ line. The aetiology of DNA damage in spermatozoa involves a cascade of changes that progress from the induction of oxidative stress and oxidized DNA base adduct formation to DNA fragmentation and cell death. Preparation of spermatozoa on discontinuous density gradients aggravates the problem by stimulating the formation of 8OHdG in live cells. However, the development of novel methods and optimized thresholds for diagnosing oxidative DNA damage in human spermatozoa should assist in the clinical management of this pathology.

Identification of a novel testis-specific primate antigen

Publisher: Elsevier BV

Date: 08-1997

DOI: 10.1016/S0165-0378(97)90400-9

The role of the molecular chaperone heat shock protein A2 (HSPA2) in regulating human sperm-egg recognition

Publisher: Medknow

Date: 2015

DOI: 10.4103/1008-682X.151395

COVID‐19 and human spermatozoa—Potential risks for infertility and sexual transmission?

Publisher: Wiley

Date: 05-08-2020

DOI: 10.1111/ANDR.12859

Persistence of the parabens in soil and their potential toxicity to earthworms

Publisher: Elsevier BV

Date: 04-2021

DOI: 10.1016/J.ETAP.2020.103574

The source and significance of DNA damage in human spermatozoa; a commentary on diagnostic strategies and straw man fallacies

Publisher: Oxford University Press (OUP)

Date: 04-2013

DOI: 10.1093/MOLEHR/GAT025

Abstract: This article considers the origins of DNA damage in human spermatozoa, the methods that are available to monitor this aspect of semen quality and the clinical significance of such measurements. DNA damage in spermatozoa appears to be largely oxidative in nature, inversely correlated with levels of nuclear protamination and frequently associated with the activation of a truncated apoptotic pathway. DNA base adducts formed as a result of oxidative attack are released from the spermatozoa into the extracellular space through the action of a glycosylase, OGG1. This creates an abasic site, which is not resolved until fertilization because spermatozoa do not possess the molecular machinery needed to continue the base excision repair pathway. The abasic sites so generated in human spermatozoa are readily detected by SCSA or the Comet assay however, no signal is detectable with TUNEL. This is because spermatozoa lack the enzyme (APE1) needed to create the free 3' hydroxyl groups required by this detection system. Nevertheless, spermatozoa do eventually become TUNEL positive as they enter the perimortem. The American Society of Reproductive Medicine Practice Committee has suggested that DNA damage in spermatozoa should not be assessed because the correlation with pregnancy is inconsistent across independent studies. However, this is a straw man argument. The reason why such assays should be undertaken is not just that they reflect the underlying quality of spermatogenesis but, more importantly, that the DNA damage they reveal may have detrimental effects on the developmental normality of the embryo and the health of possible future children.

Reactive oxygen species and sperm function-in sickness and in health

Publisher: Wiley

Date: 12-11-2012

DOI: 10.2164/JANDROL.112.016535

Abstract: The ability of spermatozoa to generate reactive oxygen species (ROS) has been appreciated since the 1940s. It is a universal property of mature spermatozoa from all mammalian species and a major contributor to the oxidative stress responsible for defective sperm function. The mechanisms by which oxidative stress limits the functional competence of mammalian spermatozoa involve the peroxidation of lipids, the induction of oxidative DNA damage, and the formation of protein adducts. ROS production in these cells involves electron leakage from the sperm mitochondria, triggered by a multitude of factors that impede electron flow along the electron transport chain. The net result of mitochondrial ROS generation is to damage these organelles and initiate an intrinsic apoptotic cascade, as a consequence of which spermatozoa lose their motility, DNA integrity, and vitality. This pathway of programmed senescence also results in the exteriorization of phosphatidylserine, which may facilitate the silent phagocytosis of these cells in the aftermath of insemination, in turn influencing the female tract immune response to sperm antigens and future fertility. Despite the vulnerability of sperm to oxidative stress, it is also clear that normal sperm function depends on low levels of ROS generation in order to promote the signal transduction pathways associated with capacitation. Modulators of ROS generation by spermatozoa may therefore have clinical utility in regulating the fertilizing capacity of these cells and preventing the development of antisperm immunity. Achievement of these objectives will require a systematic evaluation of pro- and antioxidant strategies in vivo and in vitro.

Whole-body exposures to radiofrequency-electromagnetic energy can cause DNA damage in mouse spermatozoa via an oxidative mechanism

Publisher: Springer Science and Business Media LLC

Date: 25-11-2019

DOI: 10.1038/S41598-019-53983-9

Abstract: Artificially generated radiofrequency-electromagnetic energy (RF-EME) is now ubiquitous in our environment owing to the utilization of mobile phone and Wi-Fi based communication devices. While several studies have revealed that RF-EME is capable of eliciting biological stress, particularly in the context of the male reproductive system, the mechanistic basis of this biophysical interaction remains largely unresolved. To extend these studies, here we exposed unrestrained male mice to RF-EME generated via a dedicated waveguide (905 MHz, 2.2 W/kg) for 12 h per day for a period of 1, 3 or 5 weeks. The testes of exposed mice exhibited no evidence of gross histological change or elevated stress, irrespective of the RF-EME exposure regimen. By contrast, 5 weeks of RF-EME exposure adversely impacted the vitality and motility profiles of mature epididymal spermatozoa. These spermatozoa also experienced increased mitochondrial generation of reactive oxygen species after 1 week of exposure, with elevated DNA oxidation and fragmentation across all exposure periods. Notwithstanding these lesions, RF-EME exposure did not impair the fertilization competence of spermatozoa nor their ability to support early embryonic development. This study supports the utility of male germ cells as sensitive tools with which to assess the biological impacts of whole-body RF-EME exposure.

Impact of radio frequency electromagnetic radiation on DNA integrity in the male germline

Publisher: Wiley

Date: 06-2005

DOI: 10.1111/J.1365-2605.2005.00531.X

Abstract: Concern has arisen over human exposures to radio frequency electromagnetic radiation (RFEMR), including a recent report indicating that regular mobile phone use can negatively impact upon human semen quality. These effects would be particularly serious if the biological effects of RFEMR included the induction of DNA damage in male germ cells. In this study, mice were exposed to 900 MHz RFEMR at a specific absorption rate of approximately 90 mW/kg inside a waveguide for 7 days at 12 h per day. Following exposure, DNA damage to caudal epididymal spermatozoa was assessed by quantitative PCR (QPCR) as well as alkaline and pulsed-field gel electrophoresis. The treated mice were overtly normal and all assessment criteria, including sperm number, morphology and vitality were not significantly affected. Gel electrophoresis revealed no gross evidence of increased single- or double-DNA strand breakage in spermatozoa taken from treated animals. However, a detailed analysis of DNA integrity using QPCR revealed statistically significant damage to both the mitochondrial genome (p < 0.05) and the nuclear beta-globin locus (p < 0.01). This study suggests that while RFEMR does not have a dramatic impact on male germ cell development, a significant genotoxic effect on epididymal spermatozoa is evident and deserves further investigation.

Clinical assessment of human sperm function

Publisher: Informa UK Limited

Date: 1983

DOI: 10.3109/01443618309073708

Differential contribution of leucocytes and spermatozoa to the generation of reactive oxygen species in the ejaculates of oligozoospermic patients and fertile donors

Publisher: Bioscientifica

Date: 03-1992

DOI: 10.1530/JRF.0.0940451

Abstract: Cells isolated from the ejaculates of a high proportion of patients exhibiting oligozoospermia are characterized by generation rates of reactive oxygen species that considerably exceed those obtained for the normal fertile population. The purpose of this study was to resolve the cellular source of this enhanced activity. Semen s les from a cohort of oligozoospermic patients and a group of fertile controls were fractionated on discontinuous Percoll gradients to generate three cell populations (0, 50 and 100%) of differing density. For each fraction, both the steady-state and the phorbol-ester-induced chemiluminescent signals were significantly (P less than 0.001) greater for the oligozoospermic s les than for the fertile controls. In the fertile donors, leucocytes comprised the major source of reactive oxygen species, particularly in the low-density Percoll fractions in oligozoospermic patients, however, spermatozoa were identified as a second major source of reactive oxygen species. Particularly striking was an intense phorbol-ester-induced chemiluminescent signal generated by oligozoospermic spermatozoa, purified by passage through isotonic Percoll and free of leucocyte contamination, which was 167 times greater than the median signal generated by the corresponding fraction from the fertile controls (P less than 0.001). These results emphasize the importance of spermatozoa as a major source of reactive oxygen species in oligozoospermia and have implications for the diagnosis and treatment of this condition, as well as for the design of appropriate diagnostic strategies.

Prediction of the in-vitro fertilization (IVF) potential of human spermatozoa using sperm function tests: The effect of the delay between testing and IVF

Publisher: Oxford University Press (OUP)

Date: 05-1996

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A019291

Abstract: To examine the diagnostic significance of several criteria of semen quality and to determine whether their prognostic value is eroded by the time interval between assessment and the attempt at in-vitro fertilization (IVF) with embryo transfer, 73 couples undergoing IVF and embryo transfer therapy were studied. The ability of human spermatozoa to achieve fertilization in vitro was examined in relation to the conventional semen profile, sperm morphology, the computer-aided assessment of sperm movement, ionophore-induced acrosome reaction, acridine orange staining, and chemiluminescent signals induced by phorbol ester and N-formyl-methionyl-leucyl-phenylalanine (FMLP). Spermatozoa were examined both in semen and after preparation on Percoll, some weeks prior to IVF. Fertilization rates were noted to be significantly correlated with elements of sperm movement characteristics, sperm morphology, and reactive oxygen species generation. Prediction of fertilization rates in a stepwise multiple regression analysis was obtained using four variables: sperm morphology, FMLP-induced chemi-luminescence and sperm movement characteristics (beat cross frequency and straightness) (r approximately 0.5). When multiple logistic regression analysis was used to predict which s les would achieve fertilization rates above and below a 50% threshold, three variables of predictive value including linearity, average path velocity and FMLP-induced chemiluminescence were selected. Combination of these variables classified the s les achieving good or poor fertilization with an overall accuracy of 83.6%. The time interval between semen assessment and IVF had little effect on the predictive value of these tests. In conclusion, the fertilizing ability of human spermatozoa is related to sperm morphology, attributes of sperm movement and reactive oxygen species production. The time delay between testing and IVF did not appear to affect predictive accuracy.

The Amoroso Lecture: The human spermatozoon - A cell in crisis?

Publisher: Bioscientifica

Date: 1999

DOI: 10.1530/JRF.0.1150001

Abstract: A great deal of evidence has accumulated in recent years to suggest that there has been a gradual increase in male reproductive pathology over the past 30-40 years, as evidenced by increased rates of testicular cancer and declining semen quality. The hypothesis is advanced that this phenomenon is causally related to the ability of male germ cells to generate reactive oxygen metabolites. When produced in low levels, such metabolites are thought to enhance sperm function by stimulating DNA compaction and promoting a redox-regulated cAMP-mediated pathway that is central to the induction of sperm capacitation. When produced in excessive amounts, the same metabolites stimulate DNA fragmentation and a loss of sperm function associated with peroxidative damage to the sperm plasma membrane. Free radical-induced mutations in the male germ line may also be involved in the aetiology of childhood cancer and recent increases in the incidence of seminoma. In light of these considerations, establishing the mechanisms for free radical generation by the male germ line and determining the factors that influence this activity are important objectives for future research in this area.

