STUDY QUESTION
How does oxidative stress impact upon human sperm–egg interaction and in particular the formation of zona pellucida-receptor complexes on the sperm surface?
SUMMARY ANSWER
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.
WHAT IS KNOWN ALREADY
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.
STUDY DESIGN, SIZE, DURATION
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.
PARTICIPANTS/MATERIALS, SETTING, METHODS
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 samples pooled from two or more donors per replicate, with a minimum of three replicates.
MAIN RESULTS AND THE ROLE OF CHANCE
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.
LIMITATIONS, REASONS FOR CAUTION
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.
WIDER IMPLICATIONS OF THE FINDINGS
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.
STUDY FUNDING/COMPETING INTEREST(S)
This work was supported by the National Health and Medical Research Council. Grant # APP1046346. The authors have no competing interests to declare.
TRIAL REGISTRATION NUMBER
N/A.
Many aspects of the reproductive process are impacted by oxidative stress. This article summarizes the chemical nature of reactive oxygen species and their role in both the physiological regulation ...of reproductive processes and the pathophysiology of infertility.
This article lays out the fundamental principles of oxidative stress. It describes the nature of reactive oxygen species (ROS), the way in which these potentially toxic metabolites interact with cells and how they impact both cellular function and genetic integrity. The mechanisms by which ROS generation is enhanced to the point that the cells' antioxidant defence mechanisms are overwhelmed are also reviewed taking examples from both the male and female reproductive system, with a focus on gametogenesis and fertilization. The important role of external factors in exacerbating oxidative stress and impairing reproductive competence is also examined in terms of their ability to disrupt the physiological redox regulation of reproductive processes. Developing diagnostic and therapeutic strategies to cope with oxidative stress within the reproductive system will depend on the development of a deeper understanding of the nature, source, magnitude, and location of such stress in order to fashion personalized treatments that meet a given patient's clinical needs.
Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting ...our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo‐focused ion beam milling‐enabled cryo‐electron tomography to image sperm flagella from three mammalian species. We resolve in‐cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament‐bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament‐bracing structures reinforcing microtubules at the nano‐scale to accessory structures that impose micron‐scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution.
SYNOPSIS
Diverse organisms and cell types rely on evolutionarily ancient and structurally conserved motile cilia to swim through a broad range of fluid environments. Here, in situ cryo‐electron tomography uncovers structural specialisations of sperm flagella in several mammalian species.
Mammalian sperm flagella are reinforced by micron‐scale accessory structures and nano‐scale microtubule inner proteins.
The connecting piece at the base of the flagellum forms an intricate, asymmetric chamber around the sperm centrioles.
The atypical distal centriole is composed of doublet microtubules splayed out around two singlet microtubules.
Outer dense fibers are directly coupled to axonemal doublet microtubules in the principal piece, but not in the midpiece.
In situ cryo‐electron tomography uncovers structural specialisations of sperm flagella in several mammalian species.
Abstract
BACKGROUND
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.
OBJECTIVE AND RATIONALE
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.
SEARCH METHODS
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.
OUTCOMES
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.
WIDER IMPLICATIONS
The present review highlights the vulnerability of the oocyte to DNA damage and presents a number of opportunities for research to bolster the stringency of the oocyte’s endogenous defences, with implications extending to improved diagnostics and novel therapeutic applications to alleviate the burden of infertility.
The precipitous age-related decline in female fertility is intimately associated with a reduction in both the quantity and quality of the germline (oocytes). Although complex etiologies undoubtedly ...contribute to the deterioration of oocyte quality, increasing attention has focused on the pervasive impact of oxidative stress. Indeed, the prolonged lifespan of the meiotically arrested oocyte places this cell at heightened risk of oxidative lesions, which commonly manifest in dysregulation of protein homeostasis (proteostasis). Although oocytes are able to mitigate this threat
the mobilization of a sophisticated network of surveillance, repair, and proteolytic pathways, these defenses are themselves prone to age-related defects, reducing their capacity to eliminate oxidatively damaged proteins.
Here, we give consideration to the quality control mechanisms identified within the ovary that afford protection to the female germline. Our primary focus is to review recent advances in our understanding of the autophagy pathway and its contribution to promoting oocyte longevity and modulating pathophysiological responses to oxidative stress. In addition, we explore the therapeutic potential of emerging strategies to fortify autophagic activity.
The complex interplay of oxidative stress and autophagy has yet to be fully elucidated within the context of the aging oocyte and surrounding ovarian environment.
Emerging evidence provides a strong impetus to resolve the causal link between autophagy and oxidative stress-driven pathologies in the aging oocyte. Such research may ultimately inform novel therapeutic strategies to combat the age-related loss of female fertility
fortification of intrinsic autophagic activity.
