The Multi-PDZ domain protein MUPP1 as a lipid raft-associated scaffolding protein controlling the acrosome reaction in mammalian spermatozoa

The success of acrosomal exocytosis, a complex process with a variety of interrelated steps, relies on the coordinated interaction of participating signaling molecules. Since scaffolding proteins are known to spatially organize sequential signaling pathways, we examined whether the Multi-PDZ domain protein MUPP1, recently identified in mammalian spermatozoa, is functionally active in controlling acrosomal secretion in mammalian sperm cells. To address this question, permeabilized mouse sperm were loaded with inhibitory antibodies against MUPP1 as well as with a photosensitive Ca(2+) chelator which allows a controlled release of acrosomal Ca(2+). The results revealed that MUPP1 controls initial tethering and docking of the acrosomal vesicle, whereas syntaxin 2, a t-SNARE protein also expressed in the acrosomal cap of mammalian spermatozoa, appears to take part in the final process of acrosomal fusion. Interestingly, using immunogold electron microscopy, it was found that MUPP1 is detectable in the region of the periacrosomal membrane. Furthermore, in isolated detergent-insoluble glycolipid-enriched membrane domains from epididymal spermatozoa, MUPP1 was found to show a striking association with the Triton X-100 insoluble membrane fraction, which did not change significantly upon sperm capacitation or partial chemical extraction of cholesterol. This evidence points to a role of MUPP1 as a membrane raft-associated molecular organizer, and suggests that mammalian spermatozoa may use a scaffolding protein and distinct membrane subdomains to spatially organize components involved in the process of acrosomal exocytosis.     

Syntaxin and VAMP association with lipid rafts depends on cholesterol depletion in capacitating sperm cells

Sperm cells represent a special exocytotic system since mature sperm cells contain only one large secretory vesicle, the acrosome, which fuses with the overlying plasma membrane during the fertilization process. Acrosomal exocytosis is believed to be regulated by activation of SNARE proteins. In this paper, we identified specific members of the SNARE protein family, i.e., the t-SNAREs syntaxin1 and 2, and the v-SNARE VAMP, present in boar sperm cells. Both syntaxins were predominantly found in the plasma membrane whereas v-SNAREs are mainly located in the outer acrosomal membrane of these cells. Under non-capacitating conditions both syntaxins and VAMP are scattered in well-defined punctate structures over the entire sperm head. Bicarbonate-induced in vitro activation in the presence of BSA causes a relocalization of these SNAREs to a more homogeneous distribution restricted to the apical ridge area of the sperm head, exactly matching the site of sperm zona binding and subsequent induced acrosomal exocytosis. This redistribution of syntaxin and VAMP depends on cholesterol depletion and closely resembles the previously reported redistribution of lipid raft marker proteins. Detergent-resistant membrane isolation and subsequent analysis shows that a significant proportion of syntaxin emerges in the detergent-resistant membrane (raft) fraction under such conditions, which is not the case under those conditions where cholesterol depletion is blocked. The v-SNARE VAMP displays a similar cholesterol depletion-dependent lateral and raft redistribution. Taken together, our results indicate that redistribution of syntaxin and VAMP during capacitation depends on association of these SNAREs with lipid rafts and that such a SNARE-raft association may be essential for spatial control of exocytosis and/or regulation of SNARE functioning.     

Signal transduction pathways that regulate sperm capacitation and the acrosome reaction

