Sophia A. Tsipas

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

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.

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.

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.

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

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.

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.

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.

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.

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.

Functions and effects of reactive oxygen species in male fertility

Publisher: Elsevier BV

Date: 09-2020

DOI: 10.1016/J.ANIREPROSCI.2020.106456

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

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Isolation and characterization of phage AHP-1 and its combined effect with chloramphenicol to control Aeromonas hydrophila

Publisher: Springer Science and Business Media LLC

Date: 05-11-2019

DOI: 10.1007/S42770-019-00178-Z

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

Physiological and Pathological Aspects of Sperm Metabolism

Publisher: Cambridge University Press

Date: 25-05-2017

DOI: 10.1017/9781316411124.009

Proteomics of Human Spermatozoa

Publisher: Springer Berlin Heidelberg

Date: 2009

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

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.

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.

Sintering characteristics of plasma sprayed zirconia coatings containing different stabilisers

Publisher: Elsevier BV

Date: 2009

DOI: 10.1016/J.SURFCOAT.2008.09.037

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.

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.

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.

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

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.

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.

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 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.

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.

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

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 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.

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

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.

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.

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.

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.

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

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.

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.

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

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.

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.

Unexplained antepartum stillbirth: A consequence of placental aging?

Publisher: Elsevier BV

Date: 04-2013

DOI: 10.1016/J.PLACENTA.2013.01.015

Abstract: Unexplained antepartum stillbirth is a major obstetric health problem. Data demonstrate a rapid rise in risk per 1000 continuing pregnancies as gestation advances beyond 40 weeks. We review the evidence that such stillbirths are a consequence of aging related changes in the late gestation placenta. We suggest that the relatively small number of continuing pregnancies after 40 completed weeks means that negative effects of genes that produce aging affect so few pregnancies that polymorphisms in genes that produce these effects are retained in the population. Aging related changes likely represent a consequence of the damaging effects of oxidative stress, increased by cigarette smoking counteracted by the mitigating effects of oxidative defence pathways. The aging related changes are likely downstream from nutrient sensing units such as mTOR and include effects on production of telomerase and consequent shortening of telomere length. The late gestation changes occur in the context of increasing fetal growth and nutrient supply demands that can produce the rapid development of a mismatch between placental supply and fetal need resulting in fetal demise. Premature aging may also play an important role in antepartum stillbirth occurring earlier in pregnancy, especially in the context of growth restriction.

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.

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.

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

Analysis of the effects of polyphenols on human spermatozoa reveals unexpected impacts on mitochondrial membrane potential, oxidative stress and DNA integrity; implications for assisted reproductive t

Publisher: Elsevier BV

Date: 12-2016

DOI: 10.1016/J.BCP.2016.09.015

Abstract: The need to protect human spermatozoa from oxidative stress during assisted reproductive technology, has prompted a detailed analysis of the impacts of phenolic compounds on the functional integrity of these cells. Investigation of 16 in idual compounds revealed a surprising variety of negative effects including: (i) a loss of mitochondrial membrane potential (Δψm) via mechanisms that were not related to opening of the permeability transition pore but associated with a reduction in thiol expression, (ii) a decline in intracellular reduced glutathione, (iii) the stimulation of pro-oxidant activity including the induction of ROS generation from mitochondrial and non-mitochondrial sources, (iv) stimulation of lipid peroxidation, (v) the generation of oxidative DNA damage, and (vi) impaired sperm motility. For most of the polyphenolic compounds examined, the loss of motility was gradual and highly correlated with the induction of lipid peroxidation (r=0.889). The exception was gossypol, which induced a rapid loss of motility due to its inherent alkylating activity one consequence of which was a marked reduction in carboxymethyl lysine expression on the sperm tail a post-translational modification that is known to play a key role in the regulation of sperm movement. The only polyphenols that did not appear to have adverse effects on spermatozoa were resveratrol, genistein and THP at doses below 100μM. These compounds could, therefore, have some therapeutic potential in a clinical setting.

