Elizabeth G. Bromfield

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

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.

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

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.

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

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.

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

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.

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)

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.

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

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.

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

STUDY HYPOTHESIS 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. STUDY FINDING 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. WHAT IS KNOWN ALREADY 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. STUDY DESIGN, SAMPLES/MATERIALS, METHODS 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. MAIN RESULTS AND THE ROLE OF CHANCE 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). LIMITATIONS, REASONS FOR CAUTION 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. WIDER IMPLICATIONS OF THE FINDINGS 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 individuals. LARGE SCALE DATA Large scale proteomics data can be accessed through the Proteomics Identifications Database (PRIDE). STUDY FUNDING/COMPETING INTERESTS This work was supported by the National Health and Medical Research Council. Grant # APP1046346. The authors have no competing interests to declare.

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

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.

Inhibition of arachidonate 15-lipoxygenase prevents 4-hydroxynonenal-induced protein damage in male germ cells

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. Summary Sentence Cellular oxidative stress and 4HNE generation are ameliorated through the inhibition of ALOX15 in male germ cells of the mouse.

Biochemical alterations in the oocyte in support of early embryonic development

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 divide and differentiate into the trillions of cells that comprise a new individual. Traditional paradigms hold that, beyond the initial stimulation of fluctuating calcium (Ca2+) required for oocyte activation, the fertilizing spermatozoon plays limited additional roles in the early embryo. While this model has now been drawn into question in view of the recent discovery that spermatozoa deliver developmentally important classes of small noncoding RNAs and other epigenetic modulators to oocytes during fertilization, it is nevertheless apparent that the primary responsibility for oocyte activation rests with a modest store of maternally derived proteins and mRNA accumulated during oogenesis. It is, therefore, not surprising that widespread post-translational modifications, in particular phosphorylation, hold a central role in endowing these proteins with sufficient functional diversity to initiate embryonic development. Indeed, proteins targeted for such modifications have been linked to oocyte activation, recruitment of maternal mRNAs, DNA repair and resumption of the cell cycle. This review, therefore, seeks to explore the intimate relationship between Ca2+ release and the suite of molecular modifications that sweep through the oocyte to ensure the successful union of the parental germlines and ensure embryogenic fidelity.