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. Graphical Abstract 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 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 < 0.001) 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.

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Significance: 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 via 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. Recent Advances: 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. Critical Issues: 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. Future Directions: 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 via fortification of intrinsic autophagic activity.

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 samples 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. This work was supported by the National Health and Medical Research Council. Grant # APP1046346. The authors have no competing interests to declare. N/A.

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.