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

Aldehyde Dehydrogenase Plays a Pivotal Role in the Maintenance of Stallion Sperm Motility1

Publisher: Oxford University Press (OUP)

Date: 06-2016

DOI: 10.1095/BIOLREPROD.116.140509

Abstract: Although stallion spermatozoa produce significant quantities of reactive oxygen species, a lag between 4-hydroxynonenal (4HNE) adduction and the loss of motility in stallion spermatozoa suggests the presence of a robust aldehyde detoxification mechanism. Because there is a paucity of studies characterizing the role of aldehyde dehydrogenase (ALDH) in sperm functionality, the aim of this study was to ascertain the relationship between 4HNE production and motility and ALDH expression by stallion spermatozoa. PCR analysis revealed the presence of the ALDH1A3, ALDH1B1, and ALDH2 isoforms in these cells. Strong correlations (P < 0.001) were found between ALDH expression and various motility parameters of stallion spermatozoa including the percentage of progressive (r = 0.79) and rapidly motile (r = 0.79) spermatozoa, whereas repeated measurements over 24 h revealed highly significant correlations among progressive motility loss, 4HNE accumulation, and ALDH expression (P ≤ 0.001). ALDH inhibition resulted in a spontaneous increase in 4HNE levels in viable cells (21.1 ± 5.8% vs. 42.6 ± 5.2% P ≤ 0.05) and a corresponding decrease in total motility (41.7 ± 6.2% vs. 6.4 ± 2.6% P ≤ 0.001) and progressive motility (17.0 ± 4.1% vs. 0.7 ± 0.4% P ≤ 0.001) of stallion spermatozoa over 24 h. Similarly, inhibition of ALDH in 4HNE-challenged spermatozoa significantly reduced total motility over 4 h (35.4 ± 9.7% vs. 15.3 ± 5.1%, respectively P ≤ 0.05). This study contributes valuable information about the role of the ALDH enzymes in the maintenance of stallion sperm functionality, with potential diagnostic and in vitro applications for assisted reproductive technologies.

The 12 ^th International Symposium on Spermatology

Publisher: Medknow

Date: 2015

DOI: 10.4103/1008-682X.153852

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

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.

Proteomic insights into the maturation and capacitation of mammalian spermatozoa

Publisher: Informa UK Limited

Date: 12-07-2012

DOI: 10.3109/19396368.2011.639844

Abstract: Spermatozoa represent the epitome of terminally differentiated, highly specialized cells. They are transcriptionally and translationally silent and yet manage to undergo a complete functional transformation after they leave the testes, entirely fuelled by post-translational modifications occurring during epididymal maturation and capacitation. The latter have been recognized as biological processes for more than half a century. However, the biochemical mechanisms that drive these events have remained elusive, as have the pathological mechanisms that lead to defective sperm function and infertility. In the past decade the combined power of advanced proteomics, biochemistry, and functional genomics has permitted an unprecedented improvement in our understanding of sperm cell biology. We can also predict that a systems-biology approach, in concert with the new tools provided by the 'omics' revolution, will lead to dramatic gains in our understanding in the near future. As a result of such advances, insights will be generated that should ultimately lead to significant improvements in our capacity to diagnose and treat the infertile male.

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.

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.

The presence of a truncated base excision repair pathway in human spermatozoa, Mediated by OGG1

Publisher: The Company of Biologists

Date: 2013

DOI: 10.1242/JCS.121657

Abstract: DNA repair has long been considered impossible in human spermatozoa due to the high level of DNA compaction observed in these cells. However, detailed examination of the base excision repair pathway in human spermatozoa has revealed the presence of an enzyme critical to this pathway, OGG1. This glycosylase was associated with the sperm nucleus and mitochondria and could actively excise 8-hydrdoxy, 2′-deoxyguanosine, releasing this adduct into the extracellular space. This activity was significantly reduced in the presence of cadmium (II), a recognized inhibitor of OGG1, in a time- and dose- dependent manner (P& .001). Remarkably, spermatozoa do not possess the downstream components of the base excision repair pathway, APE1 and XRCC1. The absence of these proteins was particularly significant, as APE1 is required to create a 3′-hydroxyl (3′-OH) terminus at the apurinic site created by OGG1, which would be recognized by the TUNEL assay. As a result, TUNEL was unable to detect oxidatively induced DNA damage in spermatozoa following exposure to hydrogen peroxide. In the same cells, intracellular and extracellular 8OHdG could be clearly detected in a manner that was highly correlated with the outcome of SCSA (Sperm Chromatin Structure Assay). However, incubation of these cells for 48 hours revealed a time-dependent increase in TUNEL positivity, suggesting the perimortem activation of a nuclease. These results emphasize the limited capacity of mature spermatozoa to mount a DNA repair response to oxidative stress, and highlight the importance of such mechanisms in the oocyte in order to protect the embryo from paternally mediated genetic damage.

