Reorganization of mouse sperm lipid rafts by capacitation

One of the hallmarks of mammalian sperm capacitation is the loss of cholesterol from the plasma membrane. Cholesterol has been associated with the formation of detergent insoluble membrane microdomains in many cell types, and sperm from several mammalian species have been shown to contain detergent-resistant membranes (DRMs). The change in cholesterol composition of the sperm plasma membrane during capacitation raises the question of whether the contents of DRMs are altered during this process. In this study, we investigated changes in protein composition of DRMs isolated from uncapacitated or capacitated mouse sperm. TX-100 insoluble membranes were fractionated by sucrose flotation gradient centrifugation and analyzed by Western and lectin blotting, and capacitation-related differences in protein composition were identified. Following capacitation, the detergent insoluble fractions moved to lighter positions on the sucrose gradients, reflecting a global change in density or composition. We identified several individual proteins that either became enriched or depleted in DRM fractions following capacitation. These data suggest that the physiological changes in sperm motility, ability to penetrate the zona pellucida (ZP), ZP responsiveness, and other capacitation-dependent changes, may be due in part to a functional reorganization of plasma membrane microdomains.     

Proteomic and functional analysis of human sperm detergent resistant membranes

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.     

Nitric oxide synthase inhibition in human sperm affects sperm-oocyte fusion but not zona pellucida binding

There is recent evidence that mouse and human spermatozoa contain constitutive nitric oxide synthase (cNOS) and can synthesize nitric oxide. The aim of this study was to investigate whether the inhibition of human sperm cNOS could affect sperm-oocyte fusion and sperm binding to the zona pellucida (ZP). N(G)-nitro-L-arginine methyl ester (L-NAME) was used as cNOS inhibitor. Sperm-oocyte fusion was evaluated using the hamster egg penetration test (HEPT). The ZP binding was evaluated using the hemizona assay. L-NAME added from the onset of capacitation strongly inhibited sperm-oocyte fusion. This inhibitory effect was dose dependent, stereospecific, and suppressed by L-arginine in a dose-dependent manner. L-NAME also inhibited sperm-oocyte fusion in the HEPT enhanced with progesterone (P), where P (5 microM) was added for 15 min to capacitated sperm. A lesser but significant inhibition was also observed when sperm suspensions were exposed to L-NAME following capacitation in both versions of HEPT. On the contrary, L-NAME did not affect ZP binding. In conclusion, the present study provides the evidence that cNOS plays a role in the human sperm's capacity to fuse with oocyte but not in the ZP binding.     

Interaction of human spermatozoa with the human zona pellucida and zona-free hamster oocyte following capacitation by exposure to human cervical mucus

The functional capacity for sperm interaction with the human zona pellucida and zona-free hamster oocyte was tested after human spermatozoa were capacitated by passage through a column of human cervical mucus. The results were compared with those obtained when spermatozoa from an aliquot of the same semen sample were capacitated by the standard laboratory methods involving sequential washing by dilution and centrifugation of the semen. Washed-capacitated sperm suspensions were more successful than mucus-capacitated sperm in attaching to the zona-free hamster oocyte and in fusing with its oolemma. However, the ability of mucus-capacitated sperm to penetrate the human zona pellucida was equal to washed capacitated sperm. These experiments demonstrate an approach that may be useful in comparative studies of human sperm capacitation in vivo and in vitro.     

Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid

Sperm gain full ability to bind to the zona(e) pellucida(e) (ZP) during capacitation. Since lipid rafts are implicated in cell adhesion, we determined whether capacitated sperm lipid rafts had affinity for the ZP. We demonstrated that lipid rafts, isolated as low-density detergent resistant membranes (DRMs), from capacitated pig sperm had ability to bind to homologous ZP. This binding was dependent on pig ZPB glycoprotein, a major participant in sperm binding. Capacitated sperm DRMs were also enriched in the male germ cell specific sulfogalactosylglycerolipid (SGG), which contributed to DRMs-ZP binding. Furthermore, SGG may participate in the formation of sperm DRMs due to its interaction with cholesterol, an integral component of lipid rafts, as shown by infrared spectroscopic studies. Since sperm capacitation is associated with cholesterol efflux from the sperm membrane, we questioned whether the formation of DRMs was compromised in capacitated sperm. Our studies indeed revealed that capacitation induced increased levels of sperm DRMs, with an enhanced ZP affinity. These results corroborated the implication of lipid rafts and SGG in cell adhesion and strongly suggested that the enhanced ZP binding ability of capacitated sperm may be attributed to increased levels and a greater ZP affinity of lipid rafts in the sperm plasma membrane.     

Identification of the molecular chaperone, heat shock protein 1 (chaperonin 10), in the reproductive tract and in capacitating spermatozoa in the male mouse

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.     

Surface protein changes in goat spermatozoa during capacitation

Polypeptides of goat sperm surface before and after capacitation were examined by radiolabelling and immunologically using polyclonal antisera. Radioiodination revealed five protein bands having mol wt of 14.8, 72.4, 81, 100 and 128 kDa in uncapacitated ejaculated spermatozoa and only three bands of 23.4, 27 and 72.4 KDa in capacitated spermatozoa. The protein band with mol wt 72.4 kDa was only feebly iodinated in uncapacitated sperm surface but in capacitated spermatozoa it was heavily labelled. Western blot analysis of detergent-extracted proteins using gamma-globulin fraction of antisera raised against purified goat sperm plasma membrane revealed six antigens (17.8, 29.1, 33.4, 45.6, 85.1, 123.2 kDa) in uncapacitated spermatozoa, four (26, 32.1, 40.1, 45.6 kDa) in capacitated spermatozoa and only one (45.6 kDa) in acrosome-reacted spermatozoa. High mol wt proteins were more numerous on the surface of uncapacitated spermatozoa while the capacitated spermatozoa had relatively low mol wt proteins. An apparent effect of capacitation is the metabolism and reorganisation of proteins on goat sperm surface. Polypeptides on capacitated sperm surface revealed through radiolabelling and polyclonal antisera may have a likely receptor(s) role in the recognition and binding to homologous zona pellucida during fertilization.     

The chaperonin containing TCP1 complex (CCT/TRiC) is involved in mediating sperm-oocyte interaction

Sperm-oocyte interactions are among the most remarkable processes in cell biology. These cellular recognition events are initiated by an exquisitely specific adhesion of free-swimming spermatozoa to the zona pellucida, an acellular matrix that surrounds the ovulated oocyte. Decades of research focusing on this interaction have led to the establishment of a widely held paradigm that the zona pellucida receptor is a single molecular entity that is constitutively expressed on the sperm cell surface. In contrast, we have employed the techniques of blue native-polyacrylamide gel electrophoresis, far Western blotting, and proximity ligation to secure the first direct evidence in support of a novel hypothesis that zona binding is mediated by multimeric sperm receptor complex(es). Furthermore, we show that one such multimeric association, comprising the chaperonin-containing TCP1 complex (CCT/TRiC) and a zona-binding protein, zona pellucida-binding protein 2, is present on the surface of capacitated spermatozoa and could account for the zona binding activity of these cells. Collectively, these data provide an important biochemical insight into the molecular basis of sperm-zona pellucida interaction and a plausible explanation for how spermatozoa gain their ability to fertilize.     

