Changes in the organization of surface antigens during in-vitro capacitation of boar spermatozoa as detected by monoclonal antibodies

Monoclonal antibodies specific for three major plasma membrane (PM) proteins, previously referenced as PM protein 2.0, 4.85 and 5.0, and one specific for an unreferenced PM protein (Mr 80,000) were used with indirect fluorescence microscopy to detect the effects of capacitation on the localization of these PM proteins. In ejaculated or cauda spermatozoa, incubation in the capacitating medium caused the appearance of fluorescence in the flagellum and either a loss of fluorescence on the PM overlying the sperm head (PM proteins of 5.0 and Mr 80,000) or a delocalization of fluorescence on the head PM (PM proteins 2.0 and 4.85). Labelling spermatozoa with divalent antibody and then capacitating them indicated the PM protein 5.0 and that of Mr 80,000 migrated out of the head plasma membrane into the flagellar PM during capacitation. These antigens re-entered the head PM when fresh seminal plasma was added after the capacitation period or when energy metabolism was inhibited by azide. Cytochalasin D, an inhibitor of the polymerization of actin, prevented movement of PM protein 5.0 and that of Mr 80,000 of the head PM into the flagellum during incubation in the capacitation medium and prevented re-entry of these antigens from the flagellum into the head PM after incubation in this medium. Localization changes occurring with capacitation were time-dependent but independent of the method of preparing samples for microscopy. For the major PM proteins 4.85 and 5.0, a much smaller percentage of caput spermatozoa (approximately 20%) showed specific localization changes compared to those of the cauda (approximately 80%). Chelation of Ca2+ inhibited these changes in ejaculated spermatozoa and fresh seminal plasma, added to capacitated spermatozoa, restored the localization pattern characteristic of uncapacitated spermatozoa. These observations suggest that the organization of major proteins in the plasma membrane overlying the sperm head is altered during capacitation. These changes are reversible, are dependent on sperm maturation and also appear to involve actin filament interactions with the plasma membrane.     

Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon

Experiments to bind fluorescein-conjugated Arachis hypogea (peanut) agglutinin (FITC-PNA) to washed human spermatozoa demonstrated that this lectin binds to the acrosome region in air-dried preparations. Since there was no binding when labelling was performed in suspension, and comparable labelling to that seen in air-dried preparations was seen when spermatozoa treated with saponin (to lyse the plasma membrane) were labelled in suspension, the lectin must bind to an intracellular structure, probably the outer acrosomal membrane. This was confirmed by ultrastructural localization of colloidal gold-conjugated lectin in saponin-treated spermatozoa. Treatment of spermatozoa with the detergent Nonidet P-40 caused a marked change in the binding pattern: more spermatozoa showed binding in the equatorial segment of the acrosome with no binding in the anterior cap region. A comparable, less marked, change was seen when spermatozoa were incubated overnight under conditions known to support the capacitation and spontaneous acrosome reactions. Treatment with the calcium ionophore A23187 for 1 h to induce acrosome reactions artificially in uncapacitated spermatozoa resulted in the appearance of patchy acrosome fluorescence. From these experiments it is concluded that PNA binds specifically to the outer acrosomal membrane, and that FITC-PNA-labelling may be used to monitor the human sperm acrosome reaction.     

Restriction of a sperm surface antigen's mobility during capacitation.

Fluorescent Fab¹ [immunoglobulin G (IgG) fragment with antigen binding capacity from papain digestion of IgG] antibody fragments and globulin from antisera prepared against a single rabbit sperm surface membrane glycoprotein antigen (MGP) were used to study surface antigen mobility. Epididymal and ejaculated spermatozoa exhibited a redistribution of MGP surface antigen over the acrosomal region when labeled at 4°C and warmed to 37°C. Following in vivo capacitation, spermatozoa did not exhibit MGP surface antigen redistribution over the acrosome. The restricted mobility of this surface antigen implies a physiological change in plasma membrane fluidity, which may be a necessary preliminary to the acrosome reaction. Furthermore, it is suggested that the presence or absence of specific peripheral membrane proteins may control the positional relationship between mobile and nonmobile integral membrane components.     

