Brett Nixon

Dynamic Landscape of Extracellular Vesicle-Associated Proteins Is Related to Treatment Response of Patients with Metastatic Breast Cancer

Publisher: MDPI AG

Date: 16-11-2021

DOI: 10.3390/MEMBRANES11110880

Abstract: Breast cancer is the leading cause of cancer death in women. The majority of these deaths are due to disease metastasis, in which cancer cells disseminate to multiple organs and disrupt vital physiological functions. It is widely accepted that breast cancer cells secrete extracellular vesicles (EVs), which contain dynamic molecular cargo that act as versatile mediators of intercellular communication. Therefore, Evs. secreted by breast cancer cells could be involved in the development of metastatic disease and resistance to treatment. Moreover, changes in EV cargo could reflect the effects of therapy on their parent tumor cells. The aim of this feasibility study was to quantitatively profile the proteomes of Evs. isolated from blood s les taken from treatment sensitive and resistant metastatic breast cancer patients to identify proteins associated with responses. Three serial blood s les were collected from three patients with metastatic breast cancer receiving systemic therapy including a responder, a non-responder, and a mixed-responder. Evs. were isolated from plasma using size exclusion chromatography and their protein cargo was prepared for tandem mass tag (TMT)-labelling and quantitative analyses using two-dimensional high-performance liquid chromatography followed by tandem mass spectrometry. After filtering, we quantitatively identified 286 proteins with high confidence using a q value of 0.05. Of these, 149 were classified as EV associated candidate proteins and 137 as classical, high abundant plasma proteins. After comparing EV protein abundance between the responder and non-responder, we identified 35 proteins with unique de-regulated abundance patterns that was conserved at multiple time points. We propose that this proof-of-concept approach can be used to identify proteins which have potential as predictors of metastatic breast cancer response to treatment.

System Modelling of an Electric Two-Wheeled Vehicle for Energy Management Optimization Study

Publisher: Kyushu University

Date: 09-2021

DOI: 10.5109/4491656

Supplementary Data from ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma

Publisher: American Association for Cancer Research (AACR)

Date: 17-05-2023

DOI: 10.1158/0008-5472.22892044

Abstract: All Supplementary Tables

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.

Erratum: Genome analysis of the platypus reveals unique signatures of evolution

Publisher: Springer Science and Business Media LLC

Date: 09-2008

DOI: 10.1038/NATURE07253

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.

Supplementary Data from ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma

Publisher: American Association for Cancer Research (AACR)

Date: 17-05-2023

DOI: 10.1158/0008-5472.22892047

Abstract: All Supplementary Figures and their captions.

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.

Oxidative Stress in the Male Germline: A Review of Novel Strategies to Reduce 4-Hydroxynonenal Production

Publisher: MDPI AG

Date: 03-10-2018

DOI: 10.3390/ANTIOX7100132

Abstract: Germline oxidative stress is intimately linked to several reproductive pathologies including a failure of sperm-egg recognition. The lipid aldehyde 4-hydroxynonenal (4HNE) is particularly damaging to the process of sperm-egg recognition as it compromises the function and the stability of several germline proteins. Considering mature spermatozoa do not have the capacity for de novo protein translation, 4HNE modification of proteins in the mature gametes has uniquely severe consequences for protein homeostasis, cell function and cell survival. In somatic cells, 4HNE overproduction has been attributed to the action of lipoxygenase enzymes that facilitate the oxygenation and degradation of ω-6 polyunsaturated fatty acids (PUFAs). Accordingly, the arachidonate 15-lipoxygenase (ALOX15) enzyme has been intrinsically linked with 4HNE production, and resultant pathophysiology in various complex conditions such as coronary artery disease and multiple sclerosis. While ALOX15 has not been well characterized in germ cells, we postulate that ALOX15 inhibition may pose a new strategy to prevent 4HNE-induced protein modifications in the male germline. In this light, this review focuses on (i) 4HNE-induced protein damage in the male germline and its implications for fertility and (ii) new methods for the prevention of lipid peroxidation in germ cells.

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.

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.

Data from ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma

Publisher: American Association for Cancer Research (AACR)

Date: 14-07-2023

DOI: 10.1158/0008-5472.C.6651055.V2

Abstract: Abstract Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring i PIK3CA /i mutations showed increased sensitivity to ONC201, whereas those harboring i TP53 /i mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. Significance: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib. /

Data from ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma

Publisher: American Association for Cancer Research (AACR)

Date: 17-05-2023

DOI: 10.1158/0008-5472.C.6651055.V1

Abstract: Abstract Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring i PIK3CA /i mutations showed increased sensitivity to ONC201, whereas those harboring i TP53 /i mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. Significance: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib. /

Jumping the gun: Smoking constituent BaP causes premature primordial follicle activation and impairs oocyte fusibility through oxidative stress

Publisher: Elsevier BV

Date: 04-2012

DOI: 10.1016/J.TAAP.2012.01.028

Abstract: Benzo(a)pyrene (BaP) is an ovotoxic constituent of cigarette smoke associated with pre-mature ovarian failure and decreased rates of conception in IVF patients. Although the overall effect of BaP on female fertility has been documented, the exact molecular mechanisms behind its ovotoxicity remain elusive. In this study we examined the effects of BaP exposure on the ovarian transcriptome, and observed the effects of in vivo exposure on oocyte dysfunction. Microarray analysis of BaP cultured neonatal ovaries revealed a complex mechanism of ovotoxicity involving a small cohort of genes associated with follicular growth, cell cycle progression, and cell death. Histomorphological and immunohistochemical analysis supported these results, with BaP exposure causing increased primordial follicle activation and developing follicle atresia in vitro and in vivo. Functional analysis of oocytes obtained from adult Swiss mice treated neonatally revealed significantly increased levels of mitochondrial ROS/lipid peroxidation, and severely reduced sperm-egg binding and fusion in both low (1.5mg/kg/daily) and high (3mg/kg/daily) dose treatments. Our results reveal a complex mechanism of BaP induced ovotoxicity involving developing follicle atresia and accelerated primordial follicle activation, and suggest short term neonatal BaP exposure causes mitochondrial leakage resulting in reduced oolemma fluidity and impaired fertilisation in adulthood. This study highlights BaP as a key compound which may be partially responsible for the documented effects of cigarette smoke on follicular development and sub-fertility.

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.

Proteomic Profiling of Mouse Epididymosomes Reveals their Contributions to Post-testicular Sperm Maturation

Publisher: Elsevier BV

Date: 03-2019

DOI: 10.1074/MCP.RA118.000946

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.

Chronic acrylamide exposure in male mice induces DNA damage to spermatozoa; Potential for amelioration by resveratrol

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1016/J.REPROTOX.2016.05.004

Abstract: Humans are chronically exposed to acrylamide since carbohydrate rich foods contain the toxicant as a result of cooking at high temperatures. While acrylamide is unreactive with DNA, it is readily oxidised to glycidamide, which adducts with DNA. This metabolism occurs via the enzyme, cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1). Acrylamide was administered to male CD1 mice for three or six months at a dose of 0.18mg/kg bodyweight/day. DNA damage was detected in germ cells and mature spermatozoa of exposed mice without compromising their overall fertility. The use of resveratrol, an antioxidant and known CYP2E1 inhibitor, was found to ameliorate the DNA damage in both germ cells and spermatozoa. However, extended resveratrol treatment (six months, 10.0mg/kg bw/week) resulted in premature activation of these cells. Thus the DNA damage found in spermatozoa after chronic acrylamide administration can be alleviated but an alternative CYP2E1 inhibitor may be required.

Male infertility: Shining a light on lipids and lipid‐ modulating enzymes in the male germline

Publisher: MDPI AG

Date: 23-01-2020

DOI: 10.3390/JCM9020327

Abstract: Despite the prevalence of male factor infertility, most cases are defined as idiopathic, thus limiting treatment options and driving increased rates of recourse to assisted reproductive technologies (ARTs). Regrettably, our current armory of ARTs does not constitute therapeutic treatments for male infertility, thus highlighting an urgent need for novel intervention strategies. In our attempts to fill this void, we have come to appreciate that the production of pathological levels of oxygen radicals within the male germline are a defining etiology of many idiopathic infertility cases. Indeed, an imbalance of reactive oxygen species can precipitate a cascade of deleterious sequelae, beginning with the peroxidation of membrane lipids and culminating in cellular dysfunction and death. Here, we shine light on the importance of lipid homeostasis, and the impact of lipid stress in the demise of the male germ cell. We also seek to highlight the utility of emerging lipidomic technologies to enhance our understanding of the erse roles that lipids play in sperm function, and to identify biomarkers capable of tracking infertility in patient cohorts. Such information should improve our fundamental understanding of the mechanistic causes of male infertility and find application in the development of efficacious treatment options.

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

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.

The rise of testicular germ cell tumours: the search for causes, risk factors and novel therapeutic targets

Publisher: F1000 Research Ltd

Date: 19-02-2013

DOI: 10.12688/F1000RESEARCH.2-55.V1

Abstract: Since the beginning of the 20th century there has been a decline in the reproductive vitality of men within the Western world. The declining sperm quantity and quality has been associated with increased overt disorders of sexual development including hypospadias, undescended testes and type II testicular germ cell tumours (TGCTs). The increase in TGCTs cannot be accounted for by genetic changes in the population. Therefore exposure to environmental toxicants appears to be a major contributor to the aetiology of TGCTs and men with a genetic predisposition are particularly vulnerable. In particular, Type II TGCTs have been identified to arise from a precursor lesion Carcinoma in situ (CIS), identified as a dysfunctional gonocyte however, the exact triggers for CIS development are currently unknown. Therefore the transition from gonocytes into spermatogonia is key to those studying TGCTs. Recently we have identified seven miRNA molecules (including members of the miR-290 family and miR-136, 463* and 743a) to be significantly changed over this transition period. These miRNA molecules are predicted to have targets within the CXCR4, PTEN, DHH, RAC and PDGF pathways, all of which have important roles in germ cell migration, proliferation and homing to the spermatogonial stem cell niche. Given the plethora of potential targets affected by each miRNA molecule, subtle changes in miRNA expression could have significant consequences e.g. tumourigenesis. The role of non-traditional oncogenes and tumour suppressors such as miRNA in TGCT is highlighted by the fact that the majority of these tumours express wild type p53, a pivotal tumour suppressor usually inactivated in cancer. While treatment of TGCTs is highly successful, the impact of these treatments on fertility means that identification of exact triggers, earlier diagnosis and alternate treatments are essential. This review examines the genetic factors and possible triggers of type II TGCT to highlight target areas for potential new treatments.

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.

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.

The chemokine CXCL12 and its receptor CXCR4 are implicated in human seminoma metastasis

Publisher: Wiley

Date: 15-03-2013

DOI: 10.1111/J.2047-2927.2013.00081.X

Abstract: Seminoma and non-seminoma tumours increasingly occur within the western population. These tumours originate from carcinoma in situ (CIS) cells, which arise from dysfunctional gonocytes. CXCL12 and its receptors, CXCR4 and CXCR7, have been implicated in migration, proliferation and survival of gonocytes and their precursors and progeny, primordial germ cells and spermatogonial stem cells respectively. We previously found evidence that several miRNA molecules predicted to modulate CXCR4 signalling are differentially expressed during the differentiation of gonocytes into spermatogonia in mice. Bioinformatic analysis predicted these miRNA to modulate CXCR4 signalling, leading us to hypothesize that CXCL12-mediated CXCR4 signalling is involved in the disrupted differentiation of gonocytes that underpins CIS formation. Indeed, we detected CXCL12 in Sertoli cells of normal human testis, and relatively high expression in tumour stroma with concomitant weak staining in dispersed tumour cells. In contrast, CXCR4 was expressed in spermatogonial and meiotic germ cells of normal testis and in the majority of tumour cells. Quantitative RT-PCR identified elevated CXCR4 transcript levels in seminoma compared with normal testis and to non-seminoma, potentially reflecting the higher proportion of dysfunctional germ cells within seminomas. In the normal testis, expression of CXCR4 downstream signalling molecules phospho-MEK1/2 and phospho-ERK1/2 correlated with CXCR4/CXCL12 expression. Strikingly, this correlation was absent in seminoma and non-seminoma s les, suggesting that CXCL12 signalling is disrupted. Proliferation rate and cell survival were not altered by CXCL12 in either seminoma (TCam-2) or non-seminoma (833ke) cell lines. However, CXCL12 exposure induced TCam-2 cell invasion though simulated basement membrane, while in contrast, we provide the novel evidence that CXCR4-expressing non-seminoma cell lines 833ke and NTera2/D1 do not invade in response to CXCL12. These findings indicate that CXCL12 expression in the human testis may selectively influence seminoma migration and metastasis, correlating with its importance in gonocyte and spermatogonial stem cell biology.