Characterization of an L-Amino Acid Oxidase in Equine Spermatozoa1

Publisher: Oxford University Press (OUP)

Date: 05-2015

DOI: 10.1095/BIOLREPROD.114.126052

Abstract: This study demonstrates for the first time the presence of an L-amino acid oxidase (LAAO) enzyme in equine spermatozoa that is able to generate significant amounts of reactive oxygen species (ROS) and create a state of oxidative stress. RT-PCR analysis revealed that the mRNA for this enzyme was present in the equine testis and spermatozoa, while immunocytochemical studies demonstrated that the mature LAAO protein was located in the sperm head, particularly in the acrosomal and postacrosomal domains. Experimental studies demonstrated that the aromatic amino acids (L-phenylalanine > L-tryptophan > L-tyrosine) were substrates for this enzyme, eliciting the dose- and time-dependent generation of ROS via mechanisms that were enhanced by cell death. This unexpected result was confirmed by analyses of ROS generation in subcellular sperm fractions, which again located a majority of LAAO activity to the sperm head. Equine cryopreservation medium was shown to contain sufficient quantities of aromatic amino acids to activate the LAAO system and generate ROS. The biological significance of this activity was established in an experiment in which physiological concentrations of aromatic amino acids were found to suppress sperm motility but only if dead spermatozoa were present in the same suspension. The combination of aromatic amino acids and nonviable cells was also found to enhance the levels of lipid peroxidation in live spermatozoa. These results suggest the potential significance of LAAO activity in generating the oxidative stress associated with the cryopreservation of equine spermatozoa. It is possible that inhibitors of this enzyme system may facilitate the development of modified cryostorage regimes for clinical validation in vivo.

The identification of mouse sperm-surface-associated proteins and characterization of their ability to act as decapacitation factors

Publisher: Oxford University Press (OUP)

Date: 02-2006

DOI: 10.1095/BIOLREPROD.105.044644

Abstract: Mammalian spermatozoa must undergo capacitation before acquiring the ability to fertilize the oocyte. This process is believed to be initiated following the release of surface-associated decapacitation factors that are elaborated by both the epididymis and the male accessory organs. Herein, we report the identification of a number of proteins that are actively released from the surface of mouse spermatozoa during capacitation in vitro. As anticipated, the addition of these factors back to suspensions of mouse spermatozoa was shown to suppress several correlates of the capacitation process. Specifically, they induced a significant, dose-dependent inhibition of the ability of spermatozoa to undergo a progesterone-induced acrosome reaction and to bind to the zona pellucida in vitro. Inhibition of these functions was associated with the suppression of tyrosine phosphorylation in the sperm plasma membrane but had no effect on the phosphorylation of internal proteins in either the sperm head or tail. This inhibitory activity was attributed to a subset of the isolated proteins compromising at least four putative decapacitation factors. These proteins were identified via tandem-mass spectrometry amino acid sequence analysis as plasma membrane fatty acid binding protein, cysteine-rich secretory protein 1 (CRISP1), phosphatidylethanolamine binding protein 1 (PBP), and an unnamed protein product that we have termed decapacitation factor 10 (DF10). Of these proteins, PBP was identified as a primary candidate for a decapacitation factor.

Recent Developments in Stallion Semen Preservation

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1016/J.JEVS.2016.06.006

Double Strand Break DNA Repair occurs via Non-Homologous End-Joining in Mouse MII Oocytes.

Publisher: Springer Science and Business Media LLC

Date: 26-06-2018

DOI: 10.1038/S41598-018-27892-2

Abstract: The unique biology of the oocyte means that accepted paradigms for DNA repair and protection are not of direct relevance to the female gamete. Instead, preservation of the integrity of the maternal genome depends on endogenous protein stores and/or mRNA transcripts accumulated during oogenesis. The aim of this study was to determine whether mature (MII) oocytes have the capacity to detect DNA damage and subsequently mount effective repair. For this purpose, DNA double strand breaks (DSB) were elicited using the topoisomerase II inhibitor, etoposide (ETP). ETP challenge led to a rapid and significant increase in DSB (P = 0.0002) and the consequential incidence of metaphase plate abnormalities (P = 0.0031). Despite this, ETP-treated MII oocytes retained their ability to participate in in vitro fertilisation, though displayed reduced developmental competence beyond the 2-cell stage (P = 0.02). To account for these findings, we analysed the efficacy of DSB resolution, revealing a significant reduction in DSB lesions 4 h post-ETP treatment. Notably, this response was completely abrogated by pharmacological inhibition of key elements (DNA-PKcs and DNA ligase IV) of the canonical non-homologous end joining DNA repair pathway, thus providing the first evidence implicating this reparative cascade in the protection of the maternal genome.