Surface mapping of binding of oviductin to the plasma membrane of golden hamster spermatozoa during in vitro capacitation and acrosome reaction

Oviductins are high-molecular-weight glycoproteins synthesized and secreted by nonciliated oviductal epithelial cells and have been shown to play a role in fertilization and early embryo development. The present study was carried out to examine the in vitro binding capacity of hamster oviductin to homologous sperm and to determine the sites of its localization in untreated, capacitated, and acrosome-reacted spermatozoa. Freshly prepared epididymal and capacitated sperm as well as acrosome-reacted sperm were incubated with oviductal fluid prepared from isolated hamster oviducts, fixed and then probed with a monoclonal antibody against hamster oviductin. Results obtained with pre-embedding immunolabeling experiments revealed binding of oviductin to the acrosomal cap and the apical aspect of the postacrosomal region. Immunolabeling of both regions appeared to be more intense in capacitated spermatozoa. Acrosome-reacted sperm showed an immunoreaction of moderate intensity over the postacrosomal region. The plasma membrane overlying the equatorial segment also exhibited a weak labeling. Quantitative analysis obtained with the surface replica technique indicated that oviductin had a higher binding affinity for the acrosomal cap than the postacrosomal region and that the binding of oviductin to the latter plasma membrane domain was enhanced during capacitation. Binding of oviductin to the postacrosomal region, however, was attenuated after acrosome reaction. Immunolabeling for oviductin was found to be the weakest over the equatorial segment regardless of the experimental conditions. The binding of hamster oviductin to specific membrane domains of the homologous sperm and the changes in its distribution during capacitation and acrosome reaction may be important for the function of hamster oviductin preceding and during fertilization.     

Topographical rearrangement of a plasma membrane antigen during capacitation of rat spermatozoa in vitro

We have previously described an antigen (termed 2B1) on rat spermatozoa that is present on the plasma membrane overlying the tail domain. The antigen is mobile within the plane of the plasma membrane and a mAb to it blocks fertilization in vitro. In the present study we describe some dynamic properties of this antigen in relation to its topographical distribution. When spermatozoa were incubated in vitro in a capacitation medium and stained with 2B1 mAb/FITC-rabbit anti-mouse F(ab')2, strong fluorescence appeared over the acrosomal domain. Acute exposure of fresh spermatozoa to dissociating reagents (1 M NaCl or 5 mM 2-mercaptoethanol) or inducers of the acrosome reaction (lysolecithin + Ca2+ or A23187 + Ca2+) failed to mimic these effects. Spermatozoa prelabeled with FITC-2B1 IgG and then capacitated in the presence of excess "cold" 2B1 IgG also showed accumulation of fluorescence on the acrosomal domain, suggesting that the antigen had migrated from the tail. Migration was selective and Ca2(+)- and temperature-dependent but was not inhibited by metabolic poisons (NaF or NaN3). Motility was not obligatory for migration. Immunogold-labeling studies at the ultrastructural level showed that 2B1 antigen was restricted to the surface membrane over both the tail and the acrosomal domains and that during migration it did not change the type of membrane into which it was inserted. From a quantitative analysis of fluorescence on spermatozoa prelabeled with FITC-2B1 IgG and then capacitated, the amount of antigen that appeared on the acrosomal domain was approximately equivalent to that lost from the midpiece domain. The Mr of 2B1 antigen extracted from capacitated spermatozoa was 300-500 Da less than that extracted from noncapacitated cells, suggesting that the molecule had undergone processing concomitant with migration. These results are discussed in relation to mechanisms for targeting antigens to sites where they become physiologically active and are correctly positioned to participate in gamete recognition processes.     

Visualization and characterization of the acrosome reaction of human spermatozoa by immunolocalization with monoclonal antibody

A monoclonal antibody generated against hamster epididymal spermatozoa and recognizing an antigen within the acrosome was used in conjunction with FITC-antimouse immunoglobulin as a marker of the human acrosome during sperm development, capacitation, and the acrosome reaction. The specificity of binding of the monoclonal antibody was assessed using immunolocalization by epi-fluorescence and electron microscopy. Immunofluorescence revealed that antibody bound over the entire anterior acrosome in hamster and human spermatozoa. Ultrastructural localization indicated that antigen was predominantly present on the inner face of the outer acrosomal membrane and within the acrosomal content. Qualitative specificity was studied using a highly purified preparation of hamster acrosomes in an enzyme-linked immunosorbent assay. Since the antibody rapidly visualized human acrosomes, it was used to detect abnormal acrosome morphology of mature spermatozoa and to mark spermatids present in the ejaculate. During incubation in capacitating medium, changes in the immunofluorescence of live or methanol fixed spermatozoa were correlated with incubation interval and the ability of spermatozoa to fuse with zona-free hamster oocytes. Spermatozoa bound to zona-free hamster oocytes displayed no fluorescence, confirming that acrosome loss occurred before spermatozoa attached to the vitellus.     