Ejaculated spermatozoa must undergo physiological priming as they traverse the female reproductive tract before they can bind to the egg’s extracellular coat, the zona pellucida (ZP), undergo the acrosome reaction, and fertilize the egg. The preparatory changes are the net result of a series of biochemical and functional modifications collectively referred to as capacitation. Accumulated evidence suggests that the event that initiates capacitation is the efflux of cholesterol from the sperm plasma membrane (PM). The efflux increases permeability and fluidity of the sperm PM and causes influx of Ca²⁺ ions that starts a signaling cascade and result in sperm capacitation. The binding of capacitated spermatozoa to ZP further elevates intrasperm Ca²⁺ and starts a new signaling cascade which open up Ca²⁺ channels in the sperm PM and outer acrosomal membrane (OAM) and cause the sperm to undergo acrosomal exocytosis. The hydrolytic action of the acrosomal enzymes released at the site of sperm-egg (zona) binding, along with the hyperactivated beat pattern of the bound spermatozoon, are important factors in directing the sperm to penetrate the ZP and fertilize the egg. The role of Ca²⁺-signaling in sperm capacitation and induction of the acrosome reaction (acrosomal exocytosis) has been of wide interest. However, the precise mechanism(s) of its action remains elusive. In this article, we intend to highlight data from this and other laboratories on Ca²⁺ signaling cascades that regulate sperm functions.     

Dynamin Regulates Specific Membrane Fusion Events Necessary for Acrosomal Exocytosis in Mouse Spermatozoa

Mammalian spermatozoa must complete an acrosome reaction prior to fertilizing an oocyte. The acrosome reaction is a unique exocytotic event involving a series of prolonged membrane fusions that ultimately result in the production of membrane vesicles and release of the acrosomal contents. This event requires the concerted action of a large number of fusion-competent signaling and scaffolding proteins. Here we show that two different members of the dynamin GTPase family localize to the developing acrosome of maturing mouse germ cells. Both dynamin 1 and 2 also remain within the periacrosomal region of mature mouse spermatozoa and are thus well positioned to regulate the acrosome reaction. Two pharmacological inhibitors of dynamin, dynasore and Dyngo-4a, blocked the in vitro induction of acrosomal exocytosis by progesterone, but not by the calcium ionophore A23187, and elicited a concomitant reduction of in vitro fertilization. In vivo treatment with these inhibitors also resulted in spermatozoa displaying reduced acrosome reaction potential. Dynamin 1 and 2 phosphorylation increased on progesterone treatment, and this was also selectively blocked by dynasore. On the basis of our collective data, we propose that dynamin could regulate specific membrane fusion events necessary for acrosomal exocytosis in mouse spermatozoa.     

Reorganization of lipid rafts during capacitation of human sperm

Ejaculated mammalian sperm must complete a final maturation, termed capacitation, before they can undergo acrosomal exocytosis and fertilize an egg. In human sperm, loss of sperm sterol is an obligatory, early event in capacitation. How sterol loss leads to acrosomal responsiveness is unknown. These experiments tested the hypothesis that loss of sperm sterol affects the organization of cold detergent-resistant membrane microdomains (lipid "rafts"). The GPI-linked protein CD59, the ganglioside GM1, and the protein flotillin-2 were used as markers for lipid rafts. In uncapacitated sperm, 51% of the CD59, 41% of the GM1, and 90% of the flotillin-2 were found in the raft fraction. During capacitation, sperm lost 67% of their 3beta-hydroxysterols, and the percentages of CD59 and GM1 in the raft fraction decreased to 34% and 31%, respectively. The distribution of flotillin-2 did not change. Preventing a net loss of sperm sterol prevented the loss of CD59 and GM1 from the raft fraction. Fluorescence microscopy showed CD59 and GM1 to be distributed over the entire sperm surface. Flotillin-2 was located mainly in the posterior head and midpiece. Patching using bivalent antibodies indicated that little of the GM1 and CD59 was stably associated in the same membrane rafts. Likewise, GM1 and flotillin-2 were not associated in the same membrane rafts. In summary, lipid rafts of heterogeneous composition were identified in human sperm and the two raft components, GM1 and CD59, showed a partial sterol loss-dependent shift to the nonraft domain during capacitation.     