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

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.

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

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.

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.

Significance of Mitochondrial Reactive Oxygen Species in the Generation of Oxidative Stress in Spermatozoa

Publisher: The Endocrine Society

Date: 08-2008

DOI: 10.1210/JC.2007-2616

Abstract: Context: Male infertility has been linked with the excessive generation of reactive oxygen species (ROS) by defective spermatozoa. However, the subcellular origins of this activity are unclear. Objective: The objective of this study was to determine the importance of sperm mitochondria in creating the oxidative stress associated with defective sperm function. Method: Intracellular measurement of mitochondrial ROS generation and lipid peroxidation was performed using the fluorescent probes MitoSOX red and BODIPY C11 in conjunction with flow cytometry. Effects on sperm movement were measured by computer-assisted sperm analysis. Results: Disruption of mitochondrial electron transport flow in human spermatozoa resulted in generation of ROS from complex I (rotenone sensitive) or III (myxothiazol, antimycin A sensitive) via mechanisms that were independent of mitochondrial membrane potential. Activation of ROS generation at complex III led to the rapid release of hydrogen peroxide into the extracellular space, but no detectable peroxidative damage. Conversely, the induction of ROS on the matrix side of the inner mitochondrial membrane at complex I resulted in peroxidative damage to the midpiece and a loss of sperm movement that could be prevented by the concomitant presence of α-tocopherol. Defective human spermatozoa spontaneously generated mitochondrial ROS in a manner that was negatively correlated with motility. Simultaneous measurement of general cellular ROS generation with dihydroethidium indicated that 68% of the variability in such measurements could be explained by differences in mitochondrial ROS production. Conclusion: We conclude that the sperm mitochondria make a significant contribution to the oxidative stress experienced by defective human spermatozoa.

Oxidative damage in naturally aged mouse oocytes is exacerbated by dysregulation of proteasomal activity.

Publisher: Elsevier BV

Date: 12-2018

DOI: 10.1074/JBC.RA118.005751

New insights into the molecular mechanisms of sperm-egg interaction

Publisher: Springer Science and Business Media LLC

Date: 20-04-2007

DOI: 10.1007/S00018-007-6552-X

Abstract: At the moment of insemination millions of mammalian sperm cells are released into the female reproductive tract in order to find a single cell - the oocyte. The spermatozoa subsequently ignore the thousands of cells they make contact with during their journey to the site of fertilisation, until they reach the surface of the oocyte. At this point, they bind tenaciously to the acellular coat, known as the zona pellucida, that surrounds the oocyte and initiate the chain of cellular interactions that will 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 diagnosis of the aetiology of human infertility and the development of novel targets for fertility regulation. Herein, we describe two models indicating the plethora of highly orchestrated molecular interactions underlying successful sperm zona binding and sperm oocyte fusion.

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.

Elucidation of the signaling pathways that underpin capacitation-associated surface phosphotyrosine expression in mouse spermatozoa

Publisher: Wiley

Date: 2010

DOI: 10.1002/JCP.22090

Abstract: Recent studies from within 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. In the studies described herein we have sought to investigate the signaling pathways that underpin the tyrosine phosphorylation of sperm surface protein targets and validate the physiological significance of these pathways in relation to sperm-zona pellucida adhesion. Through selective pharmacological inhibition we have demonstrated that surface phosphotyrosine expression is unlikely to be mediated by the canonical cAMP-dependent protein kinase A (PKA) signaling cascade that has been most widely studied in relation to sperm capacitation. Rather, it appears to be primarily driven by the extracellular signal-regulated kinase (ERK) module of the mitogen-activated protein kinase (MAPK) pathway. Consistent with this notion, the main components of the ERK module (RAS, RAF1, MEK, and ERK1/2) were localized to the periacrosomal region of the head of mature mouse spermatozoa and their phosphorylation status within this region of the cell was positively modulated by capacitation. Furthermore, inhibition of several elements of this pathway suppressed sperm surface phosphotyrosine expression and induced a concomitant reduction sperm-zona pellucida interaction. Collectively, these data highlight a previously unappreciated role of the ERK module in the modification of the sperm surface during capacitation to render these cells functionally competent to engage in the process of fertilization.