DELAYED IMPLANTATION IN ROE DEER (CAPREOLUS CAPREOLUS)

Publisher: Bioscientifica

Date: 07-1974

DOI: 10.1530/JRF.0.0390225

Sperm cell biology: current perspectives and future prospects

Publisher: Medknow

Date: 22-11-2010

DOI: 10.1038/AJA.2010.155

Multiple forms of redox activity in populations of human spermatozoa

Publisher: Oxford University Press (OUP)

Date: 11-2003

DOI: 10.1093/MOLEHR/GAG086

Abstract: In this study we have examined the biochemical attributes of the redox systems that regulate human sperm function using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium, monosodium salt (WST-1), lucigenin and luminol-peroxidase as probes. WST-1 was readily reduced by human spermatozoa in the presence of an intermediate electron acceptor (IEA) or NAD(P)H. The IEA-mediated activity resembled a previously described trans-membrane NADH oxidase in being inhibited by capsaicin, superoxide dismutase (SOD) and N-ethyl maleimide, but differed in its sensitivity to p-chloromercuriphenylsulphonic acid (pCMBS). The NAD(P)H-induced WST-1 reduction resembled the superficial oxidase described previously, in its sensitivity to pCMBS, but differed in its suppression by capsaicin. Lucigenin was reduced by human spermatozoa in a manner that could be inhibited by SOD and stimulated by NAD(P)H or 12-myristate, 13-acetate phorbol ester. A23187 also stimulated human spermatozoa via a diphenylene iodonium-sensitive pathway detectable with luminol-peroxidase but not lucigenin. Defective sperm populations recovered from the low-density region of Percoll gradients were characterized by high levels of redox activity that was only discernable with lucigenin. We conclude that human spermatozoa possess multiple plasma membrane redox systems that are involved to varying extents in the physiological control and pathological disruption of sperm function. Their distinct pharmacological profiles should significantly assist attempts to resolve and characterize these systems.

Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmic sperm injection

Publisher: Oxford University Press (OUP)

Date: 07-1998

DOI: 10.1093/HUMREP/13.7.1864

Abstract: We present the first evidence that genetically damaged human spermatozoa are able to form normal pronuclei in oocytes after intracytoplasmic sperm injection (ICSI). The role of reactive oxygen species (ROS) as a cause of chromatin and DNA damage is well recognized. The same class of molecule can be found in the semen of males with severe infertility, who remained infertile until the advent of ICSI. In this study we have investigated the role of ROS in the induction of chromatin damage, DNA strand breakage and the subsequent ability of spermatozoa to decondense and form pronuclei after ICSI. Spermatozoa from normozoospermic men participating in our research programme were exposed to oxidizing environments created by co-incubation with hydrogen peroxide, reduced nicotinamide adenine dinucleotide phosphate (NADPH) or activated white cells. The subsequent ability of the spermatozoa to decondense in vitro was examined using sequential incubations in EDTA, dithiothreitol and sodium dodecyl sulphate, and the amounts of DNA strand breakage were assessed using an in-situ nick translation protocol. Finally, cells exposed to hydrogen peroxide, NADPH and activated leukocytes were microinjected into hamster oocytes, and their ability to decondense and form normal pronuclei was determined. The results indicate that human sperm chromatin becomes cross-linked under conditions of oxidative stress and exhibits increased DNA strand breakage, yet the rate of pronucleus formation is no different from that of untreated control cells. The ability of genetically damaged spermatozoa to achieve normal fertilization following ICSI has implications for the practice of this form of assisted conception therapy.

Reply

Publisher: Elsevier BV

Date: 1992

DOI: 10.1016/0002-9378(92)91873-9

Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma

Publisher: Springer Science and Business Media LLC

Date: 07-06-2021

DOI: 10.1038/S41590-021-00943-Z

The Capacitation-Apoptosis Highway: Oxysterols and Mammalian Sperm Function

Publisher: Oxford University Press (OUP)

Date: 07-2011

DOI: 10.1095/BIOLREPROD.111.092528

Evaluation of human sperm function

Publisher: Oxford University Press (OUP)

Date: 1990

DOI: 10.1093/OXFORDJOURNALS.BMB.A072423

Abstract: Our understanding of the causes of male infertility and our ability to develop an appropriate range of diagnostic tests are both dependent upon a knowledge of the cellular mechanisms responsible for the regulation of human sperm function. This review examines the intra- and extracellular factors regulating four separate components of human sperm function: sperm transport, sperm-egg recognition, the acrosome reaction and sperm-oocyte fusion. Within each of these areas the fundamental nature of the process, the control mechanisms involved, the availability of appropriate diagnostic tests and the relationship with human infertility have been considered.

Biochemical alterations in the oocyte in support of early embryonic development

Publisher: Springer Science and Business Media LLC

Date: 07-09-2016

DOI: 10.1007/S00018-016-2356-1

Abstract: Notwithstanding the enormous reproductive potential encapsulated within a mature mammalian oocyte, these cells present only a limited window for fertilization before defaulting to an apoptotic cascade known as post-ovulatory oocyte aging. The only cell with the capacity to rescue this potential is the fertilizing spermatozoon. Indeed, the union of these cells sets in train a remarkable series of events that endows the oocyte with the capacity to ide and differentiate into the trillions of cells that comprise a new in idual. Traditional paradigms hold that, beyond the initial stimulation of fluctuating calcium (Ca

A clue to Kartagener's

Publisher: Springer Science and Business Media LLC

Date: 09-1991

DOI: 10.1038/353306A0

Andrology: Analysis of the extent to which sperm movement can predict the results of ionophore-enhanced functional assays of the acrosome reaction and sperm-oocyte fusion

Publisher: Oxford University Press (OUP)

Date: 10-1994

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A138350

Abstract: This study has examined the extent to which the information generated by ionophore-enhanced bioassays of the acrosome reaction and sperm-oocyte fusion might be predicted from the computer-aided analysis of sperm motility. Strong correlations (r approximately 0.7) were observed between specific components of sperm movement in semen and the potential for A23187-induced sperm-oocyte fusion, generating a stepwise regression coefficient of R = 0.663 on the bais of two criteria, percentage progressive motility and litude of sperm lateral head displacement (ALH). The movement characteristics of the spermatozoa recovered from the Percoll gradients gave an even higher R value of 0.838 on the basis of four variables (percentage rapid, average path velocity, straightness and ALH). In contrast, the ability of human spermatozoa to undergo acrosome reaction in response to A23187 exhibited a limited correlation with sperm movement, whether these measurements were made in the original semen s le or following Percoll purification (R approximately 0.4). These results have diagnostic implications, since sperm-oocyte fusion and the acrosome reaction clearly differ in their relative dependence on sperm motility. In practical terms, it should be noted that the computer-aided analysis of sperm movement was shown to provide up to 70% of the information generated by the more laboured assessment of sperm-oocyte fusion.

Impact of a deep saturation dive on semen quality

Publisher: Wiley

Date: 04-2000

DOI: 10.1046/J.1365-2605.2000.00216.X

Abstract: The demonstration e 'Aurora' has provided an opportunity to study the impact of extreme hyperbaric conditions on male fertility. This operation involved a 33-day ing programme during which ers were exposed to a maximum pressure of 4.6 Mega Pascals (Mpa) for 7 days. At days - 4, + 27, + 34, + 82 and + 263 relative to the initiation of the e, semen s les were analysed to determine the quality of spermatogenesis and the functional competence of the spermatozoa. A dramatic fall in semen quality was observed in association with the e and by day + 82 the potential fertility of the men was seriously compromised as evidenced by oligoasthenoteratozoospermic semen profiles and the poor fertilizing potential of the spermatozoa. These studies indicate, for the first time, that the severe hyperbaric conditions associated with deep saturation es have a profound effect on male reproductive function.

Diagnostic value of the zona‐free hamster oocyte penetration test and sperm movement characteristics in oligozoospermia

Publisher: Wiley

Date: 10-1985

DOI: 10.1111/J.1365-2605.1985.TB00847.X

Abstract: A group of 27 oligozoospermic patients were followed up for 4.2 +/- 1.1 years in a prospective study designed to determine which aspects of semen quality are of value in the diagnosis of fertility. The semen analysis included the zona-free hamster oocyte penetration test, the assessment of sperm movement characteristics by time exposure photomicrography and a conventional semen profile. During the follow up period, 7 patients (25.9%) initiated a pregnancy. The identity of these fertile subjects could not have been ascertained by any single criterion of sperm movement or any feature of the conventional semen profile. The zona-free hamster egg penetration rates were also of little value in this respect since 4 of the 7 fertile patients scored 0% in this assay. Using a multivariate discriminant analysis, however, a combination of 7 criteria was identified, including hamster oocyte penetration and elements of both sperm movement and the semen profile, which could predict the fertility of these patients with 85.2% accuracy.

Use of a radioimmunoassay technique to measure the titer of anti‐cow‐zona antibodies in marmoset monkeys

Publisher: Wiley

Date: 1982

DOI: 10.1002/MRD.1120050307

The Sins of Our Forefathers: Paternal Impacts on De Novo Mutation Rate and Development

Publisher: Annual Reviews

Date: 23-11-2020

DOI: 10.1146/ANNUREV-GENET-112618-043617

Abstract: Spermatogonial stem cells (SSCs) are generally characterized by excellent DNA surveillance and repair, resulting in one of the lowest spontaneous mutation rates in the body. However, the barriers to mutagenesis can be overwhelmed under two sets of circumstances. First, replication errors may generate age-dependent mutations that provide the mutant cells with a selective advantage, leading to the clonal expansions responsible for dominant genetic diseases such as Apert syndrome and achondroplasia. The second mechanism centers on the vulnerability of the male germline to oxidative stress and the induction of oxidative DNA damage in spermatozoa. Defective repair of such oxidative damage in the fertilized oocyte results in the creation of mutations in the zygote that can influence the health and well-being of the offspring. A particular hot spot for such oxidative attack on chromosome 15 has been found to align with several mutations responsible for paternally mediated disease, including cancer, psychiatric disorders, and infertility.

Male Infertility and Oxidative Stress: A Focus on the Underlying Mechanisms

Publisher: MDPI AG

Date: 02-02-2022

DOI: 10.3390/ANTIOX11020306

Abstract: Reactive oxygen species (ROS) play a critical role in defining the functional competence of human spermatozoa. When generated in moderate amounts, ROS promote sperm capacitation by facilitating cholesterol efflux from the plasma membrane, enhancing cAMP generation, inducing cytoplasmic alkalinization, increasing intracellular calcium levels, and stimulating the protein phosphorylation events that drive the attainment of a capacitated state. However, when ROS generation is excessive and/or the antioxidant defences of the reproductive system are compromised, a state of oxidative stress may be induced that disrupts the fertilizing capacity of the spermatozoa and the structural integrity of their DNA. This article focusses on the sources of ROS within this system and examines the circumstances under which the adequacy of antioxidant protection might become a limiting factor. Seminal leukocyte contamination can contribute to oxidative stress in the ejaculate while, in the germ line, the dysregulation of electron transport in the sperm mitochondria, elevated NADPH oxidase activity, or the excessive stimulation of amino acid oxidase action are all potential contributors to oxidative stress. A knowledge of the mechanisms responsible for creating such stress within the human ejaculate is essential in order to develop better antioxidant strategies that avoid the unintentional creation of its reductive counterpart.

Chromosome studies in human in vitro fertilization

Publisher: Springer Science and Business Media LLC

Date: 04-1986

DOI: 10.1007/BF00290960

Abstract: Whether early glomerular, tubulointerstitial, vascular, and global glomerulosclerotic lesions can predict progression of diabetic nephropathy is not well defined. Here, we sought to determine whether renal structural parameters predict the development of proteinuria or ESRD after long-term follow-up. We measured several renal structures in kidney biopsies from 94 normoalbuminuric patients with longstanding type 1 diabetes using unbiased morphometric methods. Greater width of the glomerular basement membrane and higher levels of glycated hemoglobin were independent predictors of progression to diabetic nephropathy in this normoalbuminuric cohort. Moreover, none of these patients with type 1 diabetes who had glomerular basement membrane widths within the normal range developed proteinuria and/or ESRD. In conclusion, careful quantitative assessment of kidney biopsies in normoalbuminuric patients with type 1 diabetes adds substantially to the prediction of progression to clinical diabetic nephropathy.