Calmodulin signals capacitation and triggers the agonist-induced acrosome reaction in mouse spermatozoa

Capacitated acrosome-intact spermatozoa interact with specific sugar residues on neoglycoproteins (ngps) or solubilized zona pellucida (ZP), the egg's extracellular glycocalyx, prior to the initiation of a signal transduction cascade that results in the fenestration and fusion of the sperm plasma membrane and the outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents (i.e., induction of the acrosome reaction (AR)). The AR releases acrosomal contents at the site of sperm-zona binding and is thought to be a prerequisite event that allows spermatozoa to penetrate the ZP and fertilize the egg. Since Ca(2+)/calmodulin (CaM) plays a significant role in several cell signaling pathways and membrane fusion events, we have used a pharmacological approach to examine the role of CaM, a calcium-binding protein, in sperm capacitation and agonist-induced AR. Inclusion of CaM antagonists (calmodulin binding domain, calmidazolium, compound 48/80, ophiobolin A, W5, W7, and W13), either in in vitro capacitation medium or after sperm capacitation blocked the npg-/ZP-induced AR. Purified CaM largely reversed the AR blocking effects of antagonists during capacitation. Our results demonstrate that CaM plays an important role in priming (i.e., capacitation) of mouse spermatozoa as well as in the agonist-induced AR. These data allow us to propose that CaM regulates these events by modulating sperm membrane component(s).     

Identification of mouse trp homologs and lipid rafts from spermatogenic cells and sperm.

Intracellular Ca(2+) has an important regulatory role in the control of sperm motility, capacitation, and the acrosome reaction (AR). However, little is known about the molecular identity of the membrane systems that regulate Ca(2+) in sperm. In this report, we provide evidence for the expression of seven Drosophila transient receptor potential homolog genes (trp1-7) and three of their protein products (Trp1, Trp3 and Trp6) in mouse sperm. Allegedly some trps encode capacitative Ca(2+) channels. Immunoconfocal images showed that while Trp6 was present in the postacrosomal region and could be involved in sperm AR, expression of Trp1 and Trp3 was confined to the flagellum, suggesting that they may serve sperm to regulate important Ca(2+)-dependent events in addition to the AR. Likewise, one of these proteins (Trp1) co-immunolocalized with caveolin-1, a major component of caveolae, a subset of lipid rafts potentially important for signaling events and Ca(2+) flux. Furthermore, by using fluorescein-coupled cholera toxin B subunit, which specifically binds to the raft component ganglioside GM1, we identified caveolin- and Trp-independent lipid rafts residing in the plasma membrane of mature sperm. Notably, the distribution of GM1 changes drastically upon completion of the AR.     

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.     

Rat caltrin protein modulates the acrosomal exocytosis during sperm capacitation

Caltrin is a small and basic protein of the seminal vesicle secretion that inhibits sperm calcium uptake. The influence of rat caltrin on sperm physiological processes related to fertilizing competence was studied by examining its effect on 1) spontaneous acrosomal exocytosis, 2) protein tyrosine phosphorylation, and 3) sperm-egg interaction. Results show that the presence of caltrin during in vitro capacitation both reduced the rate of spontaneous acrosomal exocytosis without altering the pattern of protein tyrosine phosphorylation, and enhanced the sperm ability to bind to the zona pellucida (ZP). The significantly higher proportion of sperm with intact acrosome observed in the presence of caltrin was accompanied by a strong inhibition in the acrosomal hyaluronidase release. Enhancement of sperm-ZP binding was evident by the increase in the percentage of eggs with bound spermatozoa as well as in the number of bound sperm per egg. Similar results were obtained when the assays were performed using spermatozoa preincubated with caltrin and then washed to remove the unbound protein, indicating that the sperm-bound caltrin was the one involved in both acrosomal exocytosis inhibition and sperm-ZP binding enhancement. Caltrin bound to the sperm head was partially released during the acrosomal exocytosis induced by Ca-ionophore A23187. Indirect immunofluorescence and immunoelectron microscopy studies revealed that caltrin molecules distributed on the dorsal sperm surface disappeared after ionophore exposure, whereas those on the ventral region remained in this localization after the treatment. The present data suggest that rat caltrin molecules bound to the sperm head during ejaculation prevent the occurrence of the spontaneous acrosomal exocytosis along the female reproductive tract. Consequently, more competent spermatozoa with intact and functional acrosome would be available in the oviduct to participate in fertilization.     