An increase in oxidative protein damage is a leading contributor to the age-associated decline in oocyte quality. By removing such damaged proteins, the proteasome plays an essential role in ...maintaining the fidelity of oocyte meiosis. In this study, we established that decreased proteasome activity in naturally aged, germinal vesicle (GV) mouse oocytes positively correlates with increased protein modification by the lipid aldehyde 4-hydroxynonenal (4-HNE). Furthermore, attenuation of proteasome activity in GV oocytes of young animals was accompanied by an increase in 4-HNE–modified proteins, including α-tubulin, thereby contributing to a reduction in tubulin polymerization, microtubule stability, and integrity of oocyte meiosis. A decrease in proteasome activity was also recapitulated in the GV oocytes of young animals following exposure to oxidative insults in the form of either hydrogen peroxide (H2O2) or 4-HNE. We also observed that upon oxidative insult, 4-HNE exhibits elevated adduction to multiple proteasomal subunits. Notably, the inclusion of the antioxidant penicillamine, to limit propagation of oxidative stress cascades, led to a complete recovery of proteasome activity and enhanced clearance of 4-HNE–adducted α-tubulin during a 6-h post-treatment recovery period. This strategy also proved effective in reducing the incidence of oxidative stress–induced aneuploidy following in vitro oocyte maturation, but was ineffective for naturally aged oocytes. Taken together, our results implicate proteasome dysfunction as an important factor in the accumulation of oxidatively induced protein damage in the female germline. This discovery holds promise for the design of therapeutic interventions to address the age-dependent decline in oocyte quality.
The proteomic composition of extracellular vesicles (epididymosomes) secreted by the mouse epididymis has been determined by applying multiplexed tandem mass tag based quantification coupled with ...high resolution LC-MS/MS. This analysis confirmed that epididymosomes encapsulate an extremely rich and diverse proteomic cargo, which is commensurate with their putative role in coordinating the post-testicular maturation and storage of spermatozoa.
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Highlights
•Comparative proteomics of extracellular vesicles isolated from the mouse epididymis.•Epididymosome proteome displays pronounced segment-to-segment variation.•Epididymosomes deliver protein cargo to the sperm head.•Mechanistic insights into role of epididymosomes in sperm maturation and storage.
The functional maturation of spermatozoa that is necessary to achieve fertilization occurs as these cells transit through the epididymis, a highly specialized region of the male reproductive tract. A defining feature of this maturation process is that it occurs in the complete absence of nuclear gene transcription or de novo, protein translation in the spermatozoa. Rather, it is driven by sequential interactions between spermatozoa and the complex external milieu in which they are bathed within lumen of the epididymal tubule. A feature of this dynamic microenvironment are epididymosomes, small membrane encapsulated vesicles that are secreted from the epididymal soma. Herein, we report comparative proteomic profiling of epididymosomes isolated from different segments of the mouse epididymis using multiplexed tandem mass tag (TMT) based quantification coupled with high resolution LC-MS/MS. A total of 1640 epididymosome proteins were identified and quantified via this proteomic method. Notably, this analysis revealed pronounced segment-to-segment variation in the encapsulated epididymosome proteome. Thus, 146 proteins were identified as being differentially accumulated between caput and corpus epididymosomes, and a further 344 were differentially accumulated between corpus and cauda epididymosomes (i.e., fold change of ≤ −1.5 or ≥ 1.5; p, < 0.05). Application of gene ontology annotation revealed a substantial portion of the epididymosome proteins mapped to the cellular component of extracellular exosome and to the biological processes of transport, oxidation-reduction, and metabolism. Additional annotation of the subset of epididymosome proteins that have not previously been identified in exosomes revealed enrichment of categories associated with the acquisition of sperm function (e.g., fertilization and binding to the zona pellucida). In tandem with our demonstration that epididymosomes are able to convey protein cargo to the head of maturing spermatozoa, these data emphasize the fundamental importance of epididymosomes as key elements of the epididymal microenvironment responsible for coordinating post-testicular sperm maturation.
One of the most common lesions present in the spermatozoa of human infertility patients is an idiopathic failure of sperm-egg recognition. Although this unique cellular interaction can now be readily ...by-passed by assisted reproductive strategies such as intracytoplasmic sperm injection (ICSI), recent large-scale epidemiological studies have encouraged the cautious use of this technology and highlighted the need for further research into the mechanisms responsible for defective sperm-egg recognition. Previous work in this field has established that the sperm domains responsible for oocyte interaction are formed during spermatogenesis prior to being dynamically modified during epididymal maturation and capacitation in female reproductive tract. While the factors responsible for the regulation of these sequential maturational events are undoubtedly complex, emerging research has identified the molecular chaperone, heat shock protein A2 (HSPA2), as a key regulator of these events in human spermatozoa. HSPA2 is a testis-enriched member of the 70 kDa heat shock protein family that promotes the folding, transport, and assembly of protein complexes and has been positively correlated with in vitro fertilization (IVF) success. Furthermore, reduced expression of HSPA2 from the human sperm proteome leads to an impaired capacity for cumulus matrix dispersal, sperm-egg recognition and fertilization following both IVF and ICSI. In this review, we consider the evidence supporting the role of HSPA2 in sperm function and explore the potential mechanisms by which it is depleted in the spermatozoa of infertile patients. Such information offers novel insights into the molecular mechanisms governing sperm function.
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 reproductive consequences of global warming are not currently understood. In order to address this issue, we have examined the reproductive consequences of exposingmalemice to a mild heat stress. ...For this purpose, adultmalemice 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 DNAdamage (P<0.05), characteristic of an oxidative stress cascade. This DNA damage was detected in pachytene spermatocytes (P < 0.001) and round spermatids (P < 0.001) isolated from testes after 1 day heat recovery. Despite these lesions, the spermatozoa of heat-treated mice exhibited no differences in their ability to achieve hallmarks of capacitation or to fertilize the oocyte and support development of embryos to the blastocyst stage (all 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. Summary Sentence Environmental heating induces a state of oxidative stress in the male germ line, affecting multiple germ cell types; primarily the round spermatid and pachytene spermatocyte populations.
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