Molecular modifications of MC31/CE9, a sperm surface molecule, during sperm capacitation and the acrosome reaction in the rat: is MC31/CE9 required for fertilization?

We examined the modification of the MC31 molecule during capacitation, the acrosome reaction, and studied its role in fertilization. These studies revealed that the molecular mass of MC31 in cauda spermatozoa was approximately 28,000-26,000 Dalton (28-26 kDa). A limited change in molecular mass was seen in capacitated spermatozoa. Treatment of sperm extracts with peptide-N-glycosidase (PN glycosidase) reduced the molecular mass of MC31 in both cauda and capacitated spermatozoa from 28-26 kDa to 23-20 kDa, suggesting that MC31 from both cauda and capacitated spermatozoa is glycosylated, and indicating that capacitation induces minor posttranslational modifications in the structure of the MC31 antigen. The MC31 antigen was redistributed from the midpiece of cauda epididymal spermatozoa to the head and equatorial segment after capacitation and acrosome reaction, respectively, when traced by indirect immunofluorescence under in vitro fertilization (IVF) conditions. Some spermatozoa did not stain for the MC31 antigen and might represent spermatozoa that have shed the antigen. IVF experiments conducted to assess the effect of an anti-MC31 monoclonal antibody (mMC31) revealed that this antibody significantly (P < 0.01) inhibited fertilization of cumulus-invested zona pellucida-intact and the zona pellucida-free oocytes in a dose-dependent manner. However, sperm-oolemma binding was not affected. These findings suggest the MC31 antigen facilitates sperm-oocyte interactions.     

Role of monoclonal antibody J-23 in inducing acrosome reactions in capacitated mouse spermatozoa.

A monoclonal antibody (J-23) to the 15 kDa component on the sperm head, the acceptor, which functions in zona binding, was shown to induce the acrosome reaction in capacitated cells, but not in fresh cells. The antibody recognized its epitope in the acrosomal cap region of fresh spermatozoa and in the equatorial region on washed and capacitated spermatozoa. However, equatorial expression did not depend on the acrosome reaction, since washing fresh spermatozoa increased the percentage with equatorial fluorescence, but did not increase the percentage with reacted acrosomes. The data indicate that the acrosome reaction can be induced in capacitated spermatozoa in the absence of zona glycoproteins.     

Phospholipid composition of isolated guinea pig sperm outer acrosomal membrane and plasma membrane during capacitation in vitro

After capacitation of guinea pig spermatozoa in vitro, the plasma membrane was mechanically separated from the spermatozoa in the presence or absence of HgCl₂ and subsequently isolated by density gradient centrifugation. Examination of the spermatozoa by electron microscopy after homogenization in the presence of HgCl₂ revealed that plasma membrane was removed only from the acrosomal region and remained predominately intact posterior to the equatorial segment of the sperm head, as well as the midpiece and tail. In comparison, spermatozoa homogenized under similar buffer conditions but in the absence of HgCl₂ lose the large apical segment of the acrosome and the plasma membrane is removed essentially from the entire cell. If spermatozoa were homogenized in the absence of Hg²⁺, analysis of plasma membrane phospholipid composition revealed a complete loss of lysophosphatidylcholine (LPC) from the plasma membrane after incubation of spermatozoa in minimal capacitating medium (MCM-PL) for 2 hours. Under these culture conditions the addition of Ca²⁺ (5 mM) to the capacitated spermatozoa induced approximately 78 ± 5% (n = 3) of the motile spermatozoa to undergo acrosome reactions while still maintaining sperm motility (80 ± 5%) (n = 3). If the spermatozoa were homogenized in the presence of Hg²⁺, a time course study revealed that plasma membrane LPC loss occurred between 60 and 90 minutes of incubation. This complete loss of LPC was evident when approximately half of the capacitated spermatozoa had undergone acrosome reactions. Incubation of the spermatozoa with the metabolic and acrosome reaction inhibitor, 2-deoxyglucose (10 mM) for 2 hours, maintained the plasma membrane phospholipid composition similar to that in the noncapacitated state. These data provide evidence that changes in the plasma membrane phospholipid composition may be associated with guinea pig sperm capacitation.     