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.     

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.     

Evidence for the capacitation-associated membrane priming of mouse spermatozoa

An important feature of male fertility is the physiological priming of mammalian spermatozoa by a multifaceted process referred to as capacitation. It is a prerequisite event before spermatozoa can bind to the egg's extracellular coat, the zona pellucida, and undergo a signal transduction cascade. The net result is the fusion of the plasma membrane (PM) and underlying outer acrosomal membrane at multiple sites and the release of acrosomal contents (i.e., glycohydrolases, proteinases, etc.) at the site of sperm-zona binding. In this study, we have used an indirect immunofluorescence (IIF) assay and other staining approaches to examine capacitation-associated membrane priming of mouse spermatozoa. For IIF studies, we used affinity-purified antibodies against two glycohydrolases that cross-reacted with the acrosomal enzymes only when the uncapacitated spermatozoa were permeabilized. Incubation of spermatozoa in a medium that favors in vitro capacitation induced membrane priming that allowed the antibodies to cross-react with the acrosomal enzymes in capacitating acrosome-intact spermatozoa without permeabilization, as revealed by the appearance of several distinct fluorescent patterns, including an initial immunopositive lining over the acrosome cap to an intense immunopositive reaction throughout the acrosome. These early immunopositive patterns were followed by the appearance of intense fluorescent spots (droplets) that seem to establish contact with the PM in a time-dependent manner. Inclusion of calmodulin, a 17-kDa Ca(2+)-binding protein which promotes capacitation, in the incubation medium did not alter the overall rate of capacitation; however, its presence accelerated the initial stages of membrane priming. The potential similarities between sperm capacitation and early events of Ca(2+)-triggered membrane fusion among eukaryotes and among various stations of the secretory and endocytotic pathways are discussed.     

Surface alterations during the capacitation of mammalian spermatozoa

Capacitation is the process by which mammalian sperm acquire the ability to undergo the acrosome reaction which, in turn, is a prerequisite for sperm-egg fusion and penetration. Until recently, it was thought that capacitation involved subtle physiological and chemical changes which had no morphological counterparts even at the electron microscopic level. However, it has now been shown by a number of investigators that material associated with the plasma membrane surface is either lost or extensively redistributed during in vitro or in vivo capacitation. We have made use of lectins and antibodies as probes of the sperm surface during capacitation and the acrosome reaction. Concanavalin A (Con A), wheat germ agglutinin (WGA) and soybean agglutinin (SBA) have been used in conjunction with fluorescent tags (FITC) and ultrastructural markers (ferritin, hemocyanin) to study the surface of golden hamster, guinea pig, mouse and human spermatozoa. Con A and WGA label the plasma membrane overlying the acrosomal region quite uniformly on these species. After capacitation there is a specific loss (or masking) of lectin binding sites over the acrosomal region of the sperm head in all species examined. Antibodies prepared against sperm and specific antibodies to a cell surface protein (fibronectin) were also tagged with fluorescent or ultrastructural markers and used to label the surfaces of sperm before and after capacitation. These probes also indicate a specific loss of surface associated material over the acrosomal surface after capacitation. These results are consistent with the notion that there is a general removal of surface components during capacitation and that this denuding of the surface is a prerequisite for the following membrane fusion events involved in the acrosome reaction and sperm-egg fusion.     