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.

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.

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.

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.

The role of dopamine in the pathophysiology and treatment of apathy

Publisher: Elsevier

Date: 2016

DOI: 10.1016/BS.PBR.2016.05.007

Abstract: Disorders of diminished motivation, such as apathy, are common and prevalent across a wide range of medical conditions, including Parkinson's disease, Alzheimer's dementia, stroke, depression, and schizophrenia. Such disorders have a significant impact on morbidity and quality of life, yet their management lacks consensus and remains unsatisfactory. Here, we review laboratory and clinical evidence for the use of dopaminergic therapies in the treatment of apathy. Dopamine is a key neurotransmitter that regulates motivated decision making in humans and other species. A large corpus of evidence suggests that it plays an important role in promoting approach behavior by attributing incentive salience to reward stimuli, and facilitating the overcoming of effort costs. Furthermore, dopaminergic neurons innervate several frontostriatal structures that mediate reward-guided behavior. Based on these findings, there are a priori reasons for considering dopamine in the treatment of disorders of diminished motivation. We highlight key studies that have attempted to use dopamine to manage patients with apathy, and that collectively offer cautious evidence in favor of its efficacy. However, many of these studies are small, unblinded, and uncontrolled, and utilize subjective, questionnaire-based measures of apathy. Given the development of novel paradigms which are able to objectively dissect motivational dysfunction, we are now well positioned to quantify the effect of specific classes of dopaminergic medication on reward- and effort-based decision making in apathy. We anticipate that such paradigms will lay the foundation for future studies to evaluate new and existing treatments for disorders of motivation, using sensitive measures of apathy as primary quantifiable end points.

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.

Predicting the Outcome of Equine Artificial Inseminations Using Chilled Semen

Publisher: MDPI AG

Date: 30-03-2023

DOI: 10.3390/ANI13071203

Abstract: This study aimed to determine whether an analysis of stallion ejaculate could accurately predict the likelihood of pregnancy resulting from artificial insemination in mares. This study involved 46 inseminations of 41 mares, using 7 standardbred stallions over a 5-week period at an Australian pacing stud. Semen quality was assessed immediately after collection and again after chilling at ~5 °C for 24 h. The assessment involved evaluating ejaculate volume, sperm concentration, and motility parameters using an iSperm® Equine portable device. After the initial evaluation, a subpopulation of cells was subjected to a migration assay through a 5 µm polycarbonate filter within a Samson™ isolation chamber over a 15 min period. The cells were assessed for their concentration, motility parameters, and ability to reduce the membrane impermeant tetrazolium salt WST-1. The data, combined with the stallion and mare’s ages, were used to predict the likelihood of pregnancy, as confirmed by rectal ultrasound sonography performed 14 days post ovulation. The criteria used to predict pregnancy were optimized for each in idual stallion, resulting in an overall accuracy of 87.9% if analyzed pre-chilling and 95% if analyzed post-chilling. This study suggests that an analysis of stallion ejaculate can be used to predict the likelihood of pregnancy resulting from artificial insemination in mares with a high level of accuracy.

Investigation into the presence and functional significance of proinsulin C-peptide in the female germline.

Publisher: Oxford University Press (OUP)

