Acrosome reaction in spermatozoa from hagfish (Agnatha) Eptatretus burgeri and Eptatretus stouti: acrosomal exocytosis and identification of filamentous actin

Spermatozoa of the hagfishes Eptatretus burgeri and Eptatretus stouti, caught in the sea near Japan and North America, respectively, were found to undergo the acrosome reaction, which resulted in the formation of an acrosomal process with a filamentous core. The acrosomal region of spermatozoa of E. stouti exhibited immunofluorescent labeling using an actin antibody. The midpiece also labeled with the antibody. The acrosomal region showed a similar labeling pattern when sperm were probed with tetramethylrhodamine isothyocyanate (TRITC)-phalloidin; the midpiece did not label. Following induction of the acrosome reaction with the calcium (Ca2+) ionophore ionomycin, TRITC-phalloidin labeling was more intense in the acrosomal region, suggesting that the polymerization of actin occurs during formation of the acrosomal process, as seen in many invertebrates. The potential for sperm to undergo acrosomal exocytosis was already acquired by late spermatids. During acrosomal exocytosis, the outer acrosomal membrane and the overlying plasma membrane disappeared and were replaced by an array of vesicles; these resembled an early stage of the acrosome reaction in spermatozoa of higher vertebrates in which no formation of an acrosomal process occurs. It is phylogenetically interesting that such phenomena occur in spermatozoa of hagfish, a primitive vertebrate positioning between invertebrates and high vertebrates.     

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.     

Relationship between calcium binding sites and membrane fusion during the acrosome reaction induced by ionophore in ram spermatozoa

Ram spermatozoa treated with the ionophore A23187, to induce the acrosome reaction, were fixed in pyroantimonate-osmium in order to demonstrate calcium at an ultrastructural level. Prior to vesiculation, granules of precipitate occurred at points of contact between plasma and outer acrosomal membranes, suggesting a discrete role for calcium during membrane fusion. The earliest stages of vesiculation always occurred in the region just anterior to the equatorial segment and it was this same site where a marked concentration of precipitate, indicating calcium binding sites, could be demonstrated.     

Assessment of acrosomal status in rat spermatozoa: studies on carbohydrate and non-carbohydrate agonists

In the mouse and several other species, including man, capacitated acrosome-intact spermatozoa interact with natural [soluble zona pellucida (ZP) and progesterone (P4)] and synthetic [neoglycoproteins (ngps) and calcium (Ca(2+)) ionophore] agonists, prior to the initiation of a Ca(2+)-dependent signal transduction cascade. The net result is the fusion of the sperm plasma membrane overlying the outer acrosomal membrane at multiple sites and exocytosis of acrosomal contents [i.e., induction of the acrosome reaction (AR)]. This step is believed to be a prerequisite that enables the acrosome-reacted spermatozoon to penetrate the ZP and fertilize the egg. Although the rat is one of the most commonly used laboratory animals, very little is known about the chemical nature of agonists that induce the AR in this species. The lack of this information is primarily due to the fact that the rat sperm acrosome is a relatively thin structure. Thus, it is difficult to assess the status of the sperm acrosome in this species. In this report, we describe the use of a Coomassie brilliant blue dye staining procedure to assess the status of the rat sperm acrosome by light microscopy. The procedure is highly reproducible and has allowed us to determine the effects of carbohydrate (ngps and mouse ZP) and noncarbohydrate (P4 and Ca(2+) ionophore) agonists on capacitated spermatozoa. In addition, we have used a pharmacological approach to examine the functional significance of calmodulin (CaM), a Ca(2+)-binding protein, in induction of the AR in spermatozoa. Data presented in this report demonstrate that several ngps, solubilized mZP, P4, and Ca(2+) ionophores induce the AR in rat spermatozoa. Furthermore, we demonstrate that, whereas CaM antagonists blocked P4-induced AR, most of the inhibitors used had no significant effect on the Ca(2+) ionophore-induced (nonphysiological) AR.     