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.

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.

Developmental expression of the dynamin family of mechanoenzymes in the mouse epididymis

Publisher: Oxford University Press (OUP)

Date: 20-12-2016

DOI: 10.1095/BIOLREPROD.116.145433

Abstract: The mammalian epididymis is an exceptionally long ductal system tasked with the provision of one of the most complex intraluminal fluids found in any exocrine gland. This specialized milieu is continuously modified by the combined secretory and absorptive of the surrounding epithelium and thus finely tuned for its essential roles in promoting sperm maturation and storage. While considerable effort has been focused on defining the composition of the epididymal fluid, relatively less is known about the intracellular trafficking machinery that regulates this luminal environment. Here, we characterize the ontogeny of expression of a master regulator of this machinery, the dynamin family of mechanoenzymes. Our data show that canonical dynamin isoforms were abundantly expressed in the juvenile mouse epididymis. However, in peripubertal and adult animals dynamin takes on a heterogeneous pattern of expression such that the different isoforms displayed both cell- and segment-specific localization. Thus, dynamin 1 and 3 were predominately localized in the distal epididymal segments (corpus and cauda), where they were found within clear and principal cells, respectively. In contrast, dynamin 2 was expressed throughout the epididymis, but localized to the Golgi apparatus of the principal cells in the proximal (caput) segment and the luminal border of these cells in more distal segments. These dynamin isoforms are therefore ideally positioned to play complementary, nonredundant roles in the regulation of the epididymal milieu. In support of this hypothesis, selective inhibition of dynamin altered the profile of proteins secreted from an immortalized caput epididymal cell line.

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

Next Generation Sequencing Analysis Reveals Segmental Patterns of microRNA Expression in Mouse Epididymal Epithelial Cells

Publisher: Public Library of Science (PLoS)

Date: 13-08-2015

DOI: 10.1371/JOURNAL.PONE.0135605

miRNA and mammalian male germ cells

Publisher: Oxford University Press (OUP)

Date: 11-10-2012

DOI: 10.1093/HUMUPD/DMR041

Abstract: BACKGROUND Achieving the correct spatial and temporal expression of germ-cell-specific genes is fundamental to the production of viable healthy spermatozoa. Notably, post-transcriptional gene regulation resulting in the repression of protein translation is central to many embryonic processes, and is particularly active during spermatogenesis. In this review, we discuss microRNA (miRNA) regulation of target gene expression in relation to mammalian spermatogenesis, the establishment of testicular germ cell tumours (TGCT) and the potential use of miRNA manipulation for cancer therapy and fertility regulation. METHODS Journal databases such as PubMed were searched using key words, including miRNA, testis, spermatogenesis, germ cell, testicular cancer and cancer. RESULTS In the past decade, the deployment of small non-coding RNA molecules, including miRNA, by the cell, has been recognized as among the most important mechanisms of fine-tuning translational regulation in differentiating cell types. For key regulators of male gametogenesis, high levels of gene expression do not always correspond to elevated levels of protein expression. Cumulatively this indicates that enhancement and repression of post-transcriptional regulatory mechanisms are essential to the success of spermatogenesis. There is also growing evidence that this form of regulation contributes to the aetiology of both TGCT and spermatocytic tumours. CONCLUSIONS miRNA plays an essential role in regulation of genes during the process of spermatogenesis. Disruption of this regulation has the ability to contribute to the neoplastic development of germ cell tumours. However, targeted knockdown of specific miRNA molecules has the potential to form both anti-oncogenic reagents and underpin the basis for novel contraceptive technologies.

Non-coding RNA in spermatogenesis and epididymal maturation

Publisher: Springer Netherlands

Date: 12-12-2016

DOI: 10.1007/978-94-017-7417-8_6

Abstract: Testicular germ and somatic cells express many classes of small ncRNAs, including Dicer-independent PIWI-interacting RNAs, Dicer-dependent miRNAs, and endogenous small interfering RNA. Several studies have identified ncRNAs that are highly, exclusively, or preferentially expressed in the testis and epididymis in specific germ and somatic cell types. Temporal and spatial expression of proteins is a key requirement of successful spermatogenesis and large-scale gene transcription occurs in two key stages, just prior to transcriptional quiescence in meiosis and then during spermiogenesis just prior to nuclear silencing in elongating spermatids. More than 60 % of these transcripts are then stockpiled for subsequent translation. In this capacity ncRNAs may act to interpret and transduce cellular signals to either maintain the undifferentiated stem cell population and/or drive cell differentiation during spermatogenesis and epididymal maturation. The assignation of specific roles to the majority of ncRNA species implicated as having a role in spermatogenesis and epididymal function will underpin fundamental understanding of normal and disease states in humans such as infertility and the development of germ cell tumours.

Molecular Mechanisms Responsible for Increased Vulnerability of the Ageing Oocyte to Oxidative Damage

Publisher: Hindawi Limited

Date: 2017

DOI: 10.1155/2017/4015874

Abstract: In their midthirties, women experience a decline in fertility, coupled to a pronounced increase in the risk of aneuploidy, miscarriage, and birth defects. Although the aetiology of such pathologies are complex, a causative relationship between the age-related decline in oocyte quality and oxidative stress (OS) is now well established. What remains less certain are the molecular mechanisms governing the increased vulnerability of the aged oocyte to oxidative damage. In this review, we explore the reduced capacity of the ageing oocyte to mitigate macromolecular damage arising from oxidative insults and highlight the dramatic consequences for oocyte quality and female fertility. Indeed, while oocytes are typically endowed with a comprehensive suite of molecular mechanisms to moderate oxidative damage and thus ensure the fidelity of the germline, there is increasing recognition that the efficacy of such protective mechanisms undergoes an age-related decline. For instance, impaired reactive oxygen species metabolism, decreased DNA repair, reduced sensitivity of the spindle assembly checkpoint, and decreased capacity for protein repair and degradation collectively render the aged oocyte acutely vulnerable to OS and limits their capacity to recover from exposure to such insults. We also highlight the inadequacies of our current armoury of assisted reproductive technologies to combat age-related female infertility, emphasising the need for further research into mechanisms underpinning the functional deterioration of the ageing oocyte.

Preclinical and clinical evaluation of German-sourced ONC201 for the treatment of H3K27M-mutant diffuse intrinsic pontine glioma

Publisher: Oxford University Press (OUP)

Date: 2021

DOI: 10.1093/NOAJNL/VDAB169

Abstract: Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood brainstem tumor for which radiation is the only treatment. Case studies report a clinical response to ONC201 for patients with H3K27M-mutant gliomas. Oncoceutics (ONC201) is only available in the United States and Japan however, in Germany, DIPG patients can be prescribed and dispensed a locally produced compound—ONC201 German-sourced ONC201 (GsONC201). Pediatric oncologists face the dilemma of supporting the administration of GsONC201 as conjecture surrounds its authenticity. Therefore, we compared GsONC201 to original ONC201 manufactured by Oncoceutics Inc. Authenticity of GsONC201 was determined by high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Biological activity was shown via assessment of on-target effects, in vitro growth, proliferation, and apoptosis analysis. Patient-derived xenograft mouse models were used to assess plasma and brain tissue pharmacokinetics, pharmacodynamics, and overall survival (OS). The clinical experience of 28 H3K27M+ mutant DIPG patients who received GsONC201 (2017–2020) was analyzed. GsONC201 harbored the authentic structure, however, was formulated as a free base rather than the dihydrochloride salt used in clinical trials. GsONC201 in vitro and in vivo efficacy and drug bioavailability studies showed no difference compared to Oncoceutics ONC201. Patients treated with GsONC201 (n = 28) showed a median OS of 18 months (P = .0007). GsONC201 patients who underwent reirradiation showed a median OS of 22 months compared to 12 months for GsONC201 patients who did not (P = .012). This study confirms the biological activity of GsONC201 and documents the OS of patients who received the drug however, GsONC201 was never used as a monotherapy.

Reference Module in Biomedical Research, Encyclopedia of Reproduction Second Edition

Publisher: Elsevier

Date: 2018

DOI: 10.1016/B978-0-12-801238-3.64464-1

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.

Glioma Pathogenesis-Related 1-Like 1 Is Testis Enriched, Dynamically Modified, and Redistributed during Male Germ Cell Maturation and Has a Potential Role in Sperm-Oocyte Binding

Publisher: The Endocrine Society

Date: 10-03-2010

DOI: 10.1210/EN.2009-1255

Abstract: The glioma pathogenesis-related 1 (GLIPR1) family consists of three genes [GLIPR1, GLIPR1-like 1 (GLIPR1L1), and GLIPR1-like 2 (GLIPR1L2)] and forms a distinct subgroup within the cysteine-rich secretory protein (CRISP), antigen 5, and pathogenesis-related 1 (CAP) superfamily. CAP superfamily proteins are found in phyla ranging from plants to humans and, based largely on expression and limited functional studies, are hypothesized to have roles in carcinogenesis, immunity, cell adhesion, and male fertility. Specifically data from a number of systems suggests that sequences within the C-terminal CAP domain of CAP proteins have the ability to promote cell-cell adhesion. Herein we cloned mouse Glipr1l1 and have shown it has a testis-enriched expression profile. GLIPR1L1 is posttranslationally modified by N-linked glycosylation during spermatogenesis and ultimately becomes localized to the connecting piece of elongated spermatids and sperm. After sperm capacitation, however, GLIPR1L1 is also localized to the anterior regions of the sperm head. Zona pellucida binding assays indicate that GLIPR1L1 has a role in the binding of sperm to the zona pellucida surrounding the oocyte. These data suggest that, along with other members of the CAP superfamily and several other proteins, GLIPR1L1 is involved in the binding of sperm to the oocyte complex. Collectively these data further strengthen the role of CAP domain-containing proteins in cellular adhesion and propose a mechanism whereby CAP proteins show overlapping functional significance during fertilization.

Mouse quiescin sulfhydryl oxidases exhibit distinct epididymal luminal distribution with segment-specific sperm surface associations†

Publisher: Oxford University Press (OUP)

Date: 24-05-2018

DOI: 10.1093/BIOLRE/IOY125

Abstract: Sulfhydryl oxidation is part of the sperm maturation process essential for the acquisition of sperm fertilization competency and its structural stabilization however, the specific sulfhydryl oxidases that fulfill these roles have yet to be identified. In this study, we investigate the potential involvement of one atypical thiol oxidase family called quiescin Q6/sulfhydryl oxidase (QSOX) using the mouse epididymis as our model system. With multidisciplinary approaches, we show that QSOX isoform 1 and 2 exhibit complementary distribution throughout the epididymal duct, but that each variant possesses distinct subcellular localization within the epididymal principal cells. While QSOX2 was exclusively present in the Golgi apparatus of the caput and corpus epididymis, QSOX1c, the most profusely express QSOX1 variant, was abundantly present in the cauda luminal fluids. Moreover, immunohistochemistry studies together with proteomic identification in isolated epididymosomes provided evidence substantiating the release of QSOX2, but not QSOX1c, via an apocrine secretory pathway. Furthermore, we demonstrate for the first time, distinct association of QSOX1c and QSOX2 with the sperm acrosome and implantation fossa, during different stages of their epididymal maturation. In conclusion, our study provides the first comprehensive comparisons between QSOX1 and QSOX2 in the mouse epididymis, revealing their distinct epididymal distribution, cellular localization, mechanisms of secretion and sperm membrane association. Together, these data suggest that QSOX1 and QSOX2 have discrete biological functions in male germ cell development.