Date: 30-01-2019

DOI: 10.1093/BIOLRE/IOZ008

Abstract: Diabetes is associated with poor oocyte quality and the dysregulation of ovarian function and is thus a leading contributor to the increasing prevalence of female reproductive pathologies. Accordingly, it is well-established that insulin fulfills a key role in the regulation of several facets of female reproduction. What remains less certain is whether proinsulin C-peptide, which has recently been implicated in cellular signaling cascades, holds a functional role in the female germline. In the present study, we examined the expression of insulin, C-peptide, and its purported receptor GPR146, within the mouse ovary and oocyte. Our data establish the presence of abundant C-peptide within follicular fluid and raise the prospect that this bioactive peptide is internalized by oocytes in a G-protein coupled receptor-dependent manner. Further, our data reveal that internalized C-peptide undergoes pronounced subcellular relocalization from the ooplasm to the pronuclei postfertilization. The application of immunoprecipitation analysis and mass spectrometry identified breast cancer type 2 susceptibility protein (BRCA2), the meiotic resumption/DNA repair protein, as a primary binding partner for C-peptide within the oocyte. Collectively, these findings establish a novel accumulation profile for C-peptide in the female germline and provide the first evidence for an interaction between C-peptide and BRCA2. This interaction is particularly intriguing when considering the propensity for oocytes from diabetic women to experience aberrant meiotic resumption and perturbation of traditional DNA repair processes. This therefore provides a clear imperative for further investigation of the implications of dysregulated C-peptide production in these in iduals.

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

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.

Predicting 1 Repetition Maximum Using Handheld Dynamometry

Publisher: Wiley

Date: 06-03-2018

DOI: 10.1016/J.PMRJ.2018.02.017

Abstract: Isometric assessment of muscular function using a handheld dynamometer (HHD) is frequently used in clinic environments. However, there is controversy in terms of the validity of isometric assessment to monitor changes in dynamic performance. One repetition maximum (1RM) is considered the gold standard for evaluating dynamic strength, though clinicians do not often use 1RM testing, preferring to be cautious with clients who have preexisting impairments. If strength testing using an HHD could be used to predict 1RM, this may have significant implications for the use of isometric testing to prescribe exercise in clinical environments. To establish the relationship and agreement between 1RM and isometric strength scores measured using HHD for the biceps and quadriceps muscle groups and to determine if HHD measurements can be used to predict 1RM. Criterion standard comparison. Tertiary institution gymnasium. Convenience s le of 50 healthy adults (26 women) aged 19-33 years (mean 23.38 ± 3.11 years). Muscle strength of the biceps and quadriceps muscle groups measured by 1RM and isometric maximal voluntary contraction measured using an HHD. Statistical analysis of the relation between the measures of strength was established using Pearson correlation and a Bland-Altman plot. A linear regression analysis with included covariates (gender, age, resistance training history, and body mass index) was used to derive the prediction equations. A significant correlation was found between 1RM and HHD scores for the biceps (r = .83, P < .001) and quadriceps muscle groups (r = .82, P < .001). However, strength scores were not in agreement. Linear regression analysis found significance in predicting 1RM from all HHD scores (P < .001). Gender as a covariate significantly influenced the prediction of 1RM for the biceps (P = .005) and quadriceps (P = .003) muscle groups. There is a significant relationship between 1RM and HHD measures of strength, and measures taken using an HHD can be used to predict 1RM in the biceps and quadriceps muscle groups. The use of an HHD may therefore provide a more accessible alternative to 1RM for muscle strength assessments. Further research is warranted to determine if results are applicable in clinical populations. NA.

Proteomic Analysis of Human Spermatozoa

Publisher: Springer International Publishing

Date: 03-11-2016

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

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.

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.

CXCR4/SDF1 interaction inhibits the primordial to primary follicle transition in the neonatal mouse ovary

Publisher: Elsevier BV

Date: 05-2006

DOI: 10.1016/J.YDBIO.2006.02.012

Abstract: The molecular mechanisms behind the entry of the primordial follicle into the growing follicle pool remain poorly understood. To investigate this process further, a microarray-based comparison was undertaken between 2-day postpartum mouse ovaries consisting of primordial follicles/naked oocytes only and those with both primordial follicles and newly activated follicles (7-day postpartum). Gene candidates identified included the chemoattractive cytokine stromal derived factor-1 (SDF1) and its receptor CXCR4. SDF1 and CXCR4 have been implicated in a variety of physiological processes including the migration of embryonic germ cells to the gonads. SDF1-alpha expression increased with the developmental stage of the follicle. Embryonic expression was found to be dichotomous post-germ cell migration, with low expression in the female. Immunohistochemical studies nonetheless indicate that the autocrine pattern of expression ligand and receptor begins during embryonic life. Addition of recombinant SDF1-alpha to neonatal mouse ovaries in vitro resulted in significantly higher follicle densities than for control ovaries. TUNEL analysis indicated no detectable difference in populations of apoptotic cells of treated or control ovaries. Treated ovaries also contained a significantly lower percentage of activated follicles as determined by measurement of oocyte diameter and morphological analysis. Treatment of cultured ovaries with an inhibitor of SDF1-alpha, AMD3100, ablated the effect of SDF1-alpha. By retaining follicles in an unactivated state, SDF1/CXCR4 signaling may play an important role in maintaining the size and longevity of the primordial follicle pool.