Differential release of soluble and matrix components: evidence for intermediate states of secretion during spontaneous acrosomal exocytosis in mouse sperm

Although its exact role in fertilization is unknown, the acrosome is a very important, exocytotic organelle overlying the anterior aspect of sperm from many species. Structurally and functionally, the acrosome can be considered to consist of soluble and particulate compartments. One component of the particulate acrosomal matrix is the zona pellucida-binding protein sp56. Our demonstration that this protein is within the acrosomal matrix and not on the sperm plasma membrane has led us to reexamine the events of acrosomal exocytosis and the role of the sperm acrosomal matrix in the fertilization process. To visualize the soluble compartment, we have utilized sperm from transgenic mice that carry soluble green fluorescent protein (GFP) in their acrosomes and, as a means to assess the exposure of acrosomal matrix components, we have tested the ability of these sperm to bind beads coated with antibodies to sp56. The loss of GFP from the acrosomes and the binding of the beads by the sperm undergoing capacitation serve as indicators of distinct stages of acrosomal exocytosis, allowing us to define intermediates of acrosomal exocytosis that occur during the course of sperm capacitation. These experiments demonstrate that the exposure and release of acrosomal proteins during spontaneous acrosomal exocytosis is not synchronous but is regulated during capacitation. Furthermore, acrosomal exocytosis under these conditions required calcium in the medium. On the basis of these findings, we propose an alternative model for acrosomal exocytosis that considers a role for these intermediates of exocytosis during capacitation and sperm-ZP interactions.     

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.     

Segregation of micron-scale membrane sub-domains in live murine sperm

Lipid rafts, membrane sub-domains enriched in sterols and sphingolipids, are controversial because demonstrations of rafts have often utilized fixed cells. We showed in living sperm that the ganglioside G(M1) localized to a micron-scale membrane sub-domain in the plasma membrane overlying the acrosome. We investigated four models proposed for membrane sub-domain maintenance. G(M1) segregation was maintained in live sperm incubated under non-capacitating conditions, and after sterol efflux, a membrane alteration necessary for capacitation. The complete lack of G(M1) diffusion to the post-acrosomal plasma membrane (PAPM) in live cells argued against the transient confinement zone model. However, within seconds after cessation of sperm motility, G(M1) dramatically redistributed several microns from the acrosomal sub-domain to the post-acrosomal, non-raft sub-domain. This redistribution was not accompanied by movement of sterols, and was induced by the pentameric cholera toxin subunit B (CTB). These data argued against a lipid-lipid interaction model for sub-domain maintenance. Although impossible to rule out a lipid shell model definitively, mice lacking caveolin-1 maintained segregation of both sterols and G(M1), arguing against a role for lipid shells surrounding caveolin-1 in sub-domain maintenance. Scanning electron microscopy of sperm freeze-dried without fixation identified cytoskeletal structures at the sub-domain boundary. Although drugs used to disrupt actin and intermediate filaments had no effect on the segregation of G(M1), we found that disulfide-bonded proteins played a significant role in sub-domain segregation. Together, these data provide an example of membrane sub-domains extreme in terms of size and stability of lipid segregation, and implicate a protein-based membrane compartmentation mechanism.     

Sperm from mice genetically deficient for the PCSK4 proteinase exhibit accelerated capacitation, precocious acrosome reaction, reduced binding to egg zona pellucida, and impaired fertilizing ability

The gene for proprotein convertase subtilisin/kexin-like 4 (PCSK4, previously known as PC4) is primarily transcribed in testicular spermatogenic cells. Its inactivation in mouse causes severe male subfertility. To better understand the reproductive function of PCSK4, we examined its subcellular localization in the testicular epithelium via immunohistochemistry, and on intact sperm via indirect immunofluorescence and immunoelectron microscopy. PCSK4 was detected in the acrosomal granules of round spermatids, in the acrosomal ridges of elongated spermatids, and on the sperm plasma membrane overlying the acrosome. We also investigated PCSK4 relevance for sperm acquisition of fertilizing ability by comparing wild-type and PCSK4-null sperm for their abilities in capacitation, acrosome reaction, and egg binding in vitro. PCSK4-null sperm underwent capacitation at a faster rate; they were induced to acrosome react by lower concentrations of zona pellucida; and their egg-binding ability was only half that of wild-type sperm. These sperm physiologic anomalies likely contribute to the severe subfertility of PCSK4-deficient male mice.     