Whither must spermatozoa wander? The future of laboratory seminology

Publisher: Medknow

Date: 2010

DOI: 10.1038/AJA.2008.42

Impact of estrogenic compounds on DNA integrity in human spermatozoa: Evidence for cross-linking and redox cycling activities

Publisher: Elsevier BV

Date: 05-2008

DOI: 10.1016/J.MRFMMM.2008.02.002

Abstract: A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (beta-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17beta-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male infertility is warranted.

Synchronous Colonic Adenocarcinoma and Extragenital Malignant Mixed Mesodermal Tumour

Publisher: SAGE Publications

Date: 12-1989

DOI: 10.1177/003693308903400609

Abstract: A synchronous occurrence of large bowel adenocarcinoma and extragenital malignant mixed mesodermal tumour (MMMT) is reported. This case represents the sixth extragenital MMMT reported in the literature.

Andrology Lab Corner*: Shedding Light on Chemiluminescence: The Application of Chemiluminescence in Diagnostic Andrology

Publisher: Wiley

Date: 08-07-2004

DOI: 10.1002/J.1939-4640.2004.TB02815.X

Influence of epididymal maturation on cyclic AMP levels in hamster spermatozoa

Publisher: Wiley

Date: 02-1989

DOI: 10.1111/J.1365-2605.1989.TB01283.X

Abstract: Whilst in their native epididymal fluid, sperm from the caput epididymis of the rat and hamster contain significantly (P less than 0.01) greater amounts of cAMP than do sperm from the cauda epididymis. The cAMP levels in both cell types from these species underwent a rapid increase concomitant with dilution to a density of 20 x 10(6)/ml. Further analyses in the hamster indicated that this increase was calcium-dependent, and could be enhanced by treatment with the calmodulin antagonist, calmidazolium. Dilution of hamster sperm to a concentration of 1 x 10(6)/ml was not associated with a rapid rise in cAMP levels. This effect was shown to be due to the dilution of a component in epididymal plasma. When incubated at this lower density, the cAMP content of hamster caput sperm remained low over a 3 h period, whilst similarly treated caudal sperm exhibited a progressive rise in cAMP levels. Thus, in contrast to other species, cAMP does not appear to play a pivotal role in acquisition of the capacity for movement during epididymal maturation in the rat and hamster. However, this nucleotide may be involved in the post-ejaculatory modifications of motility which accompany the terminal stages of capacitation.

Immunocontraceptive vaccines for human use

Publisher: Elsevier BV

Date: 10-2002

DOI: 10.1016/S0165-0378(02)00010-4

Abstract: The development of safe, effective, reversible contraceptive vaccines for the regulation of human fertility would be a significant addition to our contraceptive armamentarium. However, because we are such an out-bred species, immunological responsiveness to any given vaccine is certain to exhibit a high level of inter-in idual variation that will impact upon the efficacy, reversibility and feasibility of the approach. Nevertheless a role for vaccines undoubtedly exists as an aid to birth spacing, particularly in developing countries, and as a non-surgical means of inducing sterility in men. Currently vaccines are being researched that target one of two strategic points in the reproductive process, fertilization and the maternal recognition of pregnancy. Our ability to engineer vaccines that target fertilization is h ered by deficiencies in our knowledge of the molecular mechanisms that regulate this process. However, anti-hCG vaccines have advanced to the stage of clinical trials and appear promising.

Paternal impacts on development: identification of genomic regions vulnerable to oxidative DNA damage in human spermatozoa

Publisher: Oxford University Press (OUP)

Date: 10-2019

DOI: 10.1093/HUMREP/DEZ153

Abstract: Do all regions of the paternal genome within the gamete display equivalent vulnerability to oxidative DNA damage? Oxidative DNA damage is not randomly distributed in mature human spermatozoa but is instead targeted, with particular chromosomes being especially vulnerable to oxidative stress. Oxidative DNA damage is frequently encountered in the spermatozoa of male infertility patients. Such lesions can influence the incidence of de novo mutations in children, yet it remains to be established whether all regions of the sperm genome display equivalent susceptibility to attack by reactive oxygen species. Human spermatozoa obtained from normozoospermic males (n = 8) were split into equivalent s les and subjected to either hydrogen peroxide (H2O2) treatment or vehicle controls before extraction of oxidized DNA using a modified DNA immunoprecipitation (MoDIP) protocol. Specific regions of the genome susceptible to oxidative damage were identified by next-generation sequencing and validated in the spermatozoa of normozoospermic males (n = 18) and in patients undergoing infertility evaluation (n = 14). Human spermatozoa were obtained from normozoospermic males and ided into two identical s les prior to being incubated with either H2O2 (5 mm, 1 h) to elicit oxidative stress or an equal volume of vehicle (untreated controls). Alternatively, spermatozoa were obtained from fertility patients assessed as having high basal levels of oxidative stress within their spermatozoa. All semen s les were subjected to MoDIP to selectively isolate oxidized DNA, prior to sequencing of the resultant DNA fragments using a next-generation whole-genomic sequencing platform. Bioinformatic analysis was then employed to identify genomic regions vulnerable to oxidative damage, several of which were selected for real-time quantitative PCR (qPCR) validation. Approximately 9000 genomic regions, 150–1000 bp in size, were identified as highly vulnerable to oxidative damage in human spermatozoa. Specific chromosomes showed differential susceptibility to damage, with chromosome 15 being particularly sensitive to oxidative attack while the sex chromosomes were protected. Susceptible regions generally lay outside protamine- and histone-packaged domains. Furthermore, we confirmed that these susceptible genomic sites experienced a dramatic (2–15-fold) increase in their burden of oxidative DNA damage in patients undergoing infertility evaluation compared to normal healthy donors. The limited number of s les analysed in this study warrants external validation, as do the implications of our findings. Selection of male fertility patients was based on high basal levels of oxidative stress within their spermatozoa as opposed to specific sub-classes of male factor infertility. The identification of genomic regions susceptible to oxidation in the male germ line will be of value in focusing future analyses into the mutational load carried by children in response to paternal factors such as age, the treatment of male infertility using ART and paternal exposure to environmental toxicants. Project support was provided by the University of Newcastle’s (UoN) Priority Research Centre for Reproductive Science. M.J.X. was a recipient of a UoN International Postgraduate Research Scholarship. B.N. is the recipient of a National Health and Medical Research Council of Australia Senior Research Fellowship. Authors declare no conflict of interest.

Factors Influencing the Success of Sperm‐Cervical Mucus Interaction in Patients Exhibiting Unexplained Infertility

Publisher: Wiley

Date: 02-01-1986

DOI: 10.1002/J.1939-4640.1986.TB00857.X

Abstract: The factors regulating the success of sperm-cervical mucus interaction in 46 patients exhibiting unexplained infertility were analyzed. Within this group of patients, considerable variation in the degree of mucus penetration, which appeared to be related to the properties exhibited by the spermatozoa rather than the quality of the mucus itself, was observed. The ability of the spermatozoa to fuse with zona-free hamster oocytes reflected their capacity for mucus penetration, regardless of whether human or bovine cervical mucus was used as the target. Multiple regression analysis also indicated the importance of the movement characteristics exhibited by the spermatozoa, which alone could account for up to 85% of the variability in mucus penetration. The concentration of motile spermatozoa and their linear velocity of progression influenced the number of spermatozoa penetrating the mucus in unit time, presumably through an effect on the frequency of collisions at the cervical mucus interface. Whether these collisions resulted in mucus penetration depended upon the movement characteristics of the spermatozoa and was positively associated with a "rolling" mode of progression and the litude of lateral sperm head displacement.

Proteomic Analysis of Human Spermatozoa

Publisher: Springer International Publishing

Date: 03-11-2016

DOI: 10.1007/978-3-319-40788-3_1

Relative susceptibilities of mitochondrial and nuclear DNA to damage induced by hydrogen peroxide in two mouse germ cell lines

Publisher: Informa UK Limited

Date: 06-2001

DOI: 10.1179/135100001101536157

Abstract: The objective of this study was to determine the relative susceptibilities to the damaging effects of hydrogen peroxide of DNA in the mitochondrial and nuclear compartments of two murine germ cell lines. We used a quantitative polymerase chain reaction assay (QPCR) to measure gene- and mitochondrial-specific DNA damage and examined for the presence of alkali-labile sites using alkaline gel electrophoresis. No DNA damage was observed in a nuclear gene (beta-globin) in response to hydrogen peroxide treatment. In addition, no increase in alkali-labile sites was observed. However, mitochondrial DNA suffered extensive damage which increased in a dose-dependent manner. These results demonstrate that the nuclear DNA in these germ cell lines is relatively resistant to peroxide-mediated DNA damage, and that mitochondrial DNA is a sensitive biomarker for oxidative stress in these cells.

Direct methods for the detection of reactive oxygen species in human semen samples

Publisher: Humana Press

Date: 2012

DOI: 10.1007/978-1-61779-776-7_14

Sperm function tests and fertility

Publisher: Wiley

Date: 02-2006

DOI: 10.1111/J.1365-2605.2005.00630.X

Abstract: Traditionally, the diagnosis of male infertility has depended upon a descriptive evaluation of human semen with emphasis on the number of spermatozoa that are present in the ejaculate, their motility and their morphology. The fundamental tenet underlying this approach is that male fertility can be defined by reference to a threshold concentration of motile, morphologically normal spermatozoa that must be exceeded in order to achieve conception. Many independent studies have demonstrated that this fundamental concept is flawed and, in reality, it is not so much the absolute number of spermatozoa that determines fertility, but their functional competence. In the light of this conclusion, a range of in vitro tests have been developed to monitor various aspects of sperm function including their potential for movement, cervical mucus penetration, capacitation, zona recognition, the acrosome reaction and sperm-oocyte fusion. Such functional assays have been found to predict the fertilizing capacity of human spermatozoa in vitro and in vivo with some accuracy. Recent developments in this field include the introduction of tests to assess the degree to which human spermatozoa have suffered oxidative stress as well as the integrity of their nuclear and mitochondrial DNA. Such assessments not only yield information on the fertilizing capacity of human spermatozoa but also their ability to support normal embryonic development.

Studies on the development of diluents for the transportation and storage of human semen rat ambient temperature

Publisher: Oxford University Press (OUP)

Date: 10-1996

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A019074

Abstract: Storage of human semen s les at ambient temperature for 24 h resulted in a significant loss of sperm motility from a mean 45.1 +/- 1.8% to 13.8 +/- 1.1% (n = 148). This motility loss was associated with a significant increase in the osmolality of the seminal plasma and the induction of peroxidative damage to the spermatozoa. Both of these detrimental changes could be prevented by diluting the original semen s le 1:1 with a citrate-egg yolk, buffer (CYB). In the presence of this extender all aspects of semen quality were efficiently preserved for 24 h, including sperm movement, penetration of a cervical mucus substitute, the acrosome reaction and sperm-oocyte fusion. CYB extension also permitted the use of chemiluminescent tests of leukocyte contamination to be performed on semen s les stored for 24 h at ambient temperatures. As a preservation medium, CYB was found to be superior to alternative formulations lacking citrate and storage at ambient temperatures was preferable to 4 degrees C. Significant improvements in motility retention were also observed when CYB was supplemented with pentoxifylline, although this treatment significantly stimulated peroxidative damage in the spermatozoa. However, if the pentoxifylline was combined with antioxidants then this collateral peroxidative damage could be reduced and the performance of CYB significantly enhanced. These results have implications for the design of diluents permitting the long-term storage and transportation of human semen s les at ambient temperatures.