Impacts of obesity, maternal obesity and nicotinamide mononucleotide supplementation on sperm quality in mice

Publisher: Bioscientifica

Date: 08-2019

DOI: 10.1530/REP-18-0574

Abstract: Male fertility and sperm quality are negatively impacted by obesity. Furthermore, recent evidence has shown that male offspring from obese rat mothers also have reduced sperm quality and fertility. Here, we extend work in this area by comparing the effects of both maternal obesity and offspring post-weaning diet-induced obesity, as well as their combination, on sperm quality in mice. We additionally tested whether administration of the NAD + -booster nicotinamide mononucleotide (NMN) can ameliorate the negative effects of obesity and maternal obesity on sperm quality. We previously showed that intraperitoneal (i.p.) injection of NMN can reduce the metabolic deficits induced by maternal obesity or post-weaning dietary obesity in mice. In this study, female mice were fed a high-fat diet (HFD) for 6 weeks until they were 18% heavier than a control diet group. Thereafter, HFD and control female mice were mated with control diet males, and male offspring were weaned into groups receiving control or HFD. At 30 weeks of age, mice received 500 mg/kg body weight NMN or vehicle PBS i.p. for 21 days. As expected, adiposity was increased by both maternal and post-weaning HFD but reduced by NMN supplementation. Post-weaning HFD reduced sperm count and motility, while maternal HFD increased offspring sperm DNA fragmentation and levels of aberrant sperm chromatin. There was no evidence that the combination of post-weaning and maternal HFD exacerbated the impacts in sperm quality suggesting that they impact spermatogenesis through different mechanisms. Surprisingly NMN reduced sperm count, vitality and increased sperm oxidative DNA damage, which was associated with increased NAD + in testes. A subsequent experiment using oral NMN at 400 mg/kg body weight was not associated with reduced sperm viability, oxidative stress, mitochondrial dysfunction or increased NAD + in testes, suggesting that the negative impacts on sperm could be dependent on dose or mode of administration.

The serine protease testisin is present on the surface of capacitated stallion spermatozoa and interacts with key zona pellucida binding proteins

Publisher: Wiley

Date: 13-12-2019

DOI: 10.1111/ANDR.12569

Abstract: Serine proteases are emerging as important players in the spermatozoon's acquisition of functional competence. This study aimed to characterize the serine protease testisin (PRSS21) in stallion spermatozoa, examining its surface expression, possible origins in the testis and epididymis, and changes in response to capacitation and acrosome reaction, as well as its capacity to form high molecular weight complexes and interact with other proteins. The role of serine proteases in spontaneous capacitation and acrosome reaction of stallion spermatozoa was established using the serine protease inhibitor, AEBSF. Testisin localization, before and after exposure of stallion spermatozoa to capacitating conditions and calcium ionophore, was examined using live cell immunofluorescence and flow cytometry. Immunohistochemistry of testicular and epididymal tissues was used to further dissect the origins of sperm testisin. Testisin's participation in high molecular weight protein complexes and identification of its interacting partner proteins were investigated using Blue Native PAGE, co-immunoprecipitation, and mass spectrometry, with interrogation of protein-protein interaction databases and gene ontology analysis of partner proteins used to further explore the potential roles of the testisin-containing complex in sperm function. Testisin surface expression increased significantly in capacitated spermatozoa (p < 0.001), increased further following acrosome reaction (p < 0.01), and was localized to the equatorial region of the sperm head. Testisin was also detected in luminal fluid within the caput and corpus regions of the epididymis, epididymal spermatozoa, and epididymal epithelial cells. Testisin formed several multiprotein complexes co-immunoprecipitation revealed interactions of testisin with a multitude of zona pellucida-binding proteins, including ZPBP, ZAN, acrosin, several heat-shock proteins, and components of the TCP1 complex. Testisin appears to form part of the zona pellucida-binding complex in stallion spermatozoa and may be involved in the proteolytic cascade that prepares the sperm surface for interaction with the oocyte.

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.