Gamete membrane fusion in hamster spermatozoa with reacted equatorial segment

Mammalian spermatozoa must undergo many changes to be able to fertilize the oocyte. One of these changes, the acrosome reaction, has been established as a requisite for gamete membrane fusion to occur; it consists of the fusion and vesiculation of the sperm plasma membrane with the outer acrosomal membrane of the principal segment of the acrosome. Reaction of the equatorial segment has occasionally been observed. The objective of the present work was to determine whether the presence of the sperm plasma membrane over the equatorial segment is necessary for gamete membrane fusion to occur. Golden hamster spermatozoa were capacitated in vitro in TAPL 10K, and the maximum possible percentage of acrosome reaction was determined at 82.79% + 1.69% SD (P = 0.27; r = 0.21). Ultrastructural studies showed that 93.6% of the reacted spermatozoa in this population had their principal and equatorial segments reacted. The fertilizing ability of these spermatozoa was assayed using zona-free hamster oocytes. The percentage of fertilized ova obtained was 98.8% (308/312). Ultrastructural studies snowed the presence of spermatozoa with reacted equatorial segment inside the cytoplasm of immature oocytes. The evidence presented in this work demonstrates that the plasma membrane of spermatozoa with reacted equatorial segment retains its ability to fuse with the oocyte.     

Sperm protein (sp50) binds to acromosome and plasma membranes in a Ca2+-dependent manner: Possible role in acrosome reaction

Annexins are a family of Ca²⁺-binding proteins involved in the exocytotic process. The presence and the role of annexins in mammalian spermatozoa have not been well established. Two annexin-like proteins were obtained from guinea pig testis, a doublet of Mr 31–33 kD (p31/33) and a protein of Mr 50 kD (p50). Both proteins were able to bind to erythrocyte ghosts in a Ca²⁺-dependent fashion. Polyclonal antibodies against p31/33 reacted with two major proteins, Mrs 50 kD (sp50) and 42 kD (sp42), from mature and immature guinea pig spermatozoa. p50 and sp50 are likely the native proteins from testis and spermatozoa, respectively, and they are seemingly related. By immunofluorescence, sp50 was only found in the apical acrosome region of immature and capacitated and noncapacitated spermatozoa, and its location was intracellular. In spermatozoa undergoing acrosome reaction, sp50 was detected in the whole acrosome, while in spermatozoa that had undergone acrosome reaction sp50 was not detected. However, in the protein pattern of acrosome reaction vesicles, anti-p31/33 antibody revealed diffuse bands of Mr 35–38 kD. sp50 was able to bind to plasma membrane fragments and acrosome outer membrane from demembranated sperm in a Ca²⁺-dependent fashion. The presence of sp50 in the acrosome region, its distribution throughout the acrosome membrane just before the acrosome reaction, and its ability to bind both plasma and outer acrosome membranes in a Ca²⁺-dependent manner suggest that sp50 may participate in the acrosome reaction mechanism in guinea pig spermatozoa. © 1996 Wiley-Liss, Inc.     

Acrosome-reacted guinea pig spermatozoa become fusion competent in the presence of extracellular potassium ions

Guinea pig spermatozoa are able to undergo capacitation and the acrosome reaction in a K+-free (-deficient) medium. However, they are unable to fuse with eggs unless they are exposed to a millimolar concentration of extracellular K+ during or after the acrosome reaction. Apparently, the plasma membrane over the equatorial segment gains the ability to fuse with eggs in the presence of K+ during and/or after the acrosome reaction. Once it becomes fusible, the membrane retains its fusibility even in a K+-deficient medium. Rb+ is almost as effective as K+ in rendering the sperm membrane fusible. Li+ and Cs+ are less effective. The molecular mechanism by which K+ renders acrosome-reacted spermatozoa fusion competent is unknown, but it may involve K+-mediated efflux of H+ from the spermatozoa.     