Relationship between the movement characteristics of human spermatozoa and their ability to penetrate cervical mucus and zona-free hamster oocytes

Publisher: Bioscientifica

Date: 03-1985

DOI: 10.1530/JRF.0.0730441

Abstract: In a group of normospermic donors exhibiting hamster oocyte penetration scores of 0-100%, multiple regression analysis indicated that only 20% of the variation in fertilizing potential could be explained by differences in the movement characteristics of the spermatozoa following incubation in vitro. When the movement characteristics of the spermatozoa in semen were considered this figure was reduced to 6.8% as a result of significant differences in the motility patterns exhibited by the seminal and post-incubation sperm populations. A much closer relationship was observed between the movement characteristics of human spermatozoa in semen and their ability to penetrate cervical mucus. When differences in motile sperm densities were taken into account, 76% of the variation in cervical mucus penetration could be accounted for by the existence of linear correlations with certain aspects of sperm movement (multiple R = 0.874). Of the various attributes of sperm motility measured (linear velocity of progression, frequency of rotation, litude of sperm head displacement, % rolling and % yawing), a failure to exhibit an adequate litude of lateral sperm head displacement was consistently found to be the most significant factor determining the success of sperm-cervical mucus interaction (R2 = 0.53).

Andrology: Seminal leukocytes: Passengers, terrorists or good samaritans?

Publisher: Oxford University Press (OUP)

Date: 07-1995

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A136165

Analysis of the global methylation status of human spermatozoa and its association with the tendency of these cells to enter apoptosis

Publisher: Hindawi Limited

Date: 05-11-2013

DOI: 10.1111/AND.12033

Abstract: The methylation status of human spermatozoa has been examined in relation to the isopycnic density of these cells and their tendency to spontaneously default to an apoptotic state. DNA methylation was evaluated using three independent procedures: high-pressure liquid chromatography, flow cytometry and immunocytochemistry. All three techniques revealed that poor-quality spermatozoa recovered from the low-density region of Percoll gradients were characterised by a global hypermethylation of their DNA. Hypermethylation was visualised with an anti-5-methylcytosine antibody as punctate areas of cross-reactivity randomly distributed throughout the chromatin. Immunocytochemical evidence was also obtained suggesting that the sperm mitochondrial genome exists in a heavily methylated state, as a possible buffer against unscheduled transcription. Defective human spermatozoa were also shown to exhibit a tendency to default to an apoptotic state characterised by an increase in annexin V binding. The measurement of annexin V binding levels in in idual sperm populations was found to be highly correlated with sperm vitality (P < 0.001) and the methylation status of their DNA (P < 0.001). We conclude that the generation of defective, apoptotic human spermatozoa is associated with disorders of spermatogenesis that lead to a global hypermethylation of their nuclear DNA.

Dual purpose contraceptives: Targeting fertility and sexually transmitted disease

Publisher: Elsevier BV

Date: 03-2011

DOI: 10.1016/J.JRI.2010.12.001

Abstract: There have been no radically new forms of contraception since the pill was introduced 1960 and even this form of fertility regulation can be traced back to endocrine advances that were made in the 1920s. Whatever new forms of fertility control we introduce for the future, they should exploit the significant advances that have been made in our understanding of the reproductive system in recent years and be tailored to the needs of the 21st century. In this context, there is an urgent need to develop novel, safe, effective, dual-purpose contraceptive agents that combine the prevention of pregnancy with protection against sexually transmitted diseases (STDs). To achieve this aim we have researched a class of a topical contraceptive agent that selectively and instantaneously immobilizes millions of spermatozoa, while suppressing the infectivity of pathogenic microbes, such as Chlamydia, in the ejaculate. This approach is based upon the ability of small molecular mass organic compounds to selectively and covalently adduct key proteins in spermatozoa and pathogenic organisms and disrupt their biological function. We have also successfully developed strategies for the preparation of latent formulations that would only become activated on contact with seminal plasma. The further development and refinement of these molecules should permit a radical rethink in the way that safe, effective topical protection is provided to control both fertility and the world-wide spread of STDs.

Enhanced detection of reactive oxygen species produced by human spermatozoa with 7‐dimethyl amino‐naphthalin–1, 2‐dicarbonic acid hydrazide

Publisher: Wiley

Date: 06-1992

DOI: 10.1111/J.1365-2605.1992.TB01341.X

Abstract: A new chemiluminescence technique has been assessed for the detection of reactive oxygen species generated by purified populations of human sperm. This revised protocol involves the use of horse-radish peroxidase (HRP) in combination with a luminol analogue, 7-dimethyl amino-naphthalin-1,2-dicarbonic acid hydrazide (DNDH), that exhibits two-three times the quantal efficiency of luminol itself. The chemiluminescent signal generated with these reagents was significantly (P less than 0.001) greater than that obtained with the conventional luminol-based methodology for both the steady-state situation and following stimulation of the sperm with PMA and A23187. Dose-response analyses indicated that the DNDH/HRP chemiluminescence system could give linear standard curves with hydrogen peroxide concentrations into the nmol l-1 range. In contrast, the exponential rise in chemiluminescence recorded with luminol was not observed until hydrogen peroxide concentrations exceeded 10 mumol l-1. It is concluded that the enhanced sensitivity of the DNDH/HRP system to low levels of hydrogen peroxide should facilitate the application of chemiluminescent techniques to the diagnosis of oxidative stress in cases of male infertility.

Analysis of the surface labelling characteristics of human spermatozoa and the interaction with anti-sperm antibodies

Publisher: Bioscientifica

Date: 07-1987

DOI: 10.1530/JRF.0.0800473

Abstract: Washed ejaculated human spermatozoa were surface labelled with 125I, using solid phase (iodogen) or enzymic (lactoperoxidase) methods, while membrane components possessing terminal galactose or galactosamine residues were labelled with the galactose oxidase-sodium [3H]borohydride technique. All three procedures revealed the presence of 2 major labelled surface components. The first comprised a broad band of radioactivity migrating just behind the ion front on SDS-PAGE, which could be extracted with chloroform and methanol, suggesting a lipid-like composition. The second fraction exhibited properties consistent with a major glycoprotein component of the human sperm plasma membrane, giving a peak of radioactivity with Mr = 20,000, within which a discrete doublet of bands (Mr = 17,000 and 19,000) could be resolved by autoradiography. A more detailed analysis of the labelled protein fraction after TCA precipitation revealed a number of other surface components, the major ones of which exhibited Mr values of 30,000, 45,000, 66,000, 115,000 and 160,000. Western blot analysis was then used to determine whether any of the surface components described above interacted with the gamma-globulin fraction of antisera obtained from patients exhibiting idiopathic autoimmunity against sperm antigens. Using a purified membrane preparation as the target, antibodies were detected against numerous high molecular weight bands with Mr values similar to the major components of the human sperm surface (35,000, 45,000, 66,000, 90,000 and 150,000). The nature of the antigens targeted by these antisera did not correlate with the ability of the latter to stimulate or suppress sperm-oocyte fusion.

The influence of actinomycin D on the protein composition of mouse uterine flushings

Publisher: Bioscientifica

Date: 05-1977

DOI: 10.1530/JRF.0.0500193

Vitamin A regulation of BMP4 expression in the male germ line

Publisher: Elsevier BV

Date: 10-2005

DOI: 10.1016/J.YDBIO.2005.07.009

Abstract: The molecular mechanisms leading to male infertility in vitamin A deficient (VAD) rodents have never been fully elucidated. Here, we report an interaction between BMP4 and retinoid signaling pathways in germ cells that may help clarify the biochemical basis of VAD. Adult germ cells, in particular spermatogonia, expressed BMP4 at both the mRNA and protein levels. BMP4 expression was significantly up-regulated in the testes of VAD mice and was down-regulated in freshly isolated germ cells and VAD testes by retinol, but not retinoic acid. The retinoid-responsive gene, RARbeta, was not induced in germ cells following retinoid treatment. Examination of BMP4 promoter usage in spermatogonia and the VAD testis revealed that germ cells utilize the recently characterized BMP4 intron 2 promoter, in addition to the classical 1A and 1B promoters. The observed decrease in BMP4 in response to retinol was mediated by the 1A and intron 2 promoters of the BMP4 gene. Our results reflect a direct requirement for retinoids by germ cells for the resumption of spermatogenesis in VAD animals via mechanisms that involve the suppression of BMP4 expression.

Oocyte recovery and fertilization rates in women at various times after the administration of hCG

Publisher: Bioscientifica

Date: 03-1986

DOI: 10.1530/JRF.0.0760771

Abstract: Volunteer women requesting laparoscopic sterilization were subjected to a fixed schedule of ovulation induction and oocyte recovery. Follicle aspiration was carried out in four groups: those to whom hCG was not administered and 12, 24 or 36 h respectively after the administration of hCG. For each group oocytes were cultured in vitro for 42 h, 30 h, 18 h and 6 h respectively, before insemination with donor spermatozoa. Oocyte recovery rates improved with longer hCG-to-recovery intervals (36% with no hCG to 81% 36 h after hCG). Although there was a slight reduction in fertilization rates when oocytes were not exposed to hCG in the follicle, normal cleavage was noted in more than 50% of oocytes in all four groups. It therefore appears that the final maturation stages of the human oocyte are not dependent on the midcycle gonadotrophin surge, provided the oocyte is matured in vitro before insemination. However, it was also evident that the fertilization rates were reduced when oocytes were removed from less mature follicles, as reflected by high androstenedione/oestradiol ratios.

The Hormonal Control of Implantation

Publisher: Wiley

Date: 2008

DOI: 10.1002/9780470720479.CH4

Abstract: The hormonal control of implantation in mammalian species with and without embryonic diapause is described. In a majority of species displaying the obligate form of diapause the corpora lutea appear to exhibit a low level of steroidogenic activity throughout diapause, full luteal activity being resumed just before the initiation of implantation. Fluctuations in the plasma levels of oestrogen and progesterone during diapause may serve to prime the uterus for implantation. In species exhibiting the facultative form of diapause, such as the rat and mouse, both progesterone and nidatory oestrogen are required for the induction of implantation. In species not displaying embryonic diapause implantation will take place in the presence of progesterone alone. In the light of these considerations the selection of animal models for drug-screening purposes and possible new approaches to contraception are discussed.

Mobile Phone Radiation Induces Reactive Oxygen Species Production and DNA Damage in Human Spermatozoa In Vitro

Publisher: Public Library of Science (PLoS)

Date: 31-07-2009

DOI: 10.1371/JOURNAL.PONE.0006446

On the future of the hamster oocyte penetration assay

Publisher: Elsevier BV

Date: 07-1994

DOI: 10.1016/S0015-0282(16)56809-5

Transmembrane migration technique: Reexamination of its usefulness in sperm motility assessments

Publisher: Elsevier BV

Date: 12-1989

DOI: 10.1016/0160-5402(89)90005-3

Abstract: A study was carried out using time-exposure photomicrography to investigate the potential usefulness of the transmembrane migration technique in the assessment of drug effects on human sperm motility. Significant but weak correlations were evident between the transmembrane migration ratio (TMMR%) and both % motility (r = 0.43 p less than 0.05) and the litude of lateral sperm head displacement (r = 0.40 p less than 0.05). However, no significant correlations were evident between TMMR% and the concentration of motile spermatozoa, mean path velocity, or frequency of sperm head rotation. Exposure to 2.5 mM 2-deoxyadenosine caused significant increases in % motility, mean path velocity, and frequency of sperm head rotation, with no concomitant rise in TMMR%, while 5.0 mM caffeine caused significant elevations in both TMMR% and in % motile cells. It is concluded that the transmembrane migration technique is a relatively insensitive method for detecting subtle but important changes in the movement characteristics of human spermatozoa.