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.

Supplementary Data from ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma

Publisher: American Association for Cancer Research (AACR)

Date: 14-07-2023

DOI: 10.1158/0008-5472.23683824

Abstract: All Supplementary Tables

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.

Formation and dissociation of sperm bundles in monotremes

Publisher: Oxford University Press (OUP)

Date: 24-08-2016

DOI: 10.1095/BIOLREPROD.116.140491

Abstract: Because monotremes are the earliest offshoot of the mammalian lineage, the platypus and short-beaked echidna were studied as model animals to assess the origin and biological significance of adaptations considered unique to therian mammals: epididymal sperm maturation and subsequent capacitation. We show that spermatozoa from both species assemble into bundles of approximately 100 cells during passage through the epididymis and that an epididymal protein-secreted protein, acidic, cysteine-rich (osteonectin SPARC)-is involved in bundle formation. The bundles persisted during incubation in vitro for at least 1 h under conditions that capacitate therian spermatozoa, and then underwent a time-dependent dissociation to release spermatozoa capable of fertilization. Only after this dissociation could the spermatozoa bind to the perivitelline membrane of a hen's egg, display an altered form of motility reminiscent of hyperactivation, and be induced to undergo an acrosome reaction. It is concluded that the development of sperm bundles in the monotreme epididymis mandates that they require a time-dependent process to be capable of fertilizing an ovum. However, because this functional end point was achieved without overt changes in protein tyrosine phosphorylation (a hallmark of capacitation in therians), it is concluded that the process in monotremes is distinctly different from capacitation in therian mammals.

Profiling of epididymal small non‐protein‐coding RNA s

Publisher: Wiley

Date: 25-04-2019

DOI: 10.1111/ANDR.12640

Abstract: Our understanding of epididymal physiology and function has been transformed over the three decades in which the International Meeting Series on the Epididymis has been hosted. This transformation has occurred along many fronts, but among the most significant advances has been the unexpected discovery of the ersity of small non-protein-coding RNAs (sRNAs) expressed in the epididymal epithelium and differentially accumulated in the luminal population of spermatozoa. Here we survey recent literature pertaining to profiling the sRNA landscape of the mammalian epididymis with the goal of demonstrating the contribution that these key regulatory elements, and their associated pathways, make to epididymal physiology and sperm maturation. High throughput sequencing strategies have fueled an unprecedented advance in our understanding of RNA biology. In the last decade, such high throughput profiling tools have been increasingly applied to study the mammalian epididymis, presaging the discovery of erse classes of sRNA expressed along the length of the tract. Among the best studied sRNA classes are the microRNAs (miRNA), a sRNA species shown to act in concert with endocrine signals to fine-tune the segmental patterning of epididymal gene expression. In addition to performing this homeostatic role, epithelial cell-derived sRNAs also selectively accumulate into the epididymosomes and spermatozoa that occupy the duct lumen. This exciting discovery alludes to a novel form of intracellular communication that contributes to the establishment of the sperm epigenome and its modification under conditions of paternal stress. Compelling literature has identified sRNAs as a crucial regulatory tier that allows the epididymis to fulfill its combined roles of sperm transport, maturation, and storage. Continued research in this emerging field will contribute to our growing understanding of the etiology of male factor infertility and potentially allow for the future design of rational therapeutic options for these in iduals.

Supplementary Data from ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma

Publisher: American Association for Cancer Research (AACR)

Date: 14-07-2023

DOI: 10.1158/0008-5472.23683827

Abstract: All Supplementary Figures and their captions.

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.

ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma

Publisher: American Association for Cancer Research (AACR)

Date: 05-05-2023

DOI: 10.1158/0008-5472.CAN-23-0186

Abstract: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib.

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

Probing the Origins of 1,800 MHz Radio Frequency Electromagnetic Radiation Induced Damage in Mouse Immortalized Germ Cells and Spermatozoa in vitro.

Publisher: Frontiers Media SA

Date: 21-09-2018

DOI: 10.3389/FPUBH.2018.00270

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.

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.

Assisted breeding technology in the saltwater crocodile

Publisher: CSIRO Publishing

Date: 15-02-2021

DOI: 10.1071/RD20217

Abstract: This review reports the current status of artificial breeding technology in the Crocodylia and the future requirements for the establishment of AI in the saltwater crocodile. Although there are challenges regarding safe restraint and immobilisation, semen collection of the saltwater crocodile by manual stimulation has proven effective in yielding sufficient volume and sperm concentrations for empirical and molecular analyses of sperm preservation and physiology. Nevertheless, there is still much to learn with respect to fundamental anatomy, physiology and behaviour in both sexes, but particularly in the female. Although lessons can be learned from successful AI in the alligator, the details of this research are not readily accessible. Future research needs to focus on the proximate factors of seasonality and the underlying control of the female’s annual reproductive cycle this will require novel and innovative ways to collect blood s les without causing stress or injury, and ideally a dedicated crocodile research breeding colony. Because the saltwater crocodile is a farmed species, there is likely to be sufficient impetus for the application of assisted breeding technology to drive future productivity in the industry. These developments will also have benefits for the genetic and reproductive management of endangered captive populations.

Dynamin 2 is essential for mammalian spermatogenesis

Publisher: Springer Science and Business Media LLC

Date: 11-10-2016

DOI: 10.1038/SREP35084

Abstract: The dynamin family of proteins play important regulatory roles in membrane remodelling and endocytosis, especially within brain and neuronal tissues. In the context of reproduction, dynamin 1 (DNM1) and dynamin 2 (DNM2) have recently been shown to act as key mediators of sperm acrosome formation and function. However, little is known about the roles that these proteins play in the developing testicular germ cells. In this study, we employed a DNM2 germ cell-specific knockout model to investigate the role of DNM2 in spermatogenesis. We demonstrate that ablation of DNM2 in early spermatogenesis results in germ cell arrest during prophase I of meiosis, subsequent loss of all post-meiotic germ cells and concomitant sterility. These effects become exacerbated with age, and ultimately result in the demise of the spermatogonial stem cells and a Sertoli cell only phenotype. We also demonstrate that DNM2 activity may be temporally regulated by phosphorylation of DNM2 via the kinase CDK1 in spermatogonia, and dephosphorylation by phosphatase PPP3CA during meiotic and post-meiotic spermatogenesis.

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.

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.

Staying Alive: PI3K Pathway Promotes Primordial Follicle Activation and Survival in Response to 3MC-Induced Ovotoxicity

Publisher: Oxford University Press (OUP)

Date: 12-04-2012

DOI: 10.1093/TOXSCI/KFS137

Abstract: 3-Methylcholanthrene (3MC) is a potent ovotoxicant capable of causing premature ovarian failure through primordial follicle depletion. Despite 3MCs ovotoxicity having been established for 30 years, relatively little information exists on the mechanisms. In this study, we examined the effects of 3MC exposure on the immature ovarian follicle population. Microarray analysis revealed a complex mechanism of 3MC-induced ovotoxicity involving a number of cellular processes associated with xenobiotic metabolism, ovarian cancer, cell cycle progression, and cell death. 3MC exposure was also found to induce developing follicle atresia and aberrant primordial follicle activation via the stimulation of PI3K/Akt and mammalian target of rapamycin (mTOR) signaling pathways. Inhibition of PI3K/Akt signaling resulted in the severe depletion of the primordial follicle pool, with further analysis identifying increased Akt1-stimulated Bad phosphoinhibition in 3MC-treated primordial follicles. Our results suggest that the primordial follicle pool enters a "prosurvival" state upon 3MC exposure and that its depletion is due to a vicious cycle of primordial follicle activation in an attempt to replace developing follicles undergoing follicular atresia.

Chronic acrylamide exposure in male mice results in elevated DNA damage in the germline and heritable induction of CYP2E1 in the testes

Publisher: Oxford University Press (OUP)

Date: 31-08-2016

DOI: 10.1095/BIOLREPROD.116.139535

Abstract: Acute acrylamide exposure in male rodents results in reduced reproductive performance and dominant lethality. However, the reproductive effects of low dose chronic exposure, which better reflects the nature of human exposure, remain far less certain. Human dietary consumption of acrylamide has been estimated at an average of 1-4 µg/kg bw/day. In order to simulate this exposure, male mice were provided with acrylamide (1 µg/ml) via their drinking water continuously for six months, which was equivalent to a human dose of 10.5 µg/ kg bw/day. This exposure regime increased DNA damage in the spermatozoa, without affecting a concomitant reduction in overall fertility. The offspring of acrylamide treated mice did not have an increased incidence of skin papilloma formation following the two-stage tumor induction protocol. However, the male offspring of acrylamide treated fathers had significantly increased levels of DNA damage in their spermatozoa, despite having had no direct toxicant exposure. It was also found that the F0, and most crucially, F1 mice had increased levels of CYP2E1 protein in their germ cells. This is significant as CYP2E1 is the sole enzyme responsible for conversion of acrylamide to its harmful metabolite glycidamide. This altered expression may be due to epigenetic alterations. Additionally, the F0 and F1 mice had increased oxidative adducts in the DNA of their germ cells, which was hypothesized to arise as a byproduct of increased CYP2E1 activity. Therefore, chronic paternal acrylamide exposure in mice has consequences for their offspring, and raises concerns for the effects of acrylamide exposure in the human population and the accumulated effects with multiple generations of exposure.

Role of the epididymis in sperm competition

Publisher: Medknow

Date: 07-2007

DOI: 10.1111/J.1745-7262.2007.00284.X

Abstract: Although it is generally understood that the testes recruited kidney ducts for reproductive function during the evolution of vertebrates, little is understood of the biological significance of the adaptation. In the context of the evolution of the mammalian epididymis, this report provides evidence that a major role of the epididymis is to enhance a male's chance of achieving paternity in a competitive mating system. A unique ex le of sperm cooperation in monotremes is used as evidence that the epididymis produces sperm competition proteins to form groups of 100 sperm into bundles that have a forward motility nearly thrice that of in idual spermatozoa. As it required 3-h incubation in vitro under capacitation conditions to release motile sperm from the bundles, it is suggested that the monotremes provide an ex le of capacitation that is quite different from capacitation in higher mammals. It is suggested that variation between species in the intensity of sperm competition could explain the variation that occurs between species in the amount of post-testicular sperm maturation and storage in the epididymis, an explanation of why the human epididymis does not play as important a role in reproduction as the epididymis of most mammals.

Characteristics of the epididymal luminal environment responsible for sperm maturation and storage

Publisher: Frontiers Media SA

Date: 28-02-2018

DOI: 10.3389/FENDO.2018.00059

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.

Molecular Changes Induced by Oxidative Stress that Impair Human Sperm Motility

Publisher: MDPI AG

Date: 04-02-2020

DOI: 10.3390/ANTIOX9020134

Abstract: A state of oxidative stress (OS) and the presence of reactive oxygen species (ROS) in the male reproductive tract are strongly correlated with infertility. While physiological levels of ROS are necessary for normal sperm functioning, elevated ROS production can overwhelm the cell’s limited antioxidant defenses leading to dysfunction and loss of fertilizing potential. Among the deleterious pleiotropic impacts arising from OS, sperm motility appears to be particularly vulnerable. Here, we present a mechanistic account for how OS contributes to altered sperm motility profiles. In our model, it is suggested that the abundant polyunsaturated fatty acids (PUFAs) residing in the sperm membrane serve to sensitize the male germ cell to ROS attack by virtue of their ability to act as substrates for lipid peroxidation (LPO) cascades. Upon initiation, LPO leads to dramatic remodeling of the composition and biophysical properties of sperm membranes and, in the case of the mitochondria, this manifests in a dissipation of membrane potential, electron leakage, increased ROS production and reduced capacity for energy production. This situation is exacerbated by the production of cytotoxic LPO byproducts such as 4-hydroxynonenal, which dysregulate molecules associated with sperm bioenergetic pathways as well as the structural and signaling components of the motility apparatus. The impact of ROS also extends to lesions in the paternal genome, as is commonly seen in the defective spermatozoa of asthenozoospermic males. Concluding, the presence of OS in the male reproductive tract is strongly and positively correlated with reduced sperm motility and fertilizing potential, thus providing a rational target for the development of new therapeutic interventions.