Different functional states of ram spermatozoa analysed by partition in an aqueous two-phase system

The surface of spermatozoa plays a critical role in many stages involved in fertilisation. The plasma membrane undergoes important alterations in the male and female reproductive tract, which result in the ability of spermatozoa to fertilise eggs. One of these membrane modifications is sperm capacitation, a process by which sperm interacts with the zona pellucida receptors leading to the acrosome reaction. It has been proposed that the freezing process induces capacitation-like changes to spermatozoa, and that this premature capacitation could explain the reduction in longevity and fertilising capacity of cryopreserved mammalian spermatozoa. Our research focused on the relationship between membrane alterations occurring throughout freezing-thawing and the processes of capacitation and acrosome reaction. We used centrifugal countercurrent distribution (CCCD) analysis to compare the partition behaviour of ram spermatozoa that was either subjected to cold-shock or frozen-thawed with capacitated and acrosome reacted samples. In addition, the effect of the induced acrosome reaction on membrane integrity of ram spermatozoa was studied using biochemical markers and electron microscopy scanning. The CCCD analysis revealed important similarities between the surface characteristics of capacitated and cold-shocked sperm as well as between acrosome-reacted and frozen-thawed sperm. Cold-shocked and capacitated sperm showed an increased cell affinity for the lower dextran-rich phase as well as a decreased heterogeneity. Likewise, the induction of the acrosome reaction resulted in a loss of viability and an important decrease in cell surface heterogeneity compared to the untreated-control sample. Similar surface changes were found when semen samples were frozen with either Fiser or milk-yolk extender. These results confirm those obtained for membrane integrity by fluorescence markers. Thus, the high cell viability value found in the control sample (74.5%) was greatly decreased after cold-shock (22.2%), cryopreservation (26.38% Fiser medium, 24.8% milk-yolk medium) and acrosome reaction (6.6%), although it was preserved after inducing capacitation (46.7%). The study using electron microscopy scanning revealed dramatic structural alterations provoked by the induction of the acrosome reaction.     

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

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. J. Cell. Physiol. 224:71–83, 2010 © 2010 Wiley‐Liss, Inc.     

Capacitation dependent activation of tyrosine phosphorylation generates two sperm head plasma membrane proteins with high primary binding affinity for the zona pellucida

The recognition and binding of sperm cells to the zona pellucida (the extracellular matrix of the oocyte) are essential for fertilization and are believed to be species specific. Freshly ejaculated sperm cells do not bind to the zona pellucida. Physiologically this interaction is initiated after sperm activation in the female genital tract (capacitation) via a yet unknown mechanism, resulting in the binding of a receptor in the apical sperm plasma membrane to the zona pellucida. In order to mimic this biochemically, we isolated zona pellucida fragments from gilt ovaries to prepare an affinity column with the intact zona pellucida structure and loaded this column with solubilized apical plasma membranes of boar sperm cells before and after in vitro capacitation. With this technique we demonstrated that two plasma membrane proteins of capacitated boar sperm cells showed high affinity for zona pellucida fragments. Further analysis showed that these proteins were tyrosine phosphorylated. Plasma membrane proteins from freshly ejaculated sperm cells did not exhibit any zona pellucida binding proteins, likely because these proteins were not tyrosine phosphorylated.     

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

Although the molecular basis of sperm-oocyte interaction is unclear, recent studies have implicated two chaperone proteins, heat shock protein 1 (HSPD1; previously known as heat shock protein 60) and tumor rejection antigen gp96 (TRA1; previously known as endoplasmin), in the formation of a functional zona-receptor complex on the surface of mammalian spermatozoa. The current study was undertaken to investigate the expression of these chaperones during the ontogeny of male germ cells through spermatogenesis, epididymal sperm maturation, capacitation, and acrosomal exocytosis. In testicular sections, both HSPD1 and TRA1 were closely associated with the mitochondria of spermatogonia and primary spermatocytes. However, this labeling pattern disappeared from the male germ line during spermiogenesis to become undetectable in testicular spermatozoa. Subsequently, these chaperones could be detected in epididymal spermatozoa and in previously unreported "dense bodies" in the epididymal lumen. The latter appeared in the precise region of the epididymis (proximal corpus), where spermatozoa acquire the capacity to recognize and bind to the zona pellucida, implicating these structures in the functional remodeling of the sperm surface during epididymal maturation. Both HSPD1 and TRA1 were subsequently found to become coexpressed on the surface of live mouse spermatozoa following capacitation in vitro and were lost once these cells had undergone the acrosome reaction, as would be expected of cell surface molecules involved in sperm-egg interaction. These data reinforce the notion that these chaperones are intimately involved in the mechanisms by which mammalian spermatozoa both acquire and express their ability to recognize the zona pellucida.     