Reply: M540 bodies interfere with TUNEL analyses in human semen samples

Publisher: Oxford University Press (OUP)

Date: 17-01-2011

DOI: 10.1093/HUMREP/DEQ371

Significance of intracellular calcium and cyclic adenosine 3',5'-monophosphate in the mechanisms by which prostaglandins influence human sperm function

Publisher: Bioscientifica

Date: 07-1986

DOI: 10.1530/JRF.0.0770451

Abstract: The selective ability of PGE-1 and PGE-2 to enhance the capacity of human spermatozoa for hamster oocyte penetration was dependent upon high concentrations (1.7 mM) of extracellular Ca2+ and a prolonged (4 h) duration of exposure, and insensitive to the Ca2+ channel antagonist, verapamil. Studies with the intracellular calcium indicator Quin-2 indicated that exposure to PGE-1 and PGE-2, but not PGF-2 alpha, induced a significant rise in the levels of cytoplasmic Ca2+, suggesting that an ionophore-like action might be responsible for the ability of the E-series prostaglandins to influence sperm function. Stimulation of oocyte penetration with PGE-1 and PGE-2 was significantly enhanced when these compounds were presented to the spermatozoa in medium of high osmolarity (410 mosmol). A combination of PGE-1 in hyperosmotic medium did not significantly influence sperm function in cases of oligozoospermia, although it was effective with patients exhibiting idiopathic infertility. Exposure to high doses of PGE-1 and PGE-2, but not 19-hydroxy PGE or PGF-2 alpha, also induced a significant rise in the cyclic AMP content of human spermatozoa. This effect did not appear to be involved in the enhancement of fertilization rates because it did not exhibit the same absolute dependence on high levels of extracellular Ca2+ as did the fertilization responses and the enhancement of oocyte penetration and the elevation of cAMP were independent of each other within the patient population.

Fertilization without sperm

Publisher: Springer Science and Business Media LLC

Date: 02-1996

DOI: 10.1038/379493A0

Identification of SRC as a key PKA-stimulated tyrosine kinase involved in the capacitation-associated hyperactivation of murine spermatozoa

Publisher: The Company of Biologists

Date: 08-2006

DOI: 10.1242/JCS.03055

Abstract: Fertilization of the mammalian oocyte depends on the ability of spermatozoa to undergo a process known as capacitation as they ascend the female reproductive tract. A fundamental feature of this process is a marked increase in tyrosine phosphorylation by an unusual protein kinase A (PKA)-mediated pathway. To date, the identity of the intermediate PKA-activated tyrosine kinase driving capacitation is still unresolved. In this study, we have identified SRC as a candidate intermediate kinase centrally involved in the control of sperm capacitation. Consistent with this conclusion, the SRC kinase inhibitor SU6656 was shown to suppress both tyrosine phosphorylation and hyperactivation in murine spermatozoa. Moreover, SRC co-immunoprecipitated with PKA and this interaction was found to lead to an activating phosphorylation of SRC at position Y416. We have also used difference-in-2D-gel-electrophoresis (DIGE) in combination with mass spectrometry to identify a number of SRC substrates that become phosphorylated during capacitation including enolase, HSP90 and tubulin. Our data further suggest that the activation of SRC during capacitation is negatively controlled by C-terminal SRC kinase. The latter was localized to the acrosome and flagellum of murine spermatozoa by immunocytochemistry, whereas capacitation was associated with an inactivating serine phosphosphorylation of this inhibitory kinase.

Reactive oxygen species generation by human spermatozoa: a continuing enigma

Publisher: Wiley

Date: 11-07-2002

DOI: 10.1046/J.1365-2605.2002.03521.X

Serum vitamin D content is associated with semen parameters and serum testosterone levels in men

Publisher: Medknow

Date: 2021

DOI: 10.4103/AJA.AJA_9_20

Genomic integrity in the male germ line: evidence in support of the disposable soma hypothesis

Publisher: Bioscientifica

Date: 09-2018

DOI: 10.1530/REP-18-0202

Abstract: The Big Blue λ Select-cII selection system has been employed along with whole-exome sequencing to examine the susceptibility of the male germ line to mutation in two challenging situations (i) exposure to a chemotherapeutic regime including bleomycin, etoposide and cis -platinum (BEP) and (ii) the ageing process. A 3-week exposure to BEP induced complete azoospermia associated with a loss of developing germ cells and extensive vacuolization of Sertoli cell cytoplasm. Following cessation of treatment, spermatozoa first appeared in the caput epididymis after 6 weeks and by 12 weeks motile spermatozoa could be recovered from the cauda, although the count ( P 0.001) and motility ( P 0.01) of these cells were significantly reduced and superoxide generation was significantly elevated ( P 0.001). Despite this increase in free radical generation, no evidence of chromatin instability was detected in these spermatozoa. Furthermore, embryos obtained from females mated at this 12-week time point showed no evidence of an increased mutational load. Similarly, progressive ageing of Big Blue mice had no impact on the quality of the spermatozoa, fertility or mutation frequency in the offspring despite a significant increase in the mutational load carried by somatic tissues such as the liver ( P 0.05). We conclude that the male germ line is highly resistant to mutation in keeping with the disposable soma hypothesis, which posits that genetic integrity in the germ cells will be maintained at the expense of the soma, in light of the former’s sentinel position in safeguarding the stability of the genome.

The Male Is Significantly Implicated as the Cause of Unexplained Infertility

Publisher: Georg Thieme Verlag KG

Date: 2020

DOI: 10.1055/S-0040-1718941

Abstract: Male infertility is recognized as a relatively common, complex condition, generated by a broad array of environmental and genetic factors. Historical reliance on the conventional semen profile has tended to underestimate the true contribution of “the male factor” to human infertility. This review highlights the importance of genetic and epigenetic factors in the etiology of male infertility, identifying a range of mutations responsible for primary testicular failure and impaired fertilizing potential. More than three quarters of all de novo mutations arise in the male germline via mechanisms that involve the inefficient or defective repair of DNA damage. Understanding the range of factors capable of creating genetic turmoil in the paternal germline is essential, if we are to gain a deep understanding of the causes of male infertility, rather than just the symptoms that characterize its presence. High levels of DNA fragmentation induced by oxidative stress are part of this equation. Oxidative stress is, in turn, driven by biological (age, ejaculation frequency, varicocele, infection), lifestyle (smoking, obesity), and environmental factors (heat, other forms of electromagnetic radiation, and toxins) that can impair the fertilizing potential of the spermatozoa and influence the incidence of spontaneous mutations that may cause infertility in the offspring.

Parabens generate reactive oxygen species in human spermatozoa

Publisher: Wiley

Date: 02-05-2018

DOI: 10.1111/ANDR.12499

Abstract: Parabens are used as antimicrobial preservative agent in many commercial products including cosmetics and pharmaceuticals. Weak oestrogenic and antiandrogenic activities have been attributed to parabens in in vitro and in vivo studies. In this study, human spermatozoa were exposed to different concentrations of an equimolar paraben mixture containing methyl, ethyl, propyl and butylparaben as well as to methylparaben alone at a concentration that is typical of commercially available vaginal lubricants. The induction of oxidative stress and DNA damage was then assessed at different time points. Our results demonstrate that the paraben mixture was capable of stimulating the generation of mitochondrial and cytosolic reactive oxygen species (ROS), inhibiting sperm motility and viability in a dose-dependent manner. The ability of in idual parabens to activate ROS generation and induce oxidative DNA damage was related to alkyl chain length. At the concentration used clinically, methylparaben inhibited sperm motility after both 2 and 5 h exposure (p < 0.05) and affected cell viability (p < 0.01) while augmenting ROS production and oxidative DNA damage. However, DNA fragmentation was not evident following methylparaben exposure. Based on these results, we conclude that, at the concentrations used in commercially available formulations, parabens may impair sperm motility, enhance the generation of mitochondrial ROS and stimulate the formation of oxidative DNA adducts. Taken together, these data underline the potential cytotoxic and genotoxic impact of such compounds in a clinical setting.

Epithelial cell coculture and the induction of sperm capacitation

Publisher: Elsevier BV

Date: 06-1994

DOI: 10.1016/S0015-0282(16)56764-8

Abstract: To investigate the influence of cultured human oviductal epithelial cells on the movement characteristics of human spermatozoa. Human spermatozoa were cultured with monolayers of human epithelial or Vero cells or conditioned media derived from these cell types. The viability and movement characteristics of the cells was subsequently analyzed at 4, 24, and 48 hours. University hospital and Medical Research Council laboratories. Volunteer donors. Movement characteristics of human spermatozoa. The presence of both Vero and oviductal epithelial cells, but not conditioned media, had a general promoting effect on sperm survival, significantly enhancing sperm viability and motility for up to 48 hours of culture. In addition, the presence of oviductal epithelial cells had a specific, significant stimulatory effect on sperm capacitation, enhancing the incidence of hyperactivated motility after 4, 24, and 48 hours of culture. Significantly, this effect was not observed with cocultures containing Vero cells. The coincubation of human spermatozoa with human oviductal epithelial cells provides a convenient system for the induction and analysis of sperm capacitation.

Reactive oxygen species as mediators of sperm capacitation and pathological damage

Publisher: Wiley

Date: 05-09-2017

DOI: 10.1002/MRD.22871

Abstract: Oxidative stress plays a major role in the life and death of mammalian spermatozoa. These gametes are professional generators of reactive oxygen species (ROS), which appear to derive from three potential sources: sperm mitochondria, cytosolic L-amino acid oxidases, and plasma membrane Nicotinamide adenine dinucleotide phosphate oxidases. The oxidative stress created via these sources appears to play a significant role in driving the physiological changes associated with sperm capacitation through the stimulation of a cyclic adenosine monophosphate/Protein kinase A phosphorylation cascade, including the activation of Extracellular signal regulated kinase-like proteins, massive up-regulation of tyrosine phosphorylation in the sperm tail, as well as the induction of sterol oxidation. When generated in excess, however, ROS can induce lipid peroxidation that, in turn, disrupts membrane characteristics that are critical for the maintenance of sperm function, including the capacity to fertilize an egg. Furthermore, the lipid aldehydes generated as a consequence of lipid peroxidation bind to proteins in the mitochondrial electron transport chain, triggering yet more ROS generation in a self-perpetuating cycle. The high levels of oxidative stress created as a result of this process ultimately damage the DNA in the sperm nucleus indeed, DNA damage in the male germ line appears to be predominantly induced oxidatively, reflecting the vulnerability of these cells to such stress. Extensive evaluation of antioxidants that protect the spermatozoa against oxidative stress while permitting the normal reduction-oxidation regulation of sperm capacitation is therefore currently being undertaken, and has already proven efficacious in animal models.

Pharmacological inhibition of arachidonate 15-lipoxygenase protects human spermatozoa against oxidative stress.

Publisher: Oxford University Press (OUP)

Date: 13-03-2018

DOI: 10.1093/BIOLRE/IOY058

Abstract: One of the leading causes of male infertility is defective sperm function, a pathology that commonly arises from oxidative stress in the germline. Lipid peroxidation events in the sperm plasma membrane result in the generation of cytotoxic aldehydes such as 4-hydroxynonenal (4HNE), which accentuate the production of reactive oxygen species (ROS) and cause cellular damage. One of the key enzymes involved in the metabolism of polyunsaturated fatty acids to 4HNE in somatic cells is arachidonate 15-lipoxygenase (ALOX15). Although ALOX15 has yet to be characterized in human spermatozoa, our previous studies have revealed a strong link between ALOX15 activity and the levels of oxidative stress and 4HNE in mouse germ cell models. In view of these data, we sought to assess the function of ALOX15 in mature human spermatozoa and determine whether the pharmacological inhibition of this enzyme could influence the level of oxidative stress experienced by these cells. By driving oxidative stress in vitro with exogenous H2O2, our data reveal that 6,11-dihydro[1]benzothiopyrano[4,3-b]indole (PD146176 a selective ALOX15 inhibitor) was able to significantly reduce several deleterious, oxidative insults in spermatozoa. Indeed, PD146176 attenuated the production of ROS, as well as membrane lipid peroxidation and 4HNE production in human spermatozoa. Accordingly, ALOX15 inhibition also protected the functional competence of these cells to acrosome react and bind homologous human zonae pellucidae. Together, these results implicate ALOX15 in the propagation of oxidative stress cascades within human spermatozoa and offer insight into potential therapeutic avenues to address male in fertility that arises from oxidative stress.