The MicroRNA Signature of Mouse Spermatozoa Is Substantially Modified During Epididymal Maturation1

Publisher: Oxford University Press (OUP)

Date: 10-2015

DOI: 10.1095/BIOLREPROD.115.132209

Abstract: In recent years considerable effort has been devoted to understanding the epigenetic control of sperm development, leading to an increased appreciation of the importance of RNA interference pathways, and in particular miRNAs, as key regulators of spermatogenesis and epididymal maturation. It has also been shown that sperm are endowed with an impressive array of miRNA that have been implicated in various aspects of fertilization and embryo development. However, to date there have been no reports on whether the sperm miRNA signature is static or whether it is influenced by their prolonged maturation within the male reproductive tract. To investigate this phenomenon, we employed next-generation sequencing to systematically profile the miRNA signature of maturing mouse spermatozoa. In so doing we have provided the first evidence for the posttesticular modification of the sperm miRNA profile under normal physiological conditions. Such modifications include the apparent loss and acquisition of an impressive cohort of some 113 and 115 miRNAs, respectively, between the proximal and distal epididymal segments. Interestingly, the majority of these changes occur late in maturation and include the uptake of novel miRNA species in addition to a significant increase in many miRNAs natively expressed in immature sperm. Because sperm are not capable of de novo transcription, these findings identify the epididymis as an important site in establishing the sperm epigenome with the potential to influence the peri-conceptual environment of the female reproductive tract, contribute to the inheritance of acquired characteristics, and/or alter the developmental trajectory of the resulting offspring.

Characterisation of mouse epididymosomes reveals a complex profile of microRNAs and a potential mechanism for modification of the sperm epigenome

Publisher: Springer Science and Business Media LLC

Date: 23-08-2016

DOI: 10.1038/SREP31794

Abstract: Recent evidence has shown that the sperm epigenome is vulnerable to dynamic modifications arising from a variety of paternal environment exposures and that this legacy can serve as an important determinant of intergenerational inheritance. It has been postulated that such exchange is communicated to maturing spermatozoa via the transfer of small non-protein-coding RNAs (sRNAs) in a mechanism mediated by epididymosomes small membrane bound vesicles released by the soma of the male reproductive tract (epididymis). Here we confirm that mouse epididymosomes encapsulate an impressive cargo of microRNAs (miRNAs), a developmentally important sRNA class, the majority (~60%) of which are also represented by the miRNA signature of spermatozoa. This includes miRNAs that were found exclusively in epididymal sperm and epididymosomes, but not in the surrounding soma. We also documented substantial changes in the epididymosome miRNA cargo, including significant fold changes in almost half of the miRNAs along the length of the epididymis. Finally, we provide the first direct evidence for the transfer of several prominent miRNA species between mouse epididymosomes and spermatozoa to afford novel insight into a mechanism of intercellular communication by which the sRNA payload of sperm can be selectively modified during their post-testicular maturation.

The lipid peroxidation product 4-hydroxynonenal contributes to oxidative stress-mediated deterioration of the ageing oocyte

Publisher: Springer Science and Business Media LLC

Date: 24-07-2017

DOI: 10.1038/S41598-017-06372-Z

Abstract: An increase in intraovarian reactive oxygen species (ROS) has long been implicated in the decline in oocyte quality associated with maternal ageing. Oxidative stress (OS)-induced lipid peroxidation and the consequent generation of highly electrophilic aldehydes, such as 4-hydroxynonenal (4-HNE), represents a potential mechanism by which ROS can inflict damage in the ageing oocyte. In this study, we have established that aged oocytes are vulnerable to damage by 4-HNE resulting from increased cytosolic ROS production within the oocyte itself. Further, we demonstrated that the age-related induction of OS can be recapitulated by exposure of germinal vesicle (GV) oocytes to exogenous H 2 O 2 . Such treatments stimulated an increase in 4-HNE generation, which remained elevated during in vitro oocyte maturation to metaphase II. Additionally, exposure of GV oocytes to either H 2 O 2 or 4-HNE resulted in decreased meiotic completion, increased spindle abnormalities, chromosome misalignments and aneuploidy. In seeking to account for these data, we revealed that proteins essential for oocyte health and meiotic development, namely α-, β-, and γ-tubulin are vulnerable to adduction via 4-HNE. Importantly, 4-HNE-tubulin adduction, as well as increased aneuploidy rates, were resolved by co-treatment with the antioxidant penicillamine, demonstrating a possible therapeutic mechanism to improve oocyte quality in older females.

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.

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

Assessment of microRNA expression in mouse epididymal epithelial cells and spermatozoa by next generation sequencing

Publisher: Elsevier BV

Date: 12-2015

DOI: 10.1016/J.GDATA.2015.09.012

Epididymal CYP2E1 plays a critical role in acrylamide-induced DNA damage in spermatozoa and paternally mediated embryonic resorptions†

Publisher: Oxford University Press (OUP)

Date: 31-03-2017

DOI: 10.1093/BIOLRE/IOX021

Abstract: Acrylamide is a ubiquitous toxicant in human lives, due to its formation in many food products. Acrylamide induces dominant lethal mutations with administration of 25 mg/kg bw/day for 5 days in male mice. Cytochrome P450, family 2, subfamily E, polypeptide 1 (CYP2E1) is responsible for this dominant lethality. CYP2E1 is the only enzyme responsible for the conversion of acrylamide to the highly reactive metabolite glycidamide, which forms adducts with DNA. CYP2E1 is present predominantly in the liver, as well as the brain, kidney, intestines, and spleen. Within the male mouse reproductive tract, CYP2E1 localizes to spermatocytes. However, embryo resorptions have been demonstrated to occur only with exposure of the late stages of spermatogenesis and spermatozoa. It was determined that CYP2E1 is additionally expressed within the mouse epididymal epithelium, and this localization is responsible for acrylamide-induced dominant lethality. Further, an equivalent profile of CYP2E1 expression was identified in the human reproductive tract. While spermatozoa of both species were also established to possess CYP2E1, this did not contribute to acrylamide-induced DNA damage. In vitro studies strengthened these findings further, revealing that acrylamide exposure only induces DNA damage in human and mouse spermatozoa following metabolism by the mouse epididymal epithelial cell line (mECap18) to glycidamide. These findings emphasize, for the first time, the vital role of the epididymis in the reproductive toxicity associated with acute acrylamide exposure.

Effects of 4-nonylphenol and 17α-ethynylestradiol exposure in the Sydney rock oyster, Saccostrea glomerata: Vitellogenin induction and gonadal development

Publisher: Elsevier BV

Date: 06-2008

DOI: 10.1016/J.AQUATOX.2008.03.003

Abstract: Adult Saccostrea glomerata were exposed to environmentally relevant concentrations of 4-nonylphenol (1microg/L and 100microg/L) and 17alpha-ethynylestradiol (5ng/L and 50ng/L) in seawater over 8 weeks. Exposures were performed to assess effects on vitellogenin induction and gonadal development during reproductive conditioning. Chronic direct estrogenicity within gonadal tissue was assessed via an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression assay (ER-CALUX). Estradiol equivalents (EEQ) were greatest in the 100microg/L 4-nonylphenol exposure (28.7+/-2.3ng/g tissue EEQ) while 17alpha-ethynylestradiol at concentrations of 50ng/L were 2.2+/-1.5ng/g tissue EEQ. Results suggest 4-nonylphenol may be accumulated in tissue and is partly resistant to biotransformation maintaining its potential for chronic estrogenic action, while 17alpha-ethynylestradiol, although exhibiting greater estrogenic potency on biological endpoints possibly exerts its estrogenic action before being rapidly metabolised and/or excreted. A novel methodology was developed to assess vitellogenin using high-performance liquid chromatography (HPLC). Exposure to both 17alpha-ethynylestradiol (50ng/L) and 4-nonylphenol (100microg/L) produced increases in vitellogenin for females, whereas males exhibited increases in vitellogenin when exposed to 50ng/L 17alpha-ethynylestradiol only. Females exhibited greater vitellogenin responses than males at 50ng/L 17alpha-ethynylestradiol only. Histological examination of gonads revealed a number of in iduals exhibiting intersex (ovotestis) in 50ng/L 17alpha-ethynylestradiol exposures. Male in iduals in 1microg/L and 100microg/L 4-nonylphenol exposures and 5ng/L 17alpha-ethynylestradiol were at earlier stages of spermatogenic development than corresponding controls.

Post-testicular sperm maturation and identification of an epididymal protein in the Japanese quail (Coturnix coturnix japonica)

Publisher: Bioscientifica

Date: 03-2014

DOI: 10.1530/REP-13-0566

Abstract: The role of the avian epididymis in post-testicular development and capacitation was examined to assess whether avian spermatozoa undergo any processes similar to those characteristic of mammalian sperm development. We found no evidence of a need for quail sperm to undergo capacitation and 90% of testicular sperm could bind to a perivitelline membrane and acrosome react. However, computer-assisted sperm analysis showed that 20% of testicular sperm from the quail were capable of movement and only about 12% of the motile sperm would have a curvilinear velocity greater than the mean for sperm from the distal epididymis. Nevertheless, epididymal transit was associated with increases in mean sperm velocity and the proportion of motile sperm. Together, these findings explain why earlier workers have achieved some fertilizations following inseminations of testicular spermatozoa and also demonstrate the need for some epididymal maturation of avian spermatozoa. Analysis of the electrophoretic profile of quail epididymal luminal proteins revealed that only one major protein (∼16 kDa) is secreted by the epididymis and it was virtually the only protein secreted by the ipsilateral epididymis following unilateral orchidectomy. Mass spectrometry showed that this protein is hemoglobin this finding was confirmed using anti-hemoglobin antibodies. It is suggested that hemoglobin may support sperm metabolism in the quail epididymis, aid in motility, and/or serve as an antioxidant.

The Australian saltwater crocodile (Crocodylus porosus) provides evidence that the capacitation of spermatozoa may extend beyond the mammalian lineage

Publisher: The Royal Society

Date: 11-05-2016

DOI: 10.1098/RSPB.2016.0495

Abstract: Although mammalian spermatozoa only acquire functional maturity as they are conveyed through the male (epididymal maturation) and female (capacitation) reproductive tracts, the degree of post-testicular development necessary to achieve fertilization in other vertebrate species remains far less clear. Indeed, despite reports that the epididymis of birds and reptiles is capable of secreting proteins that bind and modify the sperm surface characteristics, it remains unclear whether capacitation is a pre-requisite for fertilization in these species. Using the ancient reptilian Australian saltwater crocodile as a model, this study was undertaken to explore whether reptile sperm do undergo capacitation-like changes following ejaculation. Our studies revealed that crocodile spermatozoa experienced a rapid and sustained, cyclic-AMP mediated increase in progressive motility following incubation under conditions optimized for the induction of capacitation in mammalian species such as the mouse and human. This response was coupled with elevated levels of phosphorylation associated with both protein kinase A and tyrosine kinase substrates, the latter of which were predominantly localized within the sperm flagellum. In findings that also accord with mammalian spermatozoa, we confirmed a homologue of outer dense fibre 2 as one of the principal substrates for tyrosine phosphorylation. Overall, our findings support the concept that crocodile spermatozoa do undergo a process that is homologous to capacitation in preparation for fertilization of an ovum.