Localization of tyrosine phosphorylated proteins in human sperm and relation to capacitation and zona pellucida binding

Mammalian sperm must undergo a process known as capacitation before fertilization can take place. A key intracellular event that occurs during capacitation is protein tyrosine phosphorylation. The objective of this study was to investigate and visualize protein tyrosine phosphorylation patterns in human sperm during capacitation and interaction with the zona pellucida. The presence of specific patterns was also assessed in relation to the fertilizing capacity of the spermatozoa after in vitro fertilization. Protein tyrosine phosphorylation was investigated by immunofluorescence. Phosphorylation increased significantly with capacitation and was localized mainly to the principal piece of human sperm. Following binding to the zona pellucida, the percentage of sperm with phosphotyrosine residues localized to both the neck and the principal piece was significantly higher in bound sperm than in capacitated sperm in suspension. When the percentage of principal piece-positive sperm present after capacitation was <7%, fertilization rates after in vitro fertilization were reduced. Different compartments of human spermatozoa undergo a specific sequence of phosphorylation during both capacitation and upon binding to the zona pellucida. Tyrosine phosphorylation in the principal and neck piece may be considered a prerequisite for fertilization in humans.     

An investigation using lectins of glycocomponents of mouse spermatozoa during capacitation and sperm-zona binding

Ten different lectins conjugated to fluorescein isothiocyanate (FITC) were used to study the distribution of surface carbohydrates on mouse spermatozoa, and to monitor the possible changes of their distribution during capacitation in vitro and sperm-egg interaction. Most of the lectins gave a restricted pattern of binding to fixed or unfixed epididymal spermatozoa. Binding was highly specific because no staining occurred in the presence of appropriate monosaccharides. Binding of UEA I, DBA and Con A was unaffected by the type of fixative used, but it was influenced by mild centrifugation. While unwashed spermatozoa showed binding mainly over the acrosomal cap and equatorial or postacrosomal regions, spermatozoa washed by mild centrifugation showed a change in the staining of the equatorial segment. Binding of 5 different lectins to spermatozoa did not change during capacitation in vitro. In contrast, capacitated spermatozoa bound to the zona pellucida exhibited a UEA I binding pattern which was strikingly different from that of the capacitated but unbound spermatozoa. We conclude that glycocomponents of specific regions of mouse spermatozoa do not change dramatically during capacitation, but do alter significantly during binding to the zona pellucida.     

In vitro studies of the golden hamster sperm acrosome reaction: completion on the zona pellucida and induction by homologous soluble zonae pellucidae

We have studied the occurrence of the golden hamster sperm acrosome reaction (AR) in vitro during interaction with the oocyte investments: the cumulus cell matrix and the zona pellucida. Hamster sperm were capacitated in a defined medium that does not induce the AR. These spermatozoa were allowed to interact with the ovum vestments, the events of which were recorded using high-speed videomicrography. Frame-by-frame analysis revealed that sperm did not complete the AR in the cumulus cell matrix, but did so on the zona pellucida. Furthermore, a higher percentage of sperm completed the AR on the zona pellucida of cumulus-invested than on cumulus-free eggs. We also investigated the effect of solubilized hamster and mouse zonae pellucidae on the hamster sperm AR. Addition of solubilized hamster zonae to capacitated sperm elicited the AR within 15 min. Solubilized mouse zonae were significantly less effective, indicating that the zona-induced AR in hamster sperm may be species specific. These results suggest that the hamster zona pellucida is an inducer of the AR in the intact or soluble form, and that the majority of spermatozoa traverse the cumulus cell matrix without completing the AR in our in vitro system.     