Immunocytological characterization of the outer acrosomal membrane (OAM) during acrosome reaction in boar

In order to study the acrosome reaction in boar, spermatozoa were incubated in a calcium-containing medium in the presence of the calcium ionophore A23187. The time course of the acrosome reaction was assessed by phase-contrast microscopy and correlated with the movement characteristics of the spermatozoa determined by means of multiple-exposure photography (MEP). Different stages of the acrosome reaction could be observed by indirect immunofluorescence using an antibody fraction raised in rabbits against the isolated outer acrosomal membrane (OAM). At the start of the acrosome reaction, a bright fluorescence located exclusively at the acrosomal cap of the sperm head could be observed, whereas after 60-120 min, the fluorescence vanished, indicating the complete loss of the OAM. However, to gain more insight into the stages of the plasma membrane and OAM during the acrosome reaction, immunoelectron-microscopical studies were performed using anti-OAM antibodies detected by the protein-A gold method. Ultrathin sections and total preparations in combination with transmission electron microscopy (TEM) confirmed, that boar spermatozoa start their acrosome reaction by a vesiculation of the plasma membrane, thus exposing the heavily labelled OAM, which is then lost as sheets or large vesicles. The newly exposed inner acrosomal membrane did not show any labelling with gold, thereby indicating clear differences in the antigenicity of both acrosomal membranes.     

Immunocytological characteriziation of the outer acrosomal membrane (OAM) during acrosome reaction in boar

Summary In order to study the acrosome reaction in boar, spermatozoa were incubated in a calcium-containing medium in the presence of the calcium ionophore A23187. The time course of the acrosome reaction was assessed by phasecontrast microscopy and correlated with the movement characteristics of the spermatozoa determined by means of multiple-exposure photography (MEP). Different stages of the acrosome reaction could be observed by indirect immunofluorescence using an antibody fraction raised in rabbits against the isolated outer acrosomal membrane (OAM). At the start of the acrosome reaction, a bright fluorescence located exclusively at the acrosomal cap of the sperm head could be observed, whereas after 60–120 min, the fluorescence vanished, indicating the complete loss of the OAM. However, to gain more insight into the stages of the plasma membrane and OAM during the acrosome reaction, immunoelectron-microscopical studies were performed using anti-OAM antibodies detected by the protein-A gold method. Ultrathin sections and total preparations in combination with transmission electron microscopy (TEM) confirmed, that boar spermatozoa start their acrosome reaction by a vesiculation of the plasma membrane, thus exposing the heavily labelled OAM, which is then lost as sheets or large vesicles. The newly exposed inner acrosomal membrane did not show any labelling with gold, thereby indicating clear differences in the antigenicity of both acrosomal membranes.     

Plasma membrane structure of bat spermatozoa: observations on epididymal and uterine spermatozoa in Myotis lucifugus

Plasma membrane structure of bat spermatozoa was examined utilizing electron microscopy of thin sections and freeze-fracture replicas. Notable membrane features observed in replicas from cauda epididymal spermatozoa included specialized particle aggregates at the junction between the acrosomal and postacrosomal region of the head (a membrane structure not previously described in mammalian spermatozoa) and another row of rod-like particles just anterior to the posterior ring. Both of these specializations in fractured plasma membranes correspond with regions where the membrane is closely apposed to underlying structures when viewed in thin sections. The postacrosomal sheath appears to be composed of an array of longitudinally oriented filamentous components. Characteristic ordering of intramembranous particles was also noted in replicas from the midpiece region and the annulus. Major changes in plasma membrane structure were not seen in spermatozoa stored in the female reproductive tract; however, the appearance of linear particle aggregations in the principal piece membrane was noted. No evidence was obtained to suggest that an acrosome reaction had occurred in spermatozoa stored in females.     

A scanning electron microscopic study of rabbit spermatozoa in the female reproductive tract following coitus

The surface morphology of rabbit spermatozoa, fixed in situ (female reproductive tract) and prepared for scanning electron microscopy by critical point drying, was studied for as many as 36 hours post coitum. The findings demonstrate that 1) spermatozoa in the reproductive tract following coitus exist as a heterogenous, morphological population and 2) with time, shifts within this population from one predominant morphology to another take place. In the fresh ejaculate, most spermatozoa have intact surfaces free of membranous disruptions. With time, a process of labilization (denudation) of the membranes covering the acrosomal region occurs in a progressively larger proportion of spermatozoa. The labilization originates by a process of vesiculation and/or vacuolation and leads to the appearance of a series of small fenestrations of perforations of the surface membranes. The perforations coalesce, and gradually larger areas of the surface membranes are eroded such that by 15 hours post coitum, the outer acrosomal membrane, as well as other acrosomal areas, are to varying degrees, directly exposed to the uterine milieu. Secretory granules, picked up by cilia and transferred to the spermatozoa become localized over the acrosomal region shortly after coitus. The possible significance of these time-dependent morphological events with the phenomena of capacitation and the "true" and "false" acrosome reactions are discussed.     