Supplementary Data from ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma

Publisher: American Association for Cancer Research (AACR)

Date: 17-05-2023

DOI: 10.1158/0008-5472.22892044.V1

Abstract: All Supplementary Tables

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

Post-testicular sperm maturation in the saltwater crocodile Crocodylus porosus: Assessing the temporal acquisition of sperm motility

Publisher: CSIRO Publishing

Date: 26-02-2021

DOI: 10.1071/RD20204

Abstract: Conservation efforts to secure the long-term survival of crocodilian species would benefit from the establishment of a frozen sperm bank in concert with artificial breeding technologies to maintain genetic ersity among captive assurance populations. Working towards this goal, our research has focused on the saltwater crocodile Crocodylus porosus as a tractable model for understanding crocodilian sperm physiology. In extending our systematic characterisation of saltwater crocodile spermatozoa, in this study we examined the development of motility during sperm transport through the excurrent duct system of the male crocodile. The results show that approximately 20% of crocodile testicular spermatozoa are immediately motile but experience a gradient of increasing motility (percentage motile and rate of movement) as they transit the male reproductive tract (epididymis). Moreover, we confirmed that, as in ejaculated crocodile spermatozoa, increased intracellular cAMP levels promoted a significant and sustained enhancement of sperm motility regardless of whether the cells were isolated from the testis or epididymis. Along with the development of artificial reproductive technologies, this research paves the way for the opportunistic recovery, storage and potential utilisation of post-mortem spermatozoa from genetically valuable animals.

New proteins identified in epididymal fluid from the platypus (Ornithorhynchus anatinus)

Publisher: CSIRO Publishing

Date: 2009

DOI: 10.1071/RD09091

Abstract: The platypus epididymal proteome is being studied because epididymal proteins are essential for male fertility in mammals and it is considered that knowledge of the epididymal proteome in an early mammal would be informative in assessing the convergence and ergence of proteins that are important in the function of the mammalian epididymis. Few of the epididymal proteins that have been identified in eutherian mammals were found in platypus caudal epididymal fluid, and the major epididymal proteins in the platypus (PXN-FBPL, SPARC and E-OR20) have never been identified in the epididymis of any other mammal.

The function of chaperone proteins in the assemblage of protein complexes involved in gamete adhesion and fusion processes

Publisher: Bioscientifica

Date: 02-2013

DOI: 10.1530/REP-12-0316

Abstract: The remarkable complexity of the molecular events governing adhesion and fusion of the male and female gametes is becoming apparent. Novel research suggests that these highly specific cellular interactions are facilitated by multiprotein complexes that are delivered to and/or assembled on the surface of the gametes by molecular chaperones in preparation for sperm–egg interaction. While the activation of these molecular chaperones and the mechanisms by which they shuttle proteins to the surface of the cell remain the subject of ongoing investigation, a compelling suggestion is that these processes are augmented by dynamic membrane microdomains or lipid rafts that migrate to the apical region of the sperm head after capacitation. Preliminary studies of the oocyte plasma membrane have also revealed the presence of lipid rafts comprising several molecular chaperones, raising the possibility that similar mechanisms may be involved in the activation of maternal fusion machinery and the regulation of oocyte plasma membrane integrity. Despite these findings, the analysis of oocyte surface multiprotein complexes is currently lacking. Further analyses of the intermediary proteins that facilitate the expression of key players in sperm–egg fusion are likely to deliver important insights into this unique event, which culminates in the cytoplasmic continuity of the male and female gametes.

Heat Shock Protein A2 (HSPA2): Regulatory Roles in Germ Cell Development and Sperm Function.

Publisher: Springer International Publishing

Date: 2017

DOI: 10.1007/978-3-319-51409-3_4

Abstract: Among the numerous families of heat shock protein (HSP) that have been implicated in the regulation of reproductive system development and function, those belonging to the 70 kDa HSP family have emerged as being indispensable for male fertility. In particular, the testis-enriched heat shock 70 kDa protein 2 (HSPA2) has been shown to be critical for the progression of germ cell differentiation during spermatogenesis in the mouse model. Beyond this developmentally important window, mounting evidence has also implicated HSPA2 in the functional transformation of the human sperm cell during their ascent of the female reproductive tract. Specifically, HSPA2 appears to coordinate the remodelling of specialised sperm domains overlying the anterior region of the sperm head compatible with their principle role in oocyte recognition. The fact that levels of the HSPA2 protein in mature spermatozoa tightly correlate with the efficacy of oocyte binding highlight its utility as a powerful prognostic biomarker of male fertility. In this chapter, we consider the unique structural and biochemical characteristics of HSPA2 that enable this heat shock protein to fulfil its prominent roles in orchestrating the morphological differentiation of male germ cells during spermatogenesis as well as their functional transformation during post-testicular sperm maturation.

Reproductive biology in egg-laying mammals.

Publisher: S. Karger AG

Date: 2008

DOI: 10.1159/000143429

Abstract: The duck-billed platypus and short-beaked echidna are iconic species in Australia. Their morphology and physiology have puzzled scientists all over the world for more than 200 years. Recent genetic studies, particularly the platypus whole-genome sequencing project, have revealed the molecular basis of some of the extraordinary characteristics of monotremes. This and other works demonstrate the great value of research on our most distantly related mammalian relatives for comparative genomics and developmental biology. In this review we focus on the reproductive biology of monotremes and discuss works that unravel genes involved in lactation, testicular descent, gamete biology and fertilization, and early development. In addition we discuss works on the evolution of the complex sex chromosome system in platypus and echidna, which has also significant impact on our general understanding of mammalian sex chromosomes and sex determination.

Roles of male reproductive tract extracellular vesicles in reproduction

Publisher: Wiley

Date: 17-09-2021

DOI: 10.1111/AJI.13338

Proteomic analysis of koala (phascolarctos cinereus) spermatozoa and prostatic bodies

Publisher: Wiley

Date: 28-08-2021

DOI: 10.1002/PMIC.202100067

Abstract: The aims of this study were to investigate the proteome of koala spermatozoa and that of the prostatic bodies with which they interact during ejaculation. For this purpose, spermatozoa and prostatic bodies were fractionated from the semen of four male koalas and analysed by HPLC MS/MS. This strategy identified 744 sperm and 1297 prostatic body proteins, which were subsequently attributed to 482 and 776 unique gene products, respectively. Gene ontology curation of the sperm proteome revealed an abundance of proteins mapping to the canonical sirtuin and 14‐3‐3 signalling pathways. By contrast, protein ubiquitination and unfolded protein response pathways dominated the equivalent analysis of proteins uniquely identified in prostatic bodies. Koala sperm proteins featured an enrichment of those mapping to the functional categories of cellular compromise/inflammatory response, whilst those of the prostatic body revealed an over‐representation of molecular chaperone and stress‐related proteins. Cross‐species comparisons demonstrated that the koala sperm proteome displays greater conservation with that of eutherians (human 93%) as opposed to reptile (crocodile 39%) and avian (rooster 27%) spermatozoa. Together, this work contributes to our overall understanding of the core sperm proteome and has identified biomarkers that may contribute to the exceptional longevity of koala spermatozoa during ex vivo storage.

Rabbit epididymal secretory proteins. I. Characterization and hormonal regulation

Publisher: Oxford University Press (OUP)

Date: 07-2002

DOI: 10.1095/BIOLREPROD67.1.133

Abstract: Analyses of s les of luminal fluid from the rete testis, distal efferent ducts, and epididymal regions 2-5 and 8 revealed that 91% of the fluid leaving the testis is reabsorbed by the efferent ducts, 79% of the remainder is reabsorbed proximal to epididymal regions 4 and 5, and there is a net secretion of fluid into the duct caudally. There is a net reabsorption by the efferent ducts of 73% of the protein leaving the testis and then a net secretion along the epididymis. SDS-PAGE of the luminal fluids indicated that four new protein bands that were not present in blood appeared in the efferent ducts, 5 in epididymal regions 1-5, 6 in regions 6 and 7, and one in region 8. Two bands in s les from the efferent ducts were absent caudally, and one band present in region 7 was absent in region 8. The rates of incorporation of (35)S-methionine into minced duct in vitro varied among regions when expressed per milligram of wet weight of tissue (region 2-5 > region 7 > region 6 > region 1 > region 8 > ductuli efferentes), and orchidectomy had little effect on the rates. Incorporation into four proteins that were secreted in vitro (M(r) 38 000, 20 000, 15 000, and 13 000) was reduced or abolished by orchidectomy and restored by testosterone therapy. The secretion of three proteins (M(r) 52 000, 23 000, and 22 000) was reduced or abolished by orchidectomy and not restored by testosterone therapy. SDS-PAGE of detergent extracts of sperm indicated that five proteins were lost and nine were gained during epididymal transit. Seven of the proteins gained were about the same molecular weight as proteins secreted by the epididymis (M(r) 94 000, 52 000, 38 000, 36 000, 22 000, 20 000, and 13 000) and were analyzed using N-terminal amino acid microsequencing.

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

Proteomic profiling of crocodile spermatozoa refutes the tenet that post-testicular maturation is restricted to mammals.

Publisher: Elsevier BV

Date: 03-2018

DOI: 10.1074/MCP.RA118.000904

The 12 ^th International Symposium on Spermatology

Publisher: Medknow

Date: 2015

DOI: 10.4103/1008-682X.153852

The Future of Biomarkers in Veterinary Medicine: Emerging Approaches and Associated Challenges

Publisher: MDPI AG

Date: 26-08-2022

DOI: 10.3390/ANI12172194

Abstract: New biomarkers promise to transform veterinary practice through rapid diagnosis of diseases, effective monitoring of animal health and improved welfare and production efficiency. However, the road from biomarker discovery to translation is not always straightforward. This review focuses on molecular biomarkers under development in the veterinary field, introduces the emerging technological approaches transforming this space and the role of ‘omics platforms in novel biomarker discovery. The vast majority of veterinary biomarkers are at preliminary stages of development and not yet ready to be deployed into clinical translation. Hence, we examine the major challenges encountered in the process of biomarker development from discovery, through validation and translation to clinical practice, including the hurdles specific to veterinary practice and to each of the ‘omics platforms–transcriptomics, proteomics, lipidomics and metabolomics. Finally, recommendations are made for the planning and execution of biomarker studies with a view to assisting the success of novel biomarkers in reaching their full potential.

Suppressor of cytokine signaling 4 (SOCS4): Moderator of ovarian primordial follicle activation

Publisher: Wiley

Date: 22-12-2011

DOI: 10.1002/JCP.22837

Abstract: Mammalian ovarian primordial follicle activation and regulation is considered as one of the most important stages of folliculogenesis and as such requires exquisite control. Selection of quiescent follicles to enter the growing pool determines the rate of supply of maturing follicles over the female reproductive lifespan. To coordinate this process a range of positive and negative input signals contribute to determine follicle fate. This study demonstrates that the cytokine Leukemia Inhibitory Factor (LIF) activates the Janus Kinase 1/Signal Transducers and Activators of Transcription 3 (JAK1/STAT3) signaling pathway in pre-granulosa cells and positively regulates primordial follicle activation. Negative regulation of the JAK/STAT pathway is controlled by the suppressor of cytokine signaling 4 (SOCS4) protein, which target members of negative feedback loops, Cardiotrophin like Cytokine (CLC), Poly (rC) Binding Protein 1 (PCBP1), and Cytosolic Malate Dehydrogenase (MDH1) to suppress follicle growth and development.

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.

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.

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.

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.

Erratum to Chronic Exposure to Acrylamide Induces DNA Damage in Male Germ Cells of Mice [Toxicological Sciences, 129, 1 (2012) 135-145] doi:10.1093/toxsci/kfs178

Publisher: Oxford University Press (OUP)

Date: 22-02-2013

DOI: 10.1093/TOXSCI/KFS311

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.