Causative mechanisms and functional correlates of MTT reduction in stallion spermatozoa.

Publisher: Bioscientifica

Date: 06-2022

DOI: 10.1530/REP-21-0464

Abstract: MTT is a commonly used cell vitality probe, due to its ability to form insoluble formazan deposits at cellular locations of intense oxidoreductase activity. Although this response is considered a reflection of mitochondrial redox activity, extra-mitochondrial sites of MTT reduction have been recognized within the spermatozoa of several mammalian species. Therefore, the aim of this study was to determine the major sites and causative mechanisms of MTT reduction in stallion spermatozoa. Our results show that stallion spermatozoa displayed substantial mitochondrial formazan deposition, as well as a single extra-mitochondrial formazan deposit in various locations on the sperm head in approximately 20% of cells. The quality and capacitation status of stallion spermatozoa were positively correlated with the presence of an extra-mitochondrial formazan granule. Additionally, extra-mitochondrial formazan deposition was suppressed by the presence of an NADPH oxidase (NOX) inhibitor (VAS2870 active against NOX2, NOX4 and NOX5), MnTMPyP (SOD mimetic) and zinc (NOX5 inhibitor) suggesting that extra-mitochondrial MTT reduction may be facilitated by NOX-mediated ROS generating activity, conceivably NOX5 or NOX2. When comparing MTT to resazurin, another well-known probe used to detect metabolically active cells, MTT reduction had a higher correlation with sperm concentration and motility parameters (R 2 = 0.91), than resazurin reduction (R 2 = 0.76). We conclude that MTT reduction in stallion spermatozoa follows a species-specific pattern due to a high dependence on oxidative phosphorylation and a degree of NOX activity. As such, MTT reduction is a useful diagnostic tool to assess extra-mitochondrial redox activity, and therefore, the functional qualities of stallion spermatozoa.

Imperial College London, Royal School Of Mines

Location: United Kingdom of Great Britain and Northern Ireland

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Universidad Carlos III De Madrid Escuela Politécnica Superior

Location: Spain

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Université De Bordeaux

Location: France

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World Health Organization

Location: Switzerland

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University Of London

Location: United Kingdom of Great Britain and Northern Ireland

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University Of Newcastle Faculty Of Health

Location: Australia

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University Of Cambridge

Location: United Kingdom of Great Britain and Northern Ireland

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The University Of Edinburgh

Location: United Kingdom of Great Britain and Northern Ireland

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Bangor University

Location: United Kingdom of Great Britain and Northern Ireland

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Medical Research Council

Location: United Kingdom of Great Britain and Northern Ireland

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Linkage Infrastructure, Equipment And Facilities - Grant ID: LE120100082

Start Date: 01-2012

End Date: 12-2012

Amount: $180,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0232455

Start Date: 2001

End Date: 12-2002

Amount: $545,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP150103231

Start Date: 02-2015

End Date: 12-2018

Amount: $403,300.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP160100824

Start Date: 01-2017

End Date: 01-2020

Amount: $560,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989084

Start Date: 2009

End Date: 12-2009

Amount: $275,000.00

Funder: Australian Research Council

ARC Centres Of Excellence - Grant ID: CE0348239

Start Date: 06-2003

End Date: 12-2010

Amount: $15,878,900.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100008

Start Date: 2010

End Date: 12-2010

Amount: $350,000.00

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0453683

Start Date: 2004

End Date: 12-2010

Amount: $465,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE100100142

Start Date: 2010

End Date: 07-2011

Amount: $500,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP110100984

Start Date: 07-2011

End Date: 12-2016

Amount: $735,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0561173

Start Date: 2005

End Date: 12-2005

Amount: $207,189.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100078

Start Date: 02-2011

End Date: 12-2011

Amount: $500,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989105

Start Date: 2009

End Date: 11-2009

Amount: $495,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100191

Start Date: 05-2011

End Date: 12-2011

Amount: $250,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0347245

Start Date: 2002

End Date: 12-2002

Amount: $630,000.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP190102455

Start Date: 06-2019

End Date: 12-2023

Amount: $472,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0211014

Start Date: 2001

End Date: 12-2002

Amount: $337,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0561030

Start Date: 02-2005

End Date: 02-2006

Amount: $441,100.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0453630

Start Date: 2004

End Date: 12-2004

Amount: $274,692.00

Funder: Australian Research Council