Rab3A triggers the acrosome reaction in permeabilized human spermatozoa

The acrosome reaction is a regulated exocytotic process leading to a massive fusion between the outer acrosomal membrane and the cell membrane. In spite of the great amount of information available related to the acrosome reaction in several species, there is a remarkable paucity about the role of monomeric guanosine triphosphatases (GTPases) of the Rab family-well-established participants in exocytosis in other cell types-in the acrosome reaction. Western blot and immunofluorescence analysis indicate that Rab3A is present in human spermatozoa and localizes to the acrosomal region in the sperm head. One difficulty in studying the role of proteins in intact cells is the fact that they are unable to cross the cell membrane. Therefore, we established a working model of streptolysin O-permeabilized human spermatozoa. Permeabilized spermatozoa were able to respond in a regulated way to different stimuli, such as G protein activators and calcium. An acrosomal reaction was also triggered by a Rab3A peptide corresponding to the effector region. More important, recombinant Rab3A protein in the GTP-bound form caused acrosome exocytosis. The same protein loaded with GDP or Rab11 in the GTP-bound form was inactive. Also, recombinant GDI (GDP dissociation inhibitor)-a protein that releases Rab proteins from membrane-inhibited a GTPgammaS-stimulated acrosome reaction. Our results indicate that 1) permeabilized spermatozoa can be used to study the role of macromolecules in the acrosome reaction, 2) Rab3A is present in human spermatozoa, and 3) Rab3A or another Rab3 isoform is involved in the exocytosis of the acrosomal granule in human spermatozoa.     

In vitro capacitation of dog spermatozoa as assessed by chlortetracycline staining

We developed an assay for detecting capacitation and acrosome status in dog spermatozoa using chlortetracycline (CTC) as a fluorescent probe. Sperm cells were stained after incubation in modified canine capacitation medium (mCCM). Calcium ionophore A23187 permitted the induction of acrosomal exocytosis of capacitated sperm cells. Spermac staining and transmission electron microscopy were used as control tests to detect acrosome-reacted spermatozoa. Three different patterns of CTC distribution in the spermatozoa were found. These patterns were similar to those observed in other mammalian species. The CTC test was used to monitor the time course of the capacitation process in dogs. It was found that the kinetics of capacitation in canine sperm cells are similar to those observed in other mammals.     

The monomeric GTP binding protein,rab3a,is associated with the acrosome in mouse sperm

Exocytosis of the sperm acrosome is an obligate precursor to successful egg penetration and subsequent fertilization. In most mammals, acrosomal exocytosis occurs at a precise time, after sperm binding to the zona pellucida of the egg, and is induced by a specific component of the zona pellucida. It may be considered an example of regulated secretion with the acrosome of the sperm analogous to a single secretory vesicle. Monomeric G proteins of the rab3 subfamily, specifically rab3a, have been shown to be important regulators of exocytosis in secretory cells, and we hypothesized that these proteins may regulate acrosomal exocytosis. Using α[³²P] GTP binding to Immobilon blotted mouse sperm proteins, the presence of three or more monomeric GTP binding proteins was identified with M_r = 22, 24, and 26 × 10³. Alpha[³²P] GTP binding could be competed by GTP and GDP, but not GMP, ATP, or ADP. Anti‐peptide antibodies specific for rab3a were used to identify the 24 kDa G protein as rab3a. Using immunocytochemistry, rab3a was localized to the head of acrosome‐intact sperm and was lost during acrosomal exocytosis. It was identified in membrane and cytosolic fractions of sperm with the predominant form being membrane‐bound, and its membrane association did not change upon capacitation. Immunogold labeling and electron microscopy demonstrated a subcellular localization in clusters to the periacrosomal membranes and cytoplasm. These data identify the presence of rab3a in acrosomal membranes of mouse sperm and suggest that rab3a plays a role in the regulation of zona pellucida ‐induced acrosomal exocytosis. Mol. Reprod. Dev. 53:413–421, 1999. © 1999 Wiley‐Liss, Inc.