Mouse spermatocytes express CYP2E1 and respond to acrylamide exposure

Publisher: Public Library of Science (PLoS)

Date: 02-05-2014

DOI: 10.1371/JOURNAL.PONE.0094904

Rabbit epididymal secretory proteins. II. Immunolocalization and sperm association of REP38

Publisher: Oxford University Press (OUP)

Date: 07-2002

DOI: 10.1095/BIOLREPROD67.1.140

Abstract: Polyclonal antibody was used to partially characterize REP38, a major rabbit epididymal secretory protein. Western blot analyses and immunohistochemistry indicated that REP38 is only expressed in regions 5 and 6 of the epididymis (corpus epididy-midis) and is localized in the supranuclear region and microvilli of the principal cells in these regions. It was not expressed in other tissues of the body. In region 8 (cauda epididymidis), REP38 was detected in the luminal border and cytoplasm of scattered principal cells, indicating that it may be reabsorbed in this region. This protein accumulated on the sperm plasma membrane downstream of region 5 and was localized predominantly over the acrosomal and postacrosomal regions of the head and the middle piece. Although tightly bound to epididymal sperm, REP38 migrated to the equatorial segment under conditions in vivo that would promote capacitation. When tested in vitro, anti-REP38 IgG reduced the percentage of ova fertilized in a concentration-dependent manner, apparently by blocking sperm-egg fusion.

Molecular and functional characterization of the rabbit epididymal secretory protein 52, REP52

Publisher: Oxford University Press (OUP)

Date: 05-2008

DOI: 10.1095/BIOLREPROD.107.065524

Abstract: As part of a systematic study of rabbit epididymal proteins involved in sperm maturation, we have identified and characterized a novel glycoprotein (rabbit epididymal secretory protein 52 [REP52]) of 52 kDa. REP52 is synthesized and secreted in a tissue-specific manner by the mid (region 6) and distal (region 7) corpus epididymidis and associates weakly with the sperm surface overlying the principal piece of the tail. Sequencing of cloned REP52 cDNA demonstrated that this protein represents a novel member of the highly conserved fibronectin type II (FN2) module protein family. The protein appears related but not homologous to ungulate seminal plasma proteins and is the first known ex le to be identified as a rabbit epididymal secretory protein. Consistent with other members of this protein family, REP52 possessed a high level of sequence identity within the FN2 module-encoding domains, but a highly variable N-terminal sequence that failed to show significant homology with published sequences. By analogy with evidence from studies of the ungulate seminal plasma proteins it is hypothesized that the tandemly arranged FN2 modules could facilitate the association of REP52 with sperm phosphatidylcholine residues on the outer leaflet of the sperm tail. It is also considered likely that these domains represent key elements for the function of this novel protein, a conclusion supported by the fact that antisera raised against the REP52 protein blocked in vitro fertilization in a concentration-dependent fashion.

Cryopreservation of saltwater crocodile (Crocodylus porosus) spermatozoa

Publisher: CSIRO Publishing

Date: 2017

DOI: 10.1071/RD16511

Abstract: The aim of the present study was to develop a protocol for the successful cryopreservation of Saltwater crocodile spermatozoa. Sperm cells were frozen above liquid nitrogen vapour in phosphate-buffered saline (PBS) containing either 0.3 M trehalose, 0.3 M raffinose or 0.3 M sucrose and compared with glycerol (0.3–2.7 M). Although the highest levels of mean post-thaw motility were observed following cryopreservation in 0.3 M trehalose (7.6%) and 0.3 M sucrose (7.3%), plasma membrane integrity (PI) was best following cryopreservation in 2.7 M glycerol (52.5%). A pilot study then assessed the cytotoxicity of glycerol and sucrose prior to cryopreservation and revealed no loss of survival when spermatozoa were diluted in 0.68 M glycerol or 0.2–0.3 M sucrose once cryoprotectants were washed out with PBS or Biggers, Whitten and Whittingham medium containing sperm capacitation agents (BWWCAP). A final study refined the combined use of permeating (0.68 or 1.35 M glycerol) and non-permeating (0.2 or 0.3 M sucrose) cryoprotectants. Spermatozoa were cryopreserved in liquid nitrogen vapour at rates of approximately −21°C min−1 (fast freeze) or −6.0°C min−1 (slow freeze). Post-thaw survival was highest with a combination of 0.2 M sucrose and 0.68 M glycerol and when these cryoprotectants were washed out with BWWCAP, regardless of whether spermatozoa were frozen using a fast (motility 14.2 ± 4.7% PI 20.7 ± 2.0%) or slow (motility 12.0 ± 2.7% PI 22 ± 4%) cryopreservation rate.

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.

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.

The abundance of a transfer RNA-derived RNA fragment small RNA subpopulation is enriched in cauda spermatozoa

Publisher: AME Publishing Company

Date: 11-2020

DOI: 10.1186/S41544-020-00058-X

Abstract: The small RNA (sRNA) landscape of mammalian spermatozoa is considerably altered as these gametic cells migrate through the segment specific microenvironments of the epididymis. More specifically, the microRNA (miRNA) species of sRNA dominates the sRNA landscape of spermatozoa of the proximal caput segment of the epididymis. However, in sperm cells sourced from the distal cauda epididymal segment, the transfer RNA (tRNA)-derived RNA fragment (tRF) sRNA species is the most abundant. Here we show that the 5′ halves of fifteen mature tRNAs were used as processing substrates for the production of a specific subpopulation of tRF sRNAs, 30 to 33 nucleotides (30–33-nt) in length. A quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) approach was used to experimentally validate the sRNA sequencing identified trend of enriched abundance of this specific 30–33-nt tRF subpopulation in cauda spermatozoa. The length, and exclusive alignment of the cauda spermatozoa enriched tRF subpopulation to the 5′ half of each processed tRNA precursor, identified ANGIOGENIN (ANG) as the endonuclease likely responsible for tRF production in the mouse epididymis: a prediction confirmed via immunoblotting assessment of ANG abundance in spermatozoa sourced from the caput, corpus and cauda epididymal segments. When taken together with our previous profiling of miRNA and Piwi-interacting RNA (piRNA) sRNA abundance in spermatozoa sourced from the three segments of physiologically normal mouse epididymides, the tRF profile reported here adds greater depth of coverage to the global sRNA landscape of the mouse epididymis a roadmap constructed to assist with the future molecular characterization of sRNA-directed responses to a wide range of imposed environmental stressors.

Rabbit Epididymal Secretory Proteins. III. Molecular Cloning and Characterization of the Complementary DNA for REP381

Publisher: Oxford University Press (OUP)

Date: 07-2002

DOI: 10.1095/BIOLREPROD67.1.147

Abstract: REP38 is a rabbit epididymal secretory protein of 38 kDa that has recently been shown to interact with spermatozoa. A rabbit epididymal cDNA expression library was screened with a polyclonal antibody raised against REP38. A single clone (REP38-c1) with an open reading frame encoding a polypeptide of 666 amino acids was obtained. Cleavage of a 22-amino acid N-terminal signal peptide revealed a mature protein with a theoretical molecular mass of 74.5 kDa. Northern blot analysis revealed the presence of two cross-hybridizing transcripts of approximately 1.3 and 2.5 kilobases that appear to result from alternative mRNA splicing. This finding may explain the discrepancies between the observed (38 kDa) and deduced molecular mass of REP38. Expression of both transcripts was epididymis specific and was detected only in regions 2-6. During development, the expression of REP38-c1 mRNA was initiated between 1 and 2 mo postnatum and therefore precedes the appearance of sperm within the lumen of the epididymis. These findings are in agreement with the immunohistochemical localization of the REP38 protein. Androgen deprivation induced by orchidectomy reduced REP38-c1 mRNA levels below the limit of detection, an effect that was reversed by administration of exogenous testosterone. Although REP38-c1 mRNA was detected only in the rabbit epididymis, database searches indicated homology with two rat testis specific cDNAs, KTT4 and odf2, which encode sperm outer dense fiber proteins.

Supplementary Data from ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma

Publisher: American Association for Cancer Research (AACR)

Date: 14-07-2023

DOI: 10.1158/0008-5472.23683824.V1

Abstract: All Supplementary Tables

Reactive Oxygen Species in Acute Lymphoblastic Leukaemia: Reducing Radicals to Refine Responses

Publisher: MDPI AG

Date: 14-10-2021

DOI: 10.3390/ANTIOX10101616

Abstract: Acute lymphoblastic leukaemia (ALL) is the most common cancer diagnosed in children and adolescents. Approximately 70% of patients survive -years following diagnosis, however, for those that fail upfront therapies, survival is poor. Reactive oxygen species (ROS) are elevated in a range of cancers and are emerging as significant contributors to the leukaemogenesis of ALL. ROS modulate the function of signalling proteins through oxidation of cysteine residues, as well as promote genomic instability by damaging DNA, to promote chemotherapy resistance. Current therapeutic approaches exploit the pro-oxidant intracellular environment of malignant B and T lymphoblasts to cause irreversible DNA damage and cell death, however these strategies impact normal haematopoiesis and lead to long lasting side-effects. Therapies suppressing ROS production, especially those targeting ROS producing enzymes such as the NADPH oxidases (NOXs), are emerging alternatives to treat cancers and may be exploited to improve the ALL treatment. Here, we discuss the roles that ROS play in normal haematopoiesis and in ALL. We explore the molecular mechanisms underpinning overproduction of ROS in ALL, and their roles in disease progression and drug resistance. Finally, we examine strategies to target ROS production, with a specific focus on the NOX enzymes, to improve the treatment of ALL.

Analysis of the small non-protein-coding RNA profile of mouse spermatozoa reveals specific enrichment of piRNAs within mature spermatozoa

Publisher: Informa UK Limited

Date: 21-09-2017

DOI: 10.1080/15476286.2017.1356569

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

Data from ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma

Publisher: American Association for Cancer Research (AACR)

Date: 14-07-2023

DOI: 10.1158/0008-5472.C.6651055

Abstract: Abstract Diffuse midline gliomas (DMG), including diffuse intrinsic pontine gliomas (DIPG), are the most lethal of childhood cancers. Palliative radiotherapy is the only established treatment, with median patient survival of 9 to 11 months. ONC201 is a DRD2 antagonist and ClpP agonist that has shown preclinical and emerging clinical efficacy in DMG. However, further work is needed to identify the mechanisms of response of DIPGs to ONC201 treatment and to determine whether recurring genomic features influence response. Using a systems-biological approach, we showed that ONC201 elicits potent agonism of the mitochondrial protease ClpP to drive proteolysis of electron transport chain and tricarboxylic acid cycle proteins. DIPGs harboring i PIK3CA /i mutations showed increased sensitivity to ONC201, whereas those harboring i TP53 /i mutations were more resistant. Metabolic adaptation and reduced sensitivity to ONC201 was promoted by redox-activated PI3K/Akt signaling, which could be counteracted using the brain penetrant PI3K/Akt inhibitor, paxalisib. Together, these discoveries coupled with the powerful anti-DIPG/DMG pharmacokinetic and pharmacodynamic properties of ONC201 and paxalisib have provided the rationale for the ongoing DIPG/DMG phase II combination clinical trial NCT05009992. Significance: PI3K/Akt signaling promotes metabolic adaptation to ONC201-mediated disruption of mitochondrial energy homeostasis in diffuse intrinsic pontine glioma, highlighting the utility of a combination treatment strategy using ONC201 and the PI3K/Akt inhibitor paxalisib. /

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.

Gross and microanatomy of the male reproductive duct system of the saltwater crocodile Crocodylus porosus

Publisher: CSIRO Publishing

Date: 22-03-2021

DOI: 10.1071/RD20303

Abstract: Information on the morphology and histology of the male reproductive system of the Crocodylia species is necessary to determine the role of these tissues in the production of functional spermatozoa. Accordingly, in this study we examined the gross morphology and microanatomy of the testis and the male excurrent duct system through which spermatozoa pass before ejaculation. The data demonstrate that the reproductive system in male saltwater crocodiles comprises paired testes, which convey spermatozoa distally via the rete testis into an excurrent duct system comprising ductuli efferentes, ductuli epididymides, ductus epididymidis and ductus deferens. The epithelium delineating the male tract was dominated by non-ciliated and ciliated cells structured into a simple columnar lining of the ductuli efferentes and ductuli epididymides, through to the high pseudostratified columnar epithelium of the ductus epididymidis and ductus deferens. The morphology and histochemical staining of these ducts suggest their involvement in seminal fluid production and/or its modification, which likely contributes to the nourishment, protection and/or storage of crocodile spermatozoa. As a reflection of their common Archosaurs ancestry, the overall structural characteristics we describe for the crocodile male excurrent duct system share closer similarities to those of the Aves than other clades within the Reptilia class or Mammalia.