The role of molecular chaperones in mouse sperm-egg interactions

Publisher: Elsevier BV

Date: 08-2005

DOI: 10.1016/J.MCE.2005.06.004

Abstract: Fertilization is a unique and exquisitely choreographed cellular interaction between the male and female gamete that results in the creation of a genetically unique in idual. Despite the fundamental importance of fertilization, there remains a dearth of information about the basic biochemical mechanisms that underpin this process. One of the key issues that remain unresolved is the molecular basis of sperm-egg recognition. From the female perspective, it is well established that the sperm recognition sites reside in the zona pellucida (ZP), an acellular coat that surrounds the oocyte. In contrast, numerous studies into the cognate zona receptors residing on the sperm surface have failed to shed significant light on the biochemical identity of these molecules. Such difficulties may, in part, have arisen because investigations have traditionally been based on the precept that the zona receptor represents a single molecular entity that is constitutively expressed on the sperm surface. While such a view holds obvious appeal, it fails to account for growing evidence that gamete interaction is not mediated by a simple lock-and-key mechanism. In this review, we present a novel hypothesis in which the zona recognition site is portrayed as a multimeric molecular structure that is assembled into a functional complex during a maturation process known as 'capacitation'. Furthermore, we consider the possibility that this previously cryptic complex is assembled and delivered to the outer surface of the sperm plasma membrane through the concerted action of several members of the molecular chaperone family of proteins.

Involvement of homocysteine, homocysteine thiolactone, and paraoxonase type 1 ( PON ‐1) in the etiology of defective human sperm function

Publisher: Wiley

Date: 29-01-2016

DOI: 10.1111/ANDR.12157

Abstract: This study reports, for the first time, the significant (p ≤ 0.01) accumulation of homocysteine residues in low density, defective sperm suspensions isolated from patients attending an infertility clinic. This overabundance of homocysteine was not related to a deficiency in folate availability but may have been a reflection of the oxidative stress that characterizes such defective sperm populations. Direct addition of the homocysteine cyclic congener, homocysteine thiolactone, to human spermatozoa resulted in the rapid induction of mitochondrial reactive oxygen species (ROS) generation (p < 0.001), the stimulation of lipid peroxidation (p < 0.01), the promotion of tyrosine phosphorylation (p < 0.001), and the suppression of sperm motility (p < 0.001) in the absence of any significant impact on DNA integrity. The parent homocysteine molecule was less active and took 24 h to stimulate mitochondrial ROS production possibly because of the need to convert this compound to the corresponding thiolactone before it could exert a measureable biological effect. Thiolactone was also effective in suppressing the carboxymethylation of key proteins in the sperm tail, which are thought to be involved in the regulation of sperm movement. The major enzyme responsible for removing thiolactone from proteins, paraoxonase (PON-1), was shown to be a major target for alkylation by lipid aldehydes, such as 4-hydroxynonenal, generated as a consequence of oxidative stress. Exposure of human spermatozoa to such aldehydes resulted in a dose-dependent accumulation of homocysteine in spermatozoa (p < 0.03). These results suggest that one of the consequences of oxidative stress in mammalian spermatozoa is the inhibition of PON-1, which then enhances the availability of homocysteine thiolactone to interact with the epsilon-amino group of lysine residues on sperm proteins, triggering a raft of significant biological changes in these cells that ultimately compromise sperm function.

Identification of the insulin-like growth factor II receptor as a novel receptor for binding and invasion by Listeria monocytogenes.

Publisher: American Society for Microbiology

Date: 2006

DOI: 10.1128/IAI.74.1.566-577.2006

Abstract: The gram-positive bacterium Listeria monocytogenes causes a life-threatening disease known as listeriosis. The mechanism by which L. monocytogenes invades mammalian cells is not fully understood, but the processes involved may provide targets to prevent and treat listeriosis. Here, for the first time, we have identified the insulin-like growth factor II receptor (IGFIIR also known as the cation-independent mannose 6-phosphate receptor CI M6PR or CD222) as a novel receptor for binding and invasion of Listeria species. Random peptide phage display was employed to select a peptide sequence by panning with immobilized L. monocytogenes cells this peptide sequence corresponds to a sequence within the mannose 6-phosphate binding site of the IGFIIR. All Listeria spp. specifically bound the labeled peptide but not a control peptide, which was demonstrated using fluorescence spectrophotometry and fluorescence-activated cell sorting. Further evidence for binding of the receptor by L. monocytogenes and L. innocua was provided by affinity purification of the bovine IGFIIR from fetal calf serum by use of magnetic beads coated with cell preparations of Listeria spp. as affinity matrices. Adherence to and invasion of mammalian cells by L. monocytogenes was significantly inhibited by both the synthetic peptide and mannose 6-phosphate but not by appropriate controls. These observations indicate a role for the IGFIIR in the adherence and invasion of L. monocytogenes of mammalian cells, perhaps in combination with known mechanisms. Ligation of IGFIIR by L. monocytogenes may be a novel mechanism that contributes to the regulation of infectivity, possibly in combination with other mechanisms.

Role of the inner cell mass in controlling implantation in the mouse

Publisher: Bioscientifica

Date: 11-1976

DOI: 10.1530/JRF.0.0480403

Iatrogenic DNA damage induced in human spermatozoa during sperm preparation: Protective significance of seminal plasma

Publisher: Oxford University Press (OUP)

Date: 05-1998

DOI: 10.1093/MOLEHR/4.5.439

Abstract: Before the advent of intracytoplasmic sperm injection (ICSI) semen preparation techniques focused on the need to sustain the fertilizing potential of the spermatozoa particularly by reducing oxidative stress. However, for severely oligozoospermic patients treated by ICSI, sperm preparation protocols are used which aim to maximize sperm recovery rather than sperm function. In this study we have examined the impact of different sperm preparation techniques on oxidative stress, sperm motion and DNA integrity. Reactive oxygen species (ROS) generation was monitored using luminol-dependent chemiluminescence, seminal antioxidant activity was assessed using a total reactive antioxidant potential (TRAP) assay while sperm motility and DNA damage were evaluated using computer assisted semen analysis and in-situ nick translation respectively. The results demonstrate a significant increase in the levels of ROS generated by s les prepared by swim-up from a washed pellet compared with spermatozoa isolated directly from seminal plasma. This oxidative stress was associated with a highly significant increase in the level of DNA damage sustained by the spermatozoa while the quality of sperm motility remained largely unchanged. These results suggest that if repeated centifugation protocols are to be used to prepare spermatozoa, strategies should be developed for minimizing collateral DNA damage.

Assessment of sperm function for IVF

Publisher: Oxford University Press (OUP)

Date: 1988

DOI: 10.1093/OXFORDJOURNALS.HUMREP.A136660

Abstract: Recent advances in our ability to understand and manipulate the fundamental mechanisms regulating human sperm function have led to the development of a new generation of diagnostic techniques, designed to give objective data on the functional competence of human spermatozoa. These techniques have proved to be of value in predicting the ability of patients' spermatozoa to fertilize the human ovum in vitro and in the evaluation of techniques and reagents of relevance to the therapeutic application of IVF, including sperm preparation protocols and reagents for the artificial enhancement of sperm function. Moreover the use of sperm function tests has shed light on the biochemical nature of the lesions present in the spermatozoa of subfertile males, with particular emphasis on the role played by reactive oxygen species. As a result of such studies we are now in a position to adopt a more rational approach to the development of modified IVF protocols for the treatment of male infertility.

The Potential of the Zona Pellucida as a Target for Immunocontraception

Publisher: Wiley

Date: 03-1996

DOI: 10.1111/J.1600-0897.1996.TB00027.X

Abstract: To investigate the contraceptive potential of the zona pellucida. Generation of antibodies against native and recombinant zona glycoproteins which have then been assessed for their capacity to disrupt sperm-zona interaction in vivo and in vitro. The animal model selected for these studies was the common marmoset and the end points examined were antibody titre, ovarian cyclicity and fertility. The fact that antibodies against the major zona glycoprotein, ZP3, block both the primary and secondary phases of sperm-zona interaction suggests that this molecule might have potential for contraceptive vaccine development. Active immunization of marmoset monkeys with native porcine ZP3 or recombinant human ZP3 produced long term infertility but also precipitated a premature decline in the primordial follicle population. Future studies will have to determine whether a safe, effective vaccine can be engineered by coupling unique B-cell epitopes from ZP3 to foreign T-cell antigens.

Measurement of the sperm binding capacity of the mouse zona pellucida and its use in the estimation of anti-zona antibody titres

Publisher: Bioscientifica

Date: 11-1981

DOI: 10.1530/JRF.0.0630295

Abstract: Conditions have been defined under which the sperm binding capacity of the mouse zona pellucida can be measured. As a measure of the concentration of anti-zona antibodies the inhibition of sperm binding appears to be both repeatable and sensitive. Using this technique anti-zona antibody titres were monitored in 6 rats actively immunized with extracts of mouse ovarian tissue. These animals exhibited a rapid rise in anti-zona antibody titre following the induction of immunity, and an associated significant (P less than 0.05) decline in their fertility, both in relation to the proportions of animals exhibiting fertile matings and of matings resulting in conception. Three of the animals exhibited permanent sterility despite repeated exposure to fertile males.

Complementary effects of propranolol and nonoxynol-9 upon human sperm motility

Publisher: Elsevier BV

Date: 10-1995

DOI: 10.1016/0010-7824(95)00190-L

Abstract: The inhibitory effects of nonoxynol-9, DL- and D-propranolol upon human sperm motility were determined in vitro. All three compounds were capable of causing complete cessation of sperm movement. However, greater efficacy was achieved using combinations of nonoxynol-9 and propranolol, suggesting a complementary interaction between these compounds. Investigations of the mechanism of action of propranolol revealed that an influx of calcium accompanied the loss of motility. However, since incubation in the absence of calcium enhanced the spermicidal effects of this compound, it was concluded that this calcium influx did not constitute the primary means by which motility was disrupted. Low doses of propranolol, which did not affect motility, were found to inhibit the capacity of human spermatozoa for sperm-oocyte fusion.

Functional deletion of Txndc2 and Txndc3 increases the susceptibility of spermatozoa to age-related oxidative stress

Publisher: Elsevier BV

Date: 12-2013

DOI: 10.1016/J.FREERADBIOMED.2013.05.021

Abstract: Oxidative stress in the male germ line is known to be a key factor in both the etiology of male infertility and the high levels of DNA damage encountered in human spermatozoa. Because the latter has been associated with a variety of adverse clinical outcomes, including miscarriage and developmental abnormalities in the offspring, the mechanisms that spermatozoa use to defend themselves against oxidative stress are of great interest. In this context, the male germ line expresses three unique forms of thioredoxin, known as thioredoxin domain-containing proteins (Txndc2, Txndc3, and Txndc8). Two of these proteins, Txndc2 and Txndc3, retain association with the spermatozoa after spermiation and potentially play an important role in regulating the redox status of the mature gamete. To address this area, we have functionally deleted the sperm-specific thioredoxins from the male germ line of mice by either exon deletion (Txndc2) or mutation of the bioactive cysteines (Txndc3). The combined inactivation of these Txndc isoforms did not have an overall impact on spermatogenesis, epididymal sperm maturation, or fertility. However, Txndc deficiency in spermatozoa did lead to age-dependent changes in these cells as reflected by accelerated motility loss, high rates of DNA damage, increases in reactive oxygen species generation, enhanced formation of lipid aldehyde-protein adducts, and impaired protamination of the sperm chromatin. These results suggest that although there is considerable redundancy in the systems employed by spermatozoa to defend themselves against oxidative stress, the sperm-specific thioredoxins, Txndc2 and Txndc3, are critically important in protecting these cells against the increases in oxidative stress associated with paternal age.