Stability of the acrosome of the brush-tailed possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) in vitro and after exposure to conditions and agents known to cause capacitation or acrosome reaction of eutherian spermatozoa.

Ejaculated spermatozoa from brush-tailed possums and tammar wallabies were washed by a 'swim up' procedure into Hanks Balanced Salt Solution (HBSS), and then exposed to test solutions. Spermatozoa were incubated at 33 degrees C, or room temperature when long-term sperm survival (greater than 10 h) was required. Exposure of spermatozoa to calcium ionophore A23187, cyclic nucleotides, phosphoinositide pathway intermediates, lysophospholipids, trypsin or 'capacitating' high ionic-strength medium (380 mosmol) followed by 3% bovine serum albumin for periods up to 24 h did not induce acrosomal loss. However, there were major changes within the acrosome: large numbers of empty membrane-bound vesicles were formed, the electron density of the acrosomal matrix decreased and the acrosome swelled slightly. The origin of the vesicles is unclear but the acrosomal membranes and the plasma membrane remained intact.     

Evaluation of acrosomal status of bovine spermatozoa using concanavalin a lectin

We report here that fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) specifically labels the acrosomal region of acrosome-reacted bovine spermatozoa. This labeling is found to be useful in evaluating the acrosome status of bovine spermatozoa. When fresh bovine spermatozoa that had been fixed with 4% formaldehyde, smeared on glass slides and then air-dried were stained by FITC-ConA, weak fluorescence was observed on the acrosomal region, although almost all the spermatozoa appeared to be acrosome-intact. However, when fresh sperm suspensions were incubated with FITC-ConA and then mounted on glass slides, no fluorescence was observed on the acrosomal region. Therefore, in the ensuing experiments, both the fixation and the FITC-ConA staining of spermatozoa were done in suspension. When ethanol-treated spermatozoa, whose outer membrane may be permeabilized, were stained with FITC-ConA, the fluorescence was extensively observed on the inner acrosomal region. This fluorescence was inhibited in the presence of 0.2 M D-mannose, a competitive sugar, suggesting that FITC-ConA binds specifically to glycocomponents on the inner acrosomal membrane. We next tried to stain fresh or frozen-thawed spermatozoa from 3 different bulls that had been treated with the calcium ionophore A23187, which is known to induce acrosome reaction of bovine spermatozoa, with FITC-ConA. A significant correlation between the percentage of ConA-labeled spermatozoa and that of rose bengal stained negative ones at various time points during A23187 incubation was achieved. Furthermore, suitability of dual staining to distinguish between physiological acrosome reaction (acrosome-lost and live) and degenerative acrosomal loss (acrosome-lost and dead) using FITC-ConA and Hoechst bis-benzimide 33258 (H258) supravital stain was also confirmed. From these results, it was concluded that the FITC-ConA labeling procedure is a feasible and reliable method for the assessment of physiological acrosome reaction of bovine spermatozoa.     

The use of anti-VDAC2 antibody for the combined assessment of human sperm acrosome integrity and ionophore A23187-induced acrosome reaction

Voltage-dependent anion channel (VDAC) is mainly located in the mitochondrial outer membrane and participates in many biological processes. In mammals, three VDAC subtypes (VDAC1, 2 and 3) have been identified. Although VDAC has been extensively studied in various tissues and cells, there is little knowledge about the distribution and function of VDAC in male mammalian reproductive system. Several studies have demonstrated that VDAC exists in mammalian spermatozoa and is implicated in spermatogenesis, sperm maturation, motility and fertilization. However, there is no knowledge about the respective localization and function of three VDAC subtypes in human spermatozoa. In this study, we focused on the presence of VDAC2 in human spermatozoa and its possible role in the acrosomal integrity and acrosome reaction using specific anti-VDAC2 monoclonal antibody for the first time. The results exhibited that native VDAC2 existed in the membrane components of human spermatozoa. The co-incubation of spermatozoa with anti-VDAC2 antibody did not affect the acrosomal integrity and acrosome reaction, but inhibited ionophore A23187-induced intracellular Ca(2+) increase. Our study suggested that VDAC2 was located in the acrosomal membrane or plasma membrane of human spermatozoa, and played putative roles in sperm functions through mediating Ca(2+) transmembrane transport.     

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.     

Evaluation of the acrosomal membrane in bovine spermatozoa: effects of proteinase inhibitors

Thawed