Understanding the Villain: DMBA-Induced Preantral Ovotoxicity Involves Selective Follicular Destruction and Primordial Follicle Activation through PI3K/Akt and mTOR Signaling

Publisher: Oxford University Press (OUP)

Date: 23-07-2011

DOI: 10.1093/TOXSCI/KFR195

Abstract: 7,12-Dimethylbenz-[a]anthracene (DMBA) is an environmental carcinogen which has a potent ovotoxic affect on rat and mouse ovaries, causing complete follicular depletion resulting in premature ovarian failure. Although the overall effects of DMBA on ovarian folliculogenesis are well known, little is known about the exact molecular mechanisms behind its ovotoxicity. In this study, we characterized the mechanisms behind DMBA-induced ovotoxicity in immature follicles. Microarray analysis of neonatal mouse ovaries exposed to DMBA in vitro revealed a multilayered mechanism of DMBA-induced neonatal ovotoxicity involving a distinct cohort of genes and ovarian signaling pathways primarily associated with follicular atresia, tumorigenesis, and follicular growth. Histomorphological and immunohistological analysis supported the microarray data, showing evidence of primordial follicle activation and preantral follicle atresia both in vitro and in vivo. Further immunohistological analysis identified increased Akt1 phosphorylation, mTOR activation, and decreased FOXO3a expression in DMBA-treated primordial oocytes. Our results reveal a novel mechanism of DMBA-induced preantral ovotoxicity involving selective immature follicle destruction and primordial follicle activation involving downstream members of the PI3K/Akt and mTOR signaling pathways.

Examination of the Immunocontraceptive Potential of Recombinant Rabbit Fertilin Subunits in Rabbit

Publisher: Oxford University Press (OUP)

Date: 10-1997

DOI: 10.1095/BIOLREPROD57.4.879

Abstract: Recombinant fertilin subunits produced in a bacterial expression systems were used to test fertilin as an immunocontraceptive antigen in the European rabbit (Oryctolagus cuniculus). Wild female rabbits (n = 40) were immunized with either recombinant rabbit fertilin alpha or beta subunits by the s.c. or intra-Peyer's patch route. High titers of serum anti-fertilin polyclonal IgG antibodies were achieved in all rabbits after repeated boosts, with fertilin-specific IgG but not IgA antibodies detected in vaginal lavages of all animals. The serum IgG antibodies recognized polypeptides in detergent extracts of rabbit sperm with relative molecular masses of 48, 53, and 85 kDa on reducing SDS-PAGE gels and were shown to bind to the head region of methanol-fixed and live caudal rabbit sperm. Preincubation of rabbit sperm with these anti-fertilin IgG antibodies at concentrations of 400 micrograms/ml blocked sperm binding to zona-intact oocytes and inhibited fertilization in vitro by 60-80%. However, despite the levels of circulating and vaginal IgG antibodies achieved, only 4 immunized does failed to become pregnant out of 33 that ovulated. The remaining animals either showed no effect on fertility (n = 29) relative to control animals or failed to ovulate (n = 7). All control animals ovulated and were either fully fertile (n = 15) or were mated to infertile males (n = 4). In addition, proven-fertile male domestic rabbits (n = 3) were immunized s.c. and boosted three times with fertilin beta. Only one animal subsequently showed impaired fertility. These results show that in the rabbit, high levels of circulating sperm-reactive anti-fertilin antibodies and the presence of vaginal IgG does not ensure infertility.

A novel role for milk fat globule-EGF factor 8 protein (MFGE8) in the mediation of mouse sperm–extracellular vesicle interactions

Publisher: Wiley

Date: 09-05-2021

DOI: 10.1002/PMIC.202000079

Abstract: Spermatozoa transition to functional maturity as they are conveyed through the epididymis, a highly specialized region of the male excurrent duct system. Owing to their transcriptionally and translationally inert state, this transformation into fertilization competent cells is driven by complex mechanisms of intercellular communication with the secretory epithelium that delineates the epididymal tubule. Chief among these mechanisms are the release of extracellular vesicles (EV), which have been implicated in the exchange of varied macromolecular cargo with spermatozoa. Here, we describe the optimization of a tractable cell culture model to study the mechanistic basis of sperm–extracellular vesicle interactions. In tandem with receptor inhibition strategies, our data demonstrate the importance of milk fat globule‐EGF factor 8 (MFGE8) protein in mediating the efficient exchange of macromolecular EV cargo with mouse spermatozoa with the MFGE8 integrin‐binding Arg‐Gly‐Asp (RGD) tripeptide motif identified as being of particular importance. Specifically, complementary strategies involving MFGE8 RGD domain ablation, competitive RGD‐peptide inhibition and antibody‐masking of alpha V integrin receptors, all significantly inhibited the uptake and redistribution of EV‐delivered proteins into immature mouse spermatozoa. These collective data implicate the MFGE8 ligand and its cognate integrin receptor in the mediation of the EV interactions that underpin sperm maturation.

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.

Reproduction in Monotremes

Publisher: Elsevier

Date: 2018

DOI: 10.1016/B978-0-12-809633-8.20606-7

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.

Characterization of a novel role for the dynamin mechanoenzymes in the regulation of human sperm acrosomal exocytosis

Publisher: Oxford University Press (OUP)

Date: 30-08-2017

DOI: 10.1093/MOLEHR/GAX044

Abstract: Does dynamin regulate human sperm acrosomal exocytosis? Our studies of dynamin localization and function have implicated this family of mechanoenzymes in the regulation of progesterone-induced acrosomal exocytosis in human spermatozoa. Completion of an acrosome reaction is a prerequisite for successful fertilization in all studied mammalian species. It follows that failure to complete this unique exocytotic event represents a common aetiology in the defective spermatozoa of male infertility patients that have failed IVF in a clinical setting. Recent studies have implicated the dynamin family of mechanoenzymes as important regulators of the acrosome reaction in murine spermatozoa. The biological basis of this activity appears to rest with the ability of dynamin to polymerize around newly formed membrane vesicles and subsequently regulate the rate of fusion pore expansion. To date, however, the dynamin family of GTPases have not been studied in the spermatozoa of non-rodent species. Here, we have sought to examine the presence and functional significance of dynamin in human spermatozoa. Dynamin expression was characterized in the testis and spermatozoa of several healthy normozoospermic in iduals. In addition, we assessed the influence of selective dynamin inhibition on the competence of human spermatozoa to undergo a progesterone-induced acrosome reaction. A minimum of five biological and technical replicates were performed to investigate both inter- and intra-donor variability in dynamin expression and establish statistical significance in terms of the impact of dynamin inhibition. The expression and the localization of dynamin in the human testis, epididymis and mature spermatozoa were determined through the application of immunofluorescence, immunoblotting and/or electron microscopy. Human semen s les were fractionated via density gradient centrifugation and the resultant populations of good and poor quality spermatozoa were induced to capacitate and acrosome react in the presence or absence of selective dynamin inhibitors. The acrosome integrity of live spermatozoa was subsequently assessed via the use of fluorescently conjugated Arachis hypogea lectin (PNA). The influence of dynamin phosphorylation and the regulatory kinase(s) responsible for this modification in human spermatozoa were also assessed via the use of in situ proximity ligation assays and pharmacological inhibition. In all experiments, ≥100 spermatozoa were assessed/treatment group and all graphical data are presented as the mean values ± SEM, with statistical significance being determined by ANOVA. Dynamin 1 (DNM1) and DNM2, but not DNM3, were specifically localized to the acrosomal region of the head of human spermatozoa, an ideal position from which to regulate acrosomal exocytosis. In keeping with this notion, pharmacological inhibition of DNM1 and DNM2 was able to significantly suppress the rates of acrosomal exocytosis stimulated by progesterone. Furthermore, our comparison of dynamin expression in good and poor quality spermatozoa recovered from the same ejaculate, revealed a significant reduction in the amount of DNM2 in the latter subpopulation of cells. In contrast, DNM1 was detected at equivalent levels in both subpopulations of spermatozoa. Such findings are of potential significance given that the poor quality spermatozoa proved refractory to the induction of a progesterone stimulated acrosome reaction. In seeking to identify the regulatory influence of progesterone on DNM2 function, we were able to establish that the protein is a substrate for CDK1-dependent phosphorylation. The functional significance of DNM2 phosphorylation was illustrated by the fact that pharmacological inhibition of CDK1 elicited a concomitant suppression of both DNM2-Ser764 phosphorylation and the overall rates of progesterone-induced acrosomal exocytosis. N/A. This was an in vitro study performed mainly on ejaculated human spermatozoa. This experimental paradigm necessarily eliminates the physiological contributions of the female reproductive tract that would normally support capacitation and acrosomal responsiveness. This study identifies a novel causative link between dynamin activity and the ability of human spermatozoa to complete a progesterone-induced acrosome reaction. Such findings encourage a more detailed analysis of the contribution of dynamin dysregulation as an underlying aetiology in infertile males whose spermatozoa are unable to penetrate the zona pellucida. This research was supported by a National Health and Medical Research Council of Australia Project Grant (APP1103176) awarded to B.N. and E.A.M. The authors report no conflict of interest.

Immortalized mouse caput epididymal epithelial (mECap18) cell line recapitulates the in‐vivo environment

Publisher: Wiley

Date: 27-09-2023

DOI: 10.1002/PMIC.202300253

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.

Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection of Colorectal Cancer

Publisher: IntechOpen

Date: 06-02-2019

DOI: 10.5772/INTECHOPEN.80942

PI3K/mTOR is a therapeutically targetable genetic dependency in diffuse intrinsic pontine glioma

Publisher: Cold Spring Harbor Laboratory

Date: 17-04-2023

DOI: 10.1101/2023.04.17.537256

Abstract: Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma – DIPG), are uniformly fatal brain tumors that lack effective pharmacological treatment. Analysis of pooled CRISPR-Cas9 loss-of-function gene deletion screen datasets, identified PIK3CA and MTOR as targetable molecular dependencies across DIPG patient derived models, highlighting the therapeutic potential of the blood-brain barrier penetrant PI3K/Akt/mTOR inhibitor paxalisib. At the human equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic feedback resulting in increased blood glucose and insulin levels, commensurate with DIPG patients in Phase 1b clinical trials who experienced hyperglycemia/hyperinsulinemia. To exploit genetic dependences, but maintain compliance and benefit, we optimized a paxalisib treatment regimen that employed reduced dosing more frequently, in combination with the anti-hyperglycemic drug, metformin. Combining optimized dosing with metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo , a common mechanism of PI3K-inhibitor resistance, extending the survival of DIPG xenograft models. RNA sequencing and phosphoproteomic profiling of DIPG models treated with paxalisib identified increased calcium-activated PKC signaling. Using the brain penetrant PKC inhibitor, enzastaurin in combination with paxalisib, we synergistically extended the survival of orthotopic xenograft models, benefits further promoted by metformin thus, identifying a clinically relevant DIPG combinatorial approach. Diffuse intrinsic pontine glioma is a lethal childhood brain tumor. Here we identify PIK3CA as a genetic dependency targeted by the brain penetrant pan-PI3K-inhibitor paxalisib.

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.

Supplementary Data from ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma

Publisher: American Association for Cancer Research (AACR)

Date: 14-07-2023

DOI: 10.1158/0008-5472.23683827.V1

Abstract: All Supplementary Figures and their captions.