Boronate probes for the detection of hydrogen peroxide release from human spermatozoa

Publisher: Elsevier BV

Date: 04-2015

DOI: 10.1016/J.FREERADBIOMED.2015.01.015

Abstract: Human spermatozoa are compromised by production of reactive oxygen species (ROS), and detection of ROS in spermatozoa is important for the diagnosis of male infertility. The probes 2',7'-dichlorohydrofluorescein diacetate (DCFH), dihydroethidium (DHE), and MitoSOX red (MSR) are commonly used for detecting ROS by flow cytometry however, these probes lack sensitivity to hydrogen peroxide (H2O2), which is particularly damaging to mammalian sperm cells. This study reports the synthesis and use of three aryl boronate probes, peroxyfluor-1 (PF1), carboxyperoxyfluor-1, and a novel probe, 2-(2-ethoxyethoxy)ethoxyperoxyfluor-1 (EEPF1), in human spermatozoa. PF1 and EEPF1 were effective at detecting H2O2 and peroxynitrite (ONOO(-)) produced by spermatozoa when stimulated with menadione or 4-hydroxynonenal. EEPF1 was more effective at detection of ROS in spermatozoa than DCFH, DHE, or MSR furthermore it distinguished poorly motile sperm as shown by greater ROS production. EEPF1 should therefore have a significant role in the diagnosis of oxidative stress in male infertility, cryopreservation, age, lifestyle, and exposure to environmental toxicants.

Reactive oxygen species generation by human spermatozoa – reply

Publisher: Wiley

Date: 17-03-2003

DOI: 10.1046/J.1365-2605.2003.00407.X

Comparative analysis of the ability of precursor germ cells and epididymal spermatozoa to generate reactive oxygen metabolites

Publisher: Wiley

Date: 04-1997

DOI: 10.1002/(SICI)1097-010X(19970401)277:5<390::AID-JEZ5>3.0.CO;2-K

Abstract: Male germ cells at various stages of differentiation from pachytene spermatocytes to mature caudal epididymal spermatozoa were examined for their ability to generate reactive oxygen species (ROS) using sensitive chemiluminescence techniques. In general, spermatozoa were found to spontaneously generate hydrogen peroxide as they progressed through the epididymis, maximal activity being observed on the release of mature cells from the caudal region into a modified Krebs-Ringer's solution. The spontaneous production of hydrogen peroxide rose rapidly during the first 10 min after the spermatozoa had been diluted into culture medium and thereafter stabilized, neither phorbol esters nor A23187 subsequently influencing this activity. Low levels of superoxide generation were also detected in suspensions of epididymal spermatozoa, but did not correlate with maturation status. However, superoxide production could be dramatically enhanced by the addition of exogenous NADPH, in a manner that was closely correlated with the stage of epididymal development being maximal for immature cells recovered from the caput epididymis in all species. Precursor germ cells (pachytene spermatocytes, round and elongate spermatids) similarly generated chemiluminescent signals compatible with the low level generation of ROS. Superoxide generation in these cells could again be stimulated by NADPH, via mechanisms that were inversely related to the stage of germ cell differentiation, the greatest activity being observed in pachytene spermatocytes. These results demonstrate that differentiating male germ cells have the potential to generate ROS, and have implications for the redox regulation of gonadal function and the development of reproductive pathologies involving oxidative stress.

Sperm–Zona Pellucida Interaction: Molecular Mechanisms and the Potential for Contraceptive Intervention

Publisher: Springer Berlin Heidelberg

Date: 2010

DOI: 10.1007/978-3-642-02062-9_9

Abstract: At the moment of insemination, millions of mammalian sperm cells are released into the female reproductive tract with the single goal of finding the oocyte. The spermatozoa subsequently ignore the thousands of cells they make contact with during their journey to the site of fertilization, until they reach the surface of the oocyte. At this point, they bind tenaciously to the acellular coat, known as the zona pellucida, which surrounds the oocyte and orchestrate a cascade of cellular interactions that culminate in fertilization. These exquisitely cell- and species- specific recognition events are among the most strategically important cellular interactions in biology. Understanding the cellular and molecular mechanisms that underpin them has implications for the etiology of human infertility and the development of novel targets for fertility regulation. Herein we describe our current understanding of the molecular basis of successful sperm-zona pellucida binding.

Cellular basis of defective sperm function and its association with the genesis of reactive oxygen species by human spermatozoa

Publisher: Bioscientifica

Date: 11-1987

DOI: 10.1530/JRF.0.0810459

Abstract: Addition of the alent cation ionophore, A23187, to washed populations of human spermatozoa resulted in a sudden burst of production of reactive oxygen species which peaked within 3-5 min. This activity was dependent upon the presence of calcium in the external medium and was unaffected by the mitochondrial inhibitors, oligomycin, antimycin and rotenone. Studies with scavengers of reactive oxygen species revealed that, while reagents directed against singlet oxygen and the hydroxyl radical were without effect, cytochrome C reduced the response to A23187 by about 50%, suggesting that the superoxide anion radical is a major product of the activated human spermatozoon. The clinical implications of these studies stem from the considerable variation observed between in iduals in the levels of reactive oxygen species produced by the spermatozoa. This variability was shown to be inversely related to the ability of the spermatozoa to exhibit sperm-oocyte fusion on exposure to A23187 defective s les exhibited a basal level of reactive oxygen species production which was 40 times that observed with normal functional cells.

What makes a fertile sperm? Unique molecular attributes of stallion fertility

Publisher: Bioscientifica

Date: 10-2019

DOI: 10.1530/REP-19-0060

Abstract: Stallions experience lower per-cycle conception rates compared to other livestock species, largely because they are selected for breeding based on athletic prowess and not reproductive fitness. Mares are seasonal breeders, and pregnancies cannot be detected until 10–14 days post cover via transrectal ultrasonography. This means the detection of stallion fertility fluctuations is delayed by at least 2 weeks, which within the short breeding season employed by the thoroughbred horse breeding industry, can prove quite costly. For these reasons, there is increased demand for robust laboratory assays aimed at the accurate assessment of stallion fertility. This paper reviews our existing knowledge concerning the molecular mechanisms that underpin the functional competence of stallion spermatozoa, highlighting the relative importance of oxidative stress, DNA damage, sperm proteomics and RNA profile. We also consider the way in which fundamental improvements in our understanding of stallion sperm biology are informing the identification and development of possible biomarkers of fertility and thus avenues for the development of specific assays for fertility prediction.

Diagnosis of Human Sperm Function

Publisher: Wiley

Date: 08-1986

DOI: 10.1111/J.1440-169X.1986.28S1_9.X

OXIDATIVE STRESS AND REPRODUCTIVE FUNCTION: Sperm telomeres, oxidative stress, and infertility

Publisher: Bioscientifica

Date: 12-2022

DOI: 10.1530/REP-22-0189

Abstract: Oxidative stress is recognized as an underlying driving factor of both telomere dysfunction and human subfertility/infertility. This review briefly reassesses telomere integrity as a fertility biomarker before proposing a novel, mechanistic rationale for the role of oxidative stress in the seemingly paradoxical lengthening of sperm telomeres with aging. The maintenance of redox balance in the male reproductive tract is critical to sperm health and function. Physiological levels of reactive oxygen species (ROS) promote sperm capacitation, while excess ROS exposure, or depleted antioxidant defenses, yields a state of oxidative stress which disrupts their fertilizing capacity and DNA structural integrity. The guanine moiety is the most readily oxidized of the four DNA bases and gets converted to the mutagenic lesion 8-hydroxy-deoxyguanosine (8-OHdG). Numerous studies have also confirmed oxidative stress as a driving factor behind accelerated telomere shortening and dysfunction. Although a clear consensus has not been reached, clinical studies also appear to associate telomere integrity with fertility outcomes in the assisted reproductive technology setting. Intriguingly, while sperm cellular and molecular characteristics make them more susceptible to oxidative insult than any other cell type, they are also the only cell type in which telomere lengthening accompanies aging. This article focuses on the oxidative stress response pathways to propose a mechanism for the explanation of this apparent paradox.

New Insights into Sperm Physiology and Pathology

Publisher: Springer Berlin Heidelberg

Date: 2010

DOI: 10.1007/978-3-642-02062-9_7

Abstract: Infertility is a relatively common condition affecting approximately one in ten of the population. In half of these cases, a male factor is involved, making defective sperm function the largest single, defined cause of human infertility. Among other factors, recent data suggest that oxidative stress plays a major role in the etiology of this condition. Spermatozoa spontaneously produce a variety of reactive oxygen species (ROS) including the superoxide anion, hydrogen peroxide and nitric oxide. Produced in small amounts, ROS are functionally important in driving the tyrosine phosphorylation cascades associated with sperm capacitation. However, when ROS production exceeds the spermatozoa's limited antioxidant defenses, a state of oxidative stress is induced characterized by peroxidative damage to the sperm plasma membrane and DNA strand breakage in the sperm nucleus. Such oxidative stress not only disrupts the fertilizing potential of human spermatozoa but also the ability of these cells to create a normal healthy embryo. As a result, DNA damage in human spermatozoa is correlated with an increased incidence of miscarriage and various kinds of morbidity in the offspring. These insights into the pathophysiology of defective sperm function have clear implications for the diagnosis and treatment of male infertility, particularly with respect to the potential importance of antioxidant therapy. These concepts may also be relevant to the design of novel approaches to male contraception that attempt to replicate the pathological situation.

Albumin gradients do not enrich Y-bearing human spermatozoa

Publisher: Oxford University Press (OUP)

Date: 05-1997

DOI: 10.1093/HUMREP/12.5.938

Abstract: The aim of this study was to evaluate objectively whether or not discontinuous albumin gradients enrich the proportion of Y-bearing human sperm. A blinded, collaborative trial design was employed whereby a licensed centre prepared the sperm fractions using licensed procedures, coded the sperm slides and then sent them to an independent laboratory for determination of the X:Y ratio in each sperm fraction using X and Y chromosome-specific probes and double label fluorescence in-situ hybridization (FISH). The identification codes and FISH results were collated by an independent third observer. Two albumin gradient methods which are currently used by licensed centres for male sex pre-selection, protocol 3 and modified protocol 3, were tested. Essentially the same results were obtained for the two methods. Highly motile sperm fractions were recovered from the albumin gradients, and the recoveries of motile spermatozoa (1.3-8.5%) were within the optimal range reported to produce maximal enrichment of Y-bearing spermatozoa. FISH analysis, however, revealed no enrichment for Y-bearing spermatozoa with either method, and the overall X:Y ratios were not significantly different from 1.0. Some s les showed marginal enrichment of Y-bearing spermaotozoa, whereas others showed marginal enrichment of X-bearing spermaotozoa. In conclusion, this collaborative study has demonstrated that the protocol 3 and modified protocol 3 albumin gradient procedures do not enrich Y-bearing spermatozoa. The clinical use of albumin gradients for male sex preselection should be reconsidered in the light of this and other evidence.

Free radicals, lipid peroxidation and sperm function

Publisher: CSIRO Publishing

Date: 1995

DOI: 10.1071/RD9950659

Abstract: The cellular generation of reactive oxygen species was first observed in mammalian spermatozoa in the late 1940s. The field then remained dormant for 30 years until Thaddeus Mann and Roy Jones published a series of landmark papers in the 1970s in which the importance of lipid peroxidation as a mechanism for damaging mammalian spermatozoa was first intimated. The subsequent demonstration that human spermatozoa produce reactive oxygen species and are susceptible to peroxidative damage has triggered intense interest in the role of oxidative stress in the aetiology of male infertility. Moreover, data have recently been obtained to indicate that, although excessive exposure to reactive oxygen species may be harmful to spermatozoa, in physiological amounts these molecules are of importance in the control of normal sperm function. This review considers the dualistic role of reactive oxygen species and sets out the current understanding of the importance of oxidative processes in both the physiology and the pathology of the human spermatozoon. Extra keywords: human spermatozoa, reactive oxygen species.