Immunolocalization of a guinea pig sperm surface antigen recognized by a monoclonal antibody E74

E74 is a mouse monoclonal antibody raised against the acrosome-reacted guinea pig spermatozoa. This study describes immunolocalization of the E74 antigen in guinea pig spermatozoa. Immunoelectron microscopy of guinea pig spermatozoa shows that the E74 antigen is localized on the equatorial segment plasma membrane following the acrosome reaction but not associated with the surface of the acrosome-intact spermatozoa. Immunoblot analysis of Triton X-100 extract of cauda epididymal guinea pig spermatozoa following one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis shows that E74 antibody recognizes a protein with an apparent molecular weight of 45,000 dalton. Immunoblot of sperm extracts separated by two dimensional gel electrophoresis indicates a broad spot of 45,000 dalton in the 5 to 7.5 isoelectric focusing range.     

Acrosin activation follows its surface exposure and precedes membrane fusion in human sperm acrosome reaction

Evidence has accumulated suggesting multiple roles of acrosin in fertilization, including its participation in early steps of gamete recognition and binding. However, the implication of acrosin in many of these processes is not compatible with its presumptive sequestration within the sperm acrosome until a late phase of the acrosome reaction. In an earlier study (J. Tesarik, J. Drahorad, J. Peknicova, 1988, Fertil. Steril. 50, 133-141), we reported the binding of an anti-acrosin monoclonal antibody (MO-AKR.1) to the plasma membrane overlying the acrosome of human spermatozoa starting the acrosome reaction. In this study, we characterized further this antibody with regard to its reactivity with different forms of acrosin and found that it recognizes specifically an active form of this enzyme and does not react with its proenzyme form. MO-AKR.1 was thus used as a probe for in situ analysis of acrosin activation during the acrosome reaction. When suspensions of living spermatozoa were incubated with MO-AKR.1 and with another monoclonal antibody (T6) directed to an intra-acrosomal cytoskeletal protein, significantly more spermatozoa reacted with the former antibody than with the latter; this indicated that some of the spermatozoa showing acrosin immunoreactivity carried activated acrosin on the cell surface, while their acrosome was still impermeable to intra-acrosomal-directed probes. The size of this particular sperm subpopulation was increased by the action of follicular fluid (a natural acrosome reaction inducer), but not ionophore A23187 (an artificial acrosome reaction inducer); it corresponded to the proportion of spermatozoa showing acrosin immunoreactivity on the plasma membrane but neither intra-acrosomal staining nor perceptible membrane perturbations when examined by immunoelectron microscopy. When spermatozoa were pre-incubated with protease inhibitors before the addition of acrosome reaction-inducing agents, the percentage of cells binding MO-AKR.1 was markedly reduced. These data suggest that limited acrosin activation on the sperm plasma membrane is an early event in the physiological acrosome reaction.     

Redistribution of a rat sperm epididymal glycoprotein after in vitro and in vivo capacitation.

Rat epididymal glycoprotein DE (37 kDa) associates with the sperm surface during maturation and is localized over the dorsal region of the acrosome. In the present study we examine, by indirect immunofluorescence, the localization of DE after in vitro and in vivo capacitation. While 49% of sperm capacitated in vitro for 5 hr still presented fluorescence over the dorsal region, 51% showed labeling distributed over a domain that corresponds to the equatorial segment of the sperm head. This change in the localization of fluorescence was not associated with sperm deterioration or death and increased gradually as a function of capacitation time, reaching the maximum at 5 hr. The presence of labeling over the equatorial segment results from protein migration and cannot be induced by permeabilization, proteinase, or high ionic strength treatments. The omission of Ca2+ from the standard capacitation medium inhibited the relocalization of DE, and incubation with Ca2+ ionophore A23187 for induction of the acrosome reaction (AR) significantly raised the percentage of cells with DE localized over the equatorial region. Finally, while free and cumulus-associated spermatozoa recovered from the oviducts of in vivo inseminated females presented 15% and 21% of cells with redistribution respectively, all perivitelline (acrosome reacted) spermatozoa showed DE over the equatorial segment. These results indicate that epididymal protein DE migrates to the equatorial segment under in vitro and in vivo capacitating conditions and suggest a possible association between the redistribution of DE and the occurrence of the AR.     