Capacitation-dependent reorganization of microdomains in the apical sperm head plasma membrane: functional relationship with zona binding and the zona-induced acrosome reaction

For sperm to successfully fertilize an oocyte, it needs to pass through certain steps prior to, during and after initial recognition of the zona pellucida (ZP). During capacitation, the surface of the sperm head becomes remodelled, priming it to bind to the ZP and subsequently to undergo the ZP-induced acrosome reaction. During capacitation, sperm ZP-binding proteins are ordered in functional protein complexes that only emerge at the apical tip of the sperm head plasma membrane; this is also functionally the exclusive sperm surface area involved in primary ZP binding. After primary ZP binding, the same area is probably involved in the induction of the acrosome reaction. A combination of biochemical and proteomic membrane protein techniques have enabled us to dissect and highly purify the apical sperm plasma membrane area from control and capacitated sperm cells. The actual ZP-binding proteins identified predominantly belonged to the sperm membrane-associated family members of spermadhesins (AQN-3) and were present in the aggregating lipid ordered membrane microdomains (lipid rafts) that emerged during in vitro capacitation in the apical ridge area of the sperm head plasma membrane. This clustering of these rafts was dependent on the presence of bicarbonate (involved in protein kinase A activation) and on the presence of albumin (involved in cholesterol removal). Remarkably, cholesterol removal was restricted to the non-raft membrane fraction of the sperm plasma membrane, but did not cause any depletion of cholesterol in the raft membrane fraction. Interestingly, sperm SNARE proteins (both VAMP from the outer acrosomal membrane, as well syntaxin from the apical sperm head plasma membrane) shared lateral redistribution properties, along with the ZP-binding protein complex and raft marker proteins. All of these were recovered after capacitation in detergent-resistant membrane preparations from sperm thought to represent membrane lipid rafts. We inferred that the capacitation-dependent formation of an aggregated lipid ordered apical ridge surface area in the sperm head plasma membrane was not only relevant for ZP-binding, but also for the ZP-induced acrosome reaction.     

Mice carrying two t haplotypes: sperm populations with reduced Zona pellucida binding are deficient in capacitation.

Capacitation is the unique process by which mammalian sperm become capable of undergoing the acrosome reaction (AR). An approach to studying sperm capacitation is to identify mutations altering this process. Male mice carrying two t haplotypes are sterile, with poor sperm motility, reduced zona pellucida binding, and an inability to penetrate zona-free oocytes. The objective of this study was to examine sperm capacitation and its potential relationship to zona pellucida binding in mice of the same genetic strain carrying none, one, or two t haplotypes. Sperm capacitation was assessed by the B pattern of staining by chlortetracycline (CTC) and by the ability of sperm to undergo the lysophosphatidylcholine (LPC)-induced AR. The CTC assay demonstrated that sperm capacitation from t/+ mice was similar to that from +/+ mice, but sperm from t/t mice were deficient. LPC induced the AR of capacitated sperm, but not noncapacitated sperm, in a concentration-dependent manner. Sperm from t/t mice were also deficient in the LPC-induced AR. Thus, by two independent assays, sperm from t/t mice were shown to be deficient in capacitation. To determine whether a deficiency in capacitation could influence zona binding, the ability of capacitated versus noncapacitated sperm to bind to the zona pellucida was tested. The mean numbers of sperm bound per oocyte were significantly greater for capacitated sperm than for noncapacitated sperm. These results suggest that the deficient capacitation of sperm from t/t mice could be responsible for, or at least contribute to, their reduced ability to bind to the zona pellucida.     