Oxidative stress and ageing of the post-ovulatory oocyte

Publisher: Bioscientifica

Date: 12-2013

DOI: 10.1530/REP-13-0111

Abstract: With extended periods of time following ovulation, the metaphase II stage oocyte experiences deterioration in quality referred to as post-ovulatory oocyte ageing. Post-ovulatory ageing occurs both in vivo and in vitro and has been associated with reduced fertilization rates, poor embryo quality, post-implantation errors and abnormalities in the offspring. Although the physiological consequences of post-ovulatory oocyte ageing have largely been established, the molecular mechanisms controlling this process are not well defined. This review analyses the relationships between biochemical changes exhibited by the ageing oocyte and the symptoms associated with the ageing phenotype. We also discuss molecular events that are potentially involved in orchestrating post-ovulatory ageing with a particular focus on the role of oxidative stress. We propose that oxidative stress may act as the initiator for a cascade of events that create the aged oocyte phenotype. Specifically, oxidative stress has the capacity to cause a decline in levels of critical cell cycle factors such as maturation-promoting factor, impair calcium homoeostasis, induce mitochondrial dysfunction and directly damage multiple intracellular components of the oocyte such as lipids, proteins and DNA. Finally, this review addresses current strategies for delaying post-ovulatory oocyte ageing with a particular focus on the potential use of compounds such as caffeine or selected antioxidants in the development of more refined media for the preservation of oocyte integrity during IVF procedures.

RAB-Like 2 Has an Essential Role in Male Fertility, Sperm Intra-Flagellar Transport, and Tail Assembly

Publisher: Public Library of Science (PLoS)

Date: 04-10-2012

DOI: 10.1371/JOURNAL.PGEN.1002969

Prostaglandins stimulate fertilizing capacity of human spermatozoa in vitro by a calcium dependent process

Publisher: Elsevier BV

Date: 1984

DOI: 10.1016/0090-6980(84)90359-9

Declining trends in conception rates in recent birth cohorts of native Danish women: a possible role of deteriorating male reproductive health

Publisher: Wiley

Date: 11-2007

DOI: 10.1111/J.1365-2605.2007.00827.X

The Paradoxical Relationship Between Stallion Fertility and Oxidative Stress1

Publisher: Oxford University Press (OUP)

Date: 09-2014

DOI: 10.1095/BIOLREPROD.114.118539

Abstract: The relationship between stallion fertility and oxidative stress remains poorly understood. The purpose of this study was to identify criteria for thoroughbred fertility assessment by performing a logistical regression analysis using "dismount" sperm parameters as predictors and weekly per-cycle conception rate as the dependent variable. Paradoxically, positive relationships between fertility and oxidative stress were revealed, such that s les that produced pregnancies exhibited higher rates of 8-hydroxy-2'-deoxyguanosine release (1490.2% vs. 705.5 pg/ml/24 h) and lower vitality (60.5% vs. 69.6%) and acrosome integrity (40.2% vs. 50.1%) than those that did not. We hypothesized that the most fertile spermatozoa exhibited the highest levels of oxidative phosphorylation (OXPHOS), with oxidative stress simply being a by-product of intense mitochondrial activity. Accordingly, an experiment to investigate the relationship between oxidative stress and motility was conducted and revealed positive correlations between mitochondrial ROS and total motility (R² = 0.90), rapid motility (R² = 0.89), average path velocity (VAP R² = 0.59), and curvilinear velocity (VCL R² = 0.66). Similarly, lipid peroxidation was positively correlated with total motility (R² = 0.46), rapid motility (R² = 0.51), average path velocity (R² = 0.62), and VCL (R² = 0.56), supporting the aforementioned hypothesis. The relative importance of OXPHOS in supporting the motility of equine spermatozoa was contrasted with human spermatozoa, which primarily utilize glycolysis. In this study, mitochondrial inhibition significantly reduced the velocity (P < 0.01) and ATP (P < 0.05) content of equine, but not human, spermatozoa, emphasizing the former's relative dependence on OXPHOS. The equine is the first mammal in which such a positive relationship between oxidative stress and functionality has been observed, with implications for the management of stallion fertility in vitro and in vivo.

Need for standardization and confirmation of STS deletions on the Y chromosome

Publisher: Elsevier BV

Date: 08-2008

DOI: 10.1016/J.FERTNSTERT.2008.05.026

The influence of anti-zona and anti-sperm antibodies on sperm-egg interactions

Publisher: Bioscientifica

Date: 07-1981

DOI: 10.1530/JRF.0.0620597

Abstract: Anti-zona antibodies are effective inhibitors of fertilization in vitro and, regardless of whether passive or active immunization techniques are used, in vivo. Antibodies raised against unfractionated zona pellucida antigens are chiefly directed against a group of carbohydrate-rich components localized on the outer surface of the zona. The interaction of anti-zona antibodies with these sites induces the formation of a surface precipitate which occludes the sperm binding sites by a process of steric hindrance, and stabilizes the zona structure against digestion by the proteolytic enzymes of the sperm head. Active immunization studies indicate that the long-term induction of infertility without adverse side effects is feasible in both laboratory rodents and primates when the zona pellucida is used as a target. Anti-sperm antibodies also exhibit a capacity for inhibiting fertilization in vivo and in vitro. To determine the most appropriate detection method to screen patients for anti-sperm antibodies several homologous and heterologous antisera were analysed by 5 different agglutination and immobilization techniques and then compared for their ability to inhibit the fertilizing capacity of human spermatozoa using the zona-free hamster egg penetration test. The results obtained with the Franklin--Dukes tube--slide test exhibited the closest correlation with the anti-fertility activity of a given antiserum this activity could be lified by the addition of complement to the medium. It is concluded that antibodies directed against the sperm head are responsible for limiting the fertilizing capacity of human spermatozoa in vitro and that it is these antibodies of which attention should be focused to unravel the role that immunological factors play in the aetiology of infertility in vivo.

Differential cell death decisions in the testis: evidence for an exclusive window of ferroptosis in round spermatids.

Publisher: Oxford University Press (OUP)

Date: 13-03-2019

DOI: 10.1093/MOLEHR/GAZ015

Abstract: Oxidative stress is a major aetiology in many pathologies, including that of male infertility. Recent evidence in somatic cells has linked oxidative stress to the induction of a novel cell death modality termed ferroptosis. However, the induction of this iron-regulated, caspase-independent cell death pathway has never been explored outside of the soma. Ferroptosis is initiated through the inactivation of the lipid repair enzyme glutathione peroxidase 4 (GPX4) and is exacerbated by the activity of arachidonate 15-lipoxygenase (ALOX15), a lipoxygenase enzyme that facilitates lipid degradation. Here, we demonstrate that male germ cells of the mouse exhibit hallmarks of ferroptosis including a caspase-independent decline in viability following exposure to oxidative stress conditions induced by the electrophile 4-hydroxynonenal or the ferroptosis activators (erastin and RSL3), as well as a reciprocal upregulation of ALOX15 and down regulation of GPX4 protein expression. Moreover, the round spermatid developmental stage may be sensitized to ferroptosis via the action of acyl-CoA synthetase long-chain family member 4 (ACSL4), which modifies membrane lipid composition in a manner favourable to lipid peroxidation. This work provides a clear impetus to explore the contribution of ferroptosis to the demise of germline cells during periods of acute stress in in vivo models.

Global Survey of Protein Expression during Gonadal Sex Determination in Mice

Publisher: Elsevier BV

Date: 12-2009

DOI: 10.1074/MCP.M900108-MCP200

A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation

Publisher: Wiley

Date: 22-04-2016

DOI: 10.1096/FJ.201500136R

Oxidative stress, placental ageing‐related pathologies and adverse pregnancy outcomes

Publisher: Wiley

Date: 27-02-2017

DOI: 10.1111/AJI.12653

Abstract: Oxidative stress (OS), an imbalance between free radical generation and antioxidant defence, is recognized as a key factor in the pathogenesis of adverse pregnancy outcomes. Although OS is a common future of normal pregnancy, persistent, overwhelming OS leads to consumption and decline of antioxidants, affecting placental antioxidant capacity and reducing systems. The accumulation of OS causes damage to lipids, proteins and DNA in the placental tissue that induces a form of accelerated ageing. Premature ageing of the placenta is associated with placental insufficiency that prevents the organ meeting the needs of the foetus, and as a consequence, the viability of the foetus is compromised. This review summarizes the literature regarding the role of OS and premature placental ageing in the pathophysiology of pregnancy complications.

Analysis of the Contraceptive Potential of Antibodies against Native and Deglycosylated Porcine ZP3 in Vivo and in Vitro1

Publisher: Oxford University Press (OUP)

Date: 04-1992

DOI: 10.1095/BIOLREPROD46.4.523

Abstract: We have undertaken a comparative analysis of the contraceptive activity of antibodies directed against the porcine sperm receptor zona pellucida antigen (ZP3) and its Mr = 32,000 polypeptide core (DGZP-32). The strategies employed for this analysis included the induction of active immunity in a primate, the common marmoset, and an in vitro fertilization protocol involving the use of viable human ova. In both experimental situations, antibodies against ZP3 were shown to exhibit contraceptive activity, leading respectively to the induction of long-term infertility in the primate model and to the complete inhibition of human fertilization in vitro. The in vivo studies also revealed that the induction of high titer antibodies against ZP3 was inevitably associated with the appearance of an ovarian pathology characterized by the progressive depletion of the primordial follicle pool within one to two years. This side effect could not be alleviated by the use of DGZP-32 as antigen since the induction of immunity against this polypeptide was also associated with the eventual appearance of an ovarian pathology identical to that observed with ZP3. Furthermore, the DGZP-32 peptide was less effective than ZP3 in inducing the formation of antibodies capable of inhibiting the fertilization of human ova in vitro. We conclude that significant problems remain with the use of deglycosylated zona peptides for the development of contraceptive vaccines and that their potential will not be realized until the epitopes responsible for the induction of infertility and the primordial follicle depletion have been identified and segregated.

Data on the concentrations of etoposide, PSC833, BAPTA-AM, and cycloheximide that do not compromise the vitality of mature mouse oocytes, parthenogenetically activated and fertilized embryos

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.DIB.2016.07.046

Round table discussion on Public health: contribution of family planning towards public health

Publisher: Elsevier BV

Date: 05-2006

DOI: 10.1016/J.MCE.2005.12.048

The rat sperm proteome characterized via IPG strip prefractionation and LC-MS/MS identification

Publisher: Wiley

Date: 06-2008

DOI: 10.1002/PMIC.200700876

Abstract: Proteomics represents a powerful tool for the analysis of mammalian spermatozoa, since these terminally differentiated cells are transcriptionally inactive and exhibit a limited dynamic range of protein expression. Here we report the identification of 5123 peptides, leading to 829 unambiguous and 2215 redundant gene products found to be present within rat spermatozoa derived from the cauda epididymis. Bioinformatics demonstrated that 60 proteins appeared to be specifically expressed in the genitourinary tract, including pyruvate dehydrogenase 1, ropporin, testis-specific serine kinase 4, testis-specific transporter, and retinol dehydrogenase 14. We also identified eight members of the ADAM family, seven of which have previously been detected in spermatozoa (ADAM2, -3, -4, -5, -6, -7, and -30) while ADAM34 has been identified in the sperm proteome for the first time. Approximately 21 gene products were found to possess isomerase activity including peptidylprolyl cis/trans isomerases that are known to be involved in germ cell differentiation and protein disulfide isomerases that have been implicated in sperm-oocyte fusion. Furthermore, 51 gene products clustered into ion-transporter activity. This inventory of gene products, the first ever 2-D LC-MS/MS analysis of rat spermatozoa, will be invaluable in directing future research into the molecular mechanisms that drive these highly specialized cells.

Unexplained antepartum stillbirth: A consequence of placental aging?

Publisher: Elsevier BV

Date: 04-2013