Immunolocalization of heat shock protein 70 (Hsp 70) in boar spermatozoa and its role during fertilization

The presence and cellular distribution of heat protein 70 (Hsp70) in ejaculated, capacitated, and acrosome-reacted boar spermatozoa was evaluated by immunofluorescence and Western blot; the role of Hsp70 during fertilization was also studied. In freshly ejaculated spermatozoa, Hsp70 immunoreactivity is present in a well-defined triangular-shaped area in the equatorial segment that seems to correspond to the equatorial sub-segment. The distribution of the fluorescent signal changes in capacitated sperm, that exhibit different patterns probably in relation to the stage of capacitation of individual cells; after acrosome reaction Hsp70 immunoreactivity is localized on both a thick sub-equatorial band and a triangle in the equatorial segment. In reacted spermatozoa, Hsp70 seems to be not only relocalized but also translocated from the inner to the outer leaflet of the sperm plasma membrane, as a significant (P < 0.05) increase in the proportion of unfixed cells showing the fluorescent signal has been recorded. No differences in Hsp70 amount between fresh, capacitated, and reacted semen were observed by Western blot. The presence of anti-Hsp70 antibody in the fertilization medium significantly reduced, in a concentration-dependent manner, the fertilization rate of both zona-intact and zona-free oocytes. The overall data demonstrate that Hsp70 is present on boar sperm with a dynamic redistribution as the sperm undergoes capacitation and acrosome reaction and suggest an important role of this protein during porcine gamete interaction.     

Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.     

Characterization of human sperm surface antigens with monoclonal antibodies

Monoclonal antibodies (McAb) against human ejaculated sperm were developed from mice immunized with sperm membrane preparations. A solid-phase radioimmunoassay, with dried sperm as antigen, was employed in McAb screening. The tissue and species specificity of monoclonal antibodies HS 2, 4 and 6 were evaluated after absorption of antibody preparations with heterologous sperm, human serum or seminal plasma or cells from other human organs. The sensitivity of HS 2, 4 and 6 antigens to trypsin exposure was determined: HS 4 antigen was highly sensitive while HS 2 and 6 were not. The regional distribution of McAb 4 on intact sperm cells was determined by immunofluorescence staining. HS 4 may be a sperm-coating antigen based on its presence on sperm and in seminal plasma. This possibility led to an investigation of its role in sperm capacitation. HS 4 antibody binding was reduced when capacitated sperm were compared with noncapacitated cells. HS 4 antibody, when present during capacitation and insemination, was without effect on sperm motility or fusion with zona-free hamster eggs. Trypsin removal of as much as 60% of HS 4 antigen from the cell population also did not impact on sperm function. To identify the molecular correlate of HS 4 antigen, membrane components were extracted from washed sperm with Nonidet P-40, concentrated by acetone precipitation and analyzed electrophoretically in SDS-urea on 10% polyacrylamide slab gels. Immunoassays on protein blots with peroxidase-coupled second antibody identified a single reactive species in the molecular weight range of 130,000. Multiple reactive components were detected in blot transfers of seminal plasma.     

Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization

We have previously defined distinct localizations of antigens on the surface of the guinea pig sperm using monoclonal antibodies. In the present study we have demonstrated that these antigen localizations are dynamic and can be altered during changes in the functional state of the sperm. Before the sperm is capable of fertilizing the egg, it must undergo capacitation and an exocytic event, the acrosome reaction. Prior to capacitation, the antigen recognized by the monoclonal antibody, PT-1, was restricted to the posterior tail region (principle piece and end piece). After incubation in capacitating media at 37 degrees C for 1 h, 100% of the sperm population showed migration of the PT-1 antigen onto the anterior tail. This redistribution of surface antigen resulted from a migration of the surface molecules originally present on the posterior tail. It did not occur in the presence of metabolic poisons or when tail-beating was prevented. It was temperature-dependent, and did not require exogenous Ca2+. Since the PT-1 antigen is freely diffusing on the posterior tail before migration, the mechanism of redistribution could involve the alteration of a presumptive membrane barrier. In addition, we observed the redistribution of a second surface antigen after the acrosome reaction. The antigen recognized by the monoclonal antibody, PH-20, was localized exclusively in the posterior head region of acrosome-intact sperm. Within 7-10 min of induction of the acrosome reaction with Ca2+ and A23187, 90-100% of the acrosome-reacted sperm population no longer demonstrated binding of the PH-20 antibody on the posterior head, but showed binding instead on the inner acrosomal membrane. This redistribution of the PH-20 antigen also resulted from the migration of pre-existing surface molecules, but did not appear to require energy. The migration of PH-20 antigen was a selective process; other antigens localized to the posterior head region did not leave the posterior head after the acrosome reaction. These rearrangements of cell surface molecules may act to regulate cell surface function during fertilization.     

Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization

This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.