Mammalian sperm acrosome: formation, contents, and function

Sperm-egg interaction is a carbohydrate-mediated species-specific event which initiates a signal transduction cascade resulting in the exocytosis of sperm acrosomal contents (i.e., the acrosome reaction). This step is believed to be a prerequisite which enables the acrosome-reacted spermatozoa to penetrate the zona pellucida (ZP) and fertilize the egg. Successful fertilization in the mouse and several other species, including man, involves several sequential steps. These are (1) sperm capacitation in the female genital tract; (2) binding of capacitated spermatozoa to the egg's extracellular coat, the ZP; (3) induction of acrosome reaction (i.e., sperm activation); (4) penetration of the ZP; and (5) fusion of spermatozoon with the egg vitelline membrane. This minireview focuses on the most important aspects of the sperm acrosome, from its formation during sperm development in the testis (spermatogenesis) to its modification in the epididymis and function following sperm-egg interaction. Special emphasis has been given to spermatogenesis, a complex process involving multiple molecular events during mitotic cell division, meiosis, and the process of spermiogenesis. The last event is the final phase when a nondividing round spermatid is transformed into the complex structure of the spermatozoon containing a well-developed acrosome. Our intention is also to briefly discuss the functional significance of the contents of the sperm acrosome during fertilization. It is important to mention that only the carbohydrate-recognizing receptor molecules (glycohydrolases, glycosyltransferases, and/or lectin-like molecules) present on the surface of capacitated spermatozoa are capable of binding to their complementary glycan chains on the ZP. The species-specific binding event starts a calcium-dependent signal transduction pathway resulting in sperm activation. The hydrolytic and proteolytic enzymes released at the site of sperm-zona interaction along with the enhanced thrust of the hyperactivated beat pattern of the bound spermatozoon, are important factors in regulating the penetration of the zona-intact egg.     

A short sperm-oocyte incubation time ZBA in the dog

The fertilising capacity of a semen sample can be predicted by evaluation of spermatozoa with in vitro tests. The zona pellucida binding assay (ZBA) accounts for several parameters and interprets the interaction between the spermatozoa and the oocyte. The present study was made in two parts. The aim of the first experiment was to evaluate whether the sperm binding capacity of oocytes varies between different oocyte pools. Each zona binding was made with oocytes from different bitches, using pooled frozen-thawed semen from the same two dogs. The sperm-oocyte complexes were incubated for 1h. There was a significant difference between the six replicates in the number of sperm bound to the zona pellucida (ZP), which indicates that the sperm binding capacity of the ZP differs between oocyte pools. The aims of the second experiment were to evaluate the effects of five different treatments of the spermatozoa on the ZBA, and to evaluate two different incubation times of the sperm-oocyte complexes. ZBAs were made with: fresh semen; semen kept chilled for 1 or 2 days prior to the ZBA; and with semen that had been frozen with or without Equex. The oocytes and spermatozoa were incubated for 1 or 4h. For fresh semen and for semen frozen without Equex, incubation for 1h resulted in a higher number of bound spermatozoa per oocyte than incubation for 4h (P<0.0001). When the effect of the different sperm treatments on the number of spermatozoa bound to the ZP was evaluated, it was found that this number was higher for fresh spermatozoa than for chilled or frozen-thawed spermatozoa both after 1 and 4h of co-incubation (P<0.0001). After 1-h incubation of the sperm-oocyte complexes, spermatozoa chilled for 1 day showed better zona binding capacity than spermatozoa chilled for 2 days, and spermatozoa frozen without Equex had a better zona binding capacity than spermatozoa frozen with Equex. Sperm motility and sperm plasma membrane integrity were higher in fresh than in chilled and frozen-thawed semen. The acrosome integrity was high in all groups of treated semen. In conclusion, 1-h incubation of the sperm-oocyte complexes seems to be sufficient for fresh and chilled semen. Further studies are required to establish the optimal incubation time for sperm-oocyte complexes when frozen-thawed semen is evaluated, as a comparison between semen frozen with Equex and semen frozen without Equex gave different results depending on whether the incubation time was 1 or 4h (in the present study), or 6h [Str?m Holst B, Larsson B, Linde-Forsberg C, Rodriguez-Martinez H. Evaluating chilled and frozen-thawed dog spermatozoa using a zona pellucida binding assay.     

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

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.