Adding Insult to Injury: Effects of Xenobiotic-Induced Preantral Ovotoxicity on Ovarian Development and Oocyte Fusibility

Publisher: Oxford University Press (OUP)

Date: 09-09-2010

DOI: 10.1093/TOXSCI/KFQ272

Abstract: Mammalian females are born with a finite number of nonrenewing primordial follicles, the majority of which remain in a quiescent state for many years. Because of their nonrenewing nature, these "resting" oocytes are particularly vulnerable to xenobiotic insult, resulting in premature ovarian senescence and the formation of dysfunctional oocytes. In this study, we characterized the mechanisms of ovotoxicity for three ovotoxic agents, 4-vinylcyclohexene diepoxide (VCD), methoxychlor (MXC), and menadione (MEN), all of which target immature follicles. Microarray analysis of neonatal mouse ovaries exposed to these xenobiotics in vitro revealed a more than twofold significant difference in transcript expression (p < 0.05) for a number of genes associated with apoptotic cell death and primordial follicle activation. Histomorphological and immunohistological analysis supported the microarray data, showing signs of primordial follicle activation and preantral follicle atresia both in vitro and in vivo. Sperm-oocyte fusion assays on oocytes obtained from adult Swiss mice treated neonatally revealed severely reduced sperm-egg binding and fusion in a dose-dependent manner for all the xenobiotic treatments. Additionally, lipid peroxidation analysis on xenobiotic-cultured oocytes indicated a dose-dependent increase in oocyte lipid peroxidation for all three xenobiotics in vitro. Our results reveal a novel mechanism of preantral ovotoxicity involving the homeostatic recruitment of primordial follicles to maintain the pool of developing follicles destroyed by xenobiotic exposure and to our knowledge provide the first documented evidence of short-term, low- and high-dose (VCD 40-80 mg/kg/day, MXC 50-100 mg/kg/day, MEN 7.5-15 mg/kg/day) neonatal exposure to xenobiotics causing long-term reactive oxygen species-induced oocyte dysfunction.

Plasma and acrosomal membrane lipid content of saltwater crocodile spermatozoa

Publisher: CSIRO Publishing

Date: 04-05-2021

DOI: 10.1071/RD21007

Abstract: This study describes the chemical lipid composition of the sperm plasma and acrosomal membranes of the saltwater crocodile Crocodylus porosus with the aim of providing new insights into sperm physiology, particularly that associated with their preservation ex vivo. The specific fatty acid composition of the sperm plasma and acrosomal membranes is documented. The mean (± s.d.) ratio of unsaturated to saturated membrane fatty acids within the plasma membrane was 2.57 ± 0.50, and was determined to be higher than a similar analysis of the lipids found in the acrosomal membrane (0.70 ± 0.10). The saltwater crocodile sperm plasma membrane also contained remarkably high levels of cholesterol (mean (± s.d.) 40.7 ± 4.5 nmol per 106 sperm cells) compared with the spermatozoa of other amniote species that have so far been documented. We suggest that this high cholesterol content could be conferring stability to the crocodile sperm membrane, allowing it to tolerate extreme osmotic fluxes and rapid changes in temperature. Our descriptive analysis now provides those interested in reptile and comparative sperm physiology an improved baseline database for interpreting biochemical changes associated with preservation pathology (e.g. cold shock and cryoinjury), epididymal sperm maturation and capacitation/acrosome reaction.

Supplementary Data from ONC201 in Combination with Paxalisib for the Treatment of H3K27-Altered Diffuse Midline Glioma

Publisher: American Association for Cancer Research (AACR)

Date: 17-05-2023

DOI: 10.1158/0008-5472.22892047.V1

Abstract: All Supplementary Figures and their captions.

Testicular descent, sperm maturation and capacitation. Lessons from our most distant relatives, the monotremes

Publisher: CSIRO Publishing

Date: 2009

DOI: 10.1071/RD09081

Abstract: The present review examines whether monotremes may help to resolve three questions relating to sperm production in mammals: why the testes descend into a scrotum in most mammals, why spermatozoa are infertile when they leave the testes and require a period of maturation in the specific milieu provided by the epididymides, and why ejaculated spermatozoa cannot immediately fertilise an ovum until they undergo capacitation within the female reproductive tract. Comparisons of monotremes with other mammals indicate that there is a need for considerable work on monotremes. It is hypothesised that testicular descent should be related to epididymal differentiation. Spermatozoa and ova from both groups share many of the proteins that are thought to be involved in gamete interaction, and although epididymal sperm maturation is significant it is probably less complex in monotremes than in other mammals. However, the monotreme epididymis is unique in forming spermatozoa into bundles of 100 with greatly enhanced motility compared with in idual spermatozoa. Bundle formation involves a highly organised interaction with epididymal proteins, and the bundles persist during incubation in vitro, except in specialised medium, in which spermatozoa separate after 2–3 h incubation. It is suggested that this represents an early form of capacitation.

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

Analysis of Epididymal Protein Synthesis and Secretion

Publisher: MyJove Corporation

Date: 25-08-2018

DOI: 10.3791/58308

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.

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.

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.

Chronic exposure to acrylamide induces DNA damage in male germ cells of mice

Publisher: Oxford University Press (OUP)

Date: 18-05-2012

DOI: 10.1093/TOXSCI/KFS178

Abstract: Acrylamide is a reproductive toxicant that has been detected in foods such as potato chips and breads. The consequences of chronic exposure to acrylamide in the human diet are unknown however, rodent experiments have shown that acute acrylamide exposure in males can lead to decreased fertility and dominant lethality. One of the possible mechanisms by which acrylamide elicits these effects is thought to be related to its metabolic conversion to glycidamide, which can form DNA adducts. To determine whether chronic acrylamide exposure produces genetic damage in male germ cells in vivo, male mice were subjected to acrylamide through their drinking water. Acrylamide was administered at 0.001, 0.01, 0.1, 1, and 10 µg/ml for up to 1 year, which was equivalent to 0.0001-2 mg/kg bodyweight/day. At 1, 3, 6, 9, and 12 months, early male germ cells were assessed for DNA damage using a Comet assay modified to detect adducts and γH2A.X expression, a marker of double-strand breaks. Acrylamide treatment did not significantly affect mouse or testis weight, and no gross morphological effects were observed in the testis. However, a significant dose-dependent increase in DNA damage was observed in germ cells following 6 months of exposure in the two highest dosage groups (1 and 10 µg/ml). After 12 months of exposure, increases in damage were detected at doses as low as 0.01 µg/ml (0.001 mg/kg bodyweight/day). The results of this study are the first to demonstrate that chronic exposure to acrylamide, at doses equivalent to human exposures, generates DNA damage in male germ cells of mice.

Electrophilic aldehyde products of lipid peroxidation selectively adduct to heat shock protein 90 and arylsulfatase A in stallion spermatozoa<sup><xref ref-type=fn rid=afn2>†</xref&

Publisher: Oxford University Press (OUP)

Date: 11-01-2017

DOI: 10.1095/BIOLREPROD.116.145292

Abstract: Oxidative stress is a major determinant of mammalian sperm function stimulating lipid peroxidation cascades that culminate in the generation of potentially cytotoxic aldehydes. The aim of this study was to assess the impact of such aldehydes on the functionality of stallion spermatozoa. The impact of exposure to exogenous acrolein (ACR) and 4-hydroxynonenal (4HNE) was manifested in a highly significant dose- and time-dependent increase in mitochondrial reactive oxygen species (ROS), total cellular ROS, a decrease in sperm motility, and a time-dependent increase in lipid peroxidation. Notably, low doses of ACR and 4HNE also caused a significant decrease in zona binding. In contrast, exogenous malondialdehyde, a commonly used marker of oxidative stress, had little impact on the various sperm parameters assessed. In accounting for the negative physiological impact of ACR and 4HNE, it was noted that both aldehydes readily adducted to sperm proteins located predominantly within the head, proximal centriole, and tail. The detoxifying activity of mitochondrial aldehyde dehydrogenase 2 appeared responsible for a lack of adduction in the midpiece however, this activity was overwhelmed by 24 h of electrophilic aldehyde exposure. Sequencing of the dominant proteins targeted for ACR and 4HNE covalent modification identified heat shock protein 90 alpha (cytosolic) class A member 1 and arylsulfatase A, respectively. These collective findings may prove useful in the identification of diagnostic biomarkers of stallion fertility and resolving the mechanistic basis of sperm dysfunction in this species.

Galactosyltransferase function during mammalian fertilization

Publisher: S. Karger AG

Date: 24-11-2001

DOI: 10.1159/000016805

Abstract: Gamete recognition has been studied extensively in the mouse. In this system, it is generally believed that sperm bind to a class of O-linked oligosaccharides on the zona pellucida glycoprotein, ZP3. The best characterized sperm receptor for ZP3 is β1,4-galactosyltransferase (GalT), which functions in a lectin-like capacity by binding to N-terminal N-acetylglucosamine residues on ZP3 oligosaccharides. Multivalent oligosaccharides on ZP3, as well as synthetic polymers terminating in N-acetylglucosamine aggregate GalT, leading to activation of a heterotrimeric G protein cascade and culminating in the acrosome reaction. Following fertilization, cortical granules release N-acetylglucosaminidase, which removes the binding site for sperm GalT and facilitates the zona block to polyspermic binding. Genetic manipulation of GalT expression has confirmed its function as a ZP3 receptor. Overexpressing GalT on sperm leads to increased binding of ZP3, increased G protein activation, and precocious acrosome reactions. In contrast, sperm from mice made null for GalT by homologous recombination are refractory to ZP3, in that they are unable to bind soluble ZP3 and fail to undergo the acrosome reaction in response to zona glycoproteins. Surprisingly, GalT null sperm still bind to the zona and achieve low rates of fertilization in vitro. This then suggests that sperm-egg binding involves receptor-ligand interactions independent of GalT and ZP3. The current model suggests that GalT functions as the ZP3 receptor that is responsible for inducing the acrosome reaction, whereas initial sperm-zona binding is dictated by other sperm surface receptors. Consistent with this, at least three other zona pellucida monosaccharides have been implicated in sperm binding, and novel sperm surface glycoproteins have been suggested to function in gamete binding. A large scaffolding protein has been identified that associates with the GalT cytoplasmic domain and may be responsible for orchestrating its signal transduction capacities that lead to the acrosome reaction.

Monotremes provide a key to understanding the evolutionary significance of epididymal sperm maturation

Publisher: Wiley

Date: 25-03-2011

DOI: 10.2164/JANDROL.110.012716

Abstract: It has been widely accepted that mammalian spermatozoa are infertile when they leave the testes and require a period of maturation in both the epididymis and the female reproductive tract before acquiring the ability to fertilize an oocyte. However, the necessity for such a complex process of posttesticular sperm maturation appears to be unique to mammals because it is well established that these processes do not directly influence the fertilizing ability of the spermatozoa of birds, reptiles, and other lower vertebrates. Because of their key evolutionary position and form of reproduction, we contend that monotremes (platypus and echidna) provide a unique model for resolving why these processes are necessary. In the present review, we examine evidence that the epididymal maturation of monotreme spermatozoa is far less complex than in other mammals. However, a unique feature of the monotreme epididymis lies in its ability to promote the formation of elaborate sperm bundles that serve to greatly enhance the cells' motility. It is suggested that this intriguing cooperative strategy used by monotreme sperm represents an early form of epididymal maturation that appears to have been elaborated upon during the evolution of higher mammals, possibly as an adaptation for sperm competition.

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.

The University Of Newcastle

Location: Australia

View Organisation

ARC Future Fellowships - Grant ID: FT140101368

Start Date: 2015

End Date: 12-2018

Amount: $882,232.00

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

Discovery Projects - Grant ID: DP1092849

Start Date: 2010

End Date: 12-2013

Amount: $270,000.00

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 Infrastructure, Equipment And Facilities - Grant ID: LE100100142

Start Date: 2010

End Date: 07-2011

Amount: $500,000.00

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

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989105

Start Date: 2009

End Date: 11-2009

Amount: $495,000.00

Funder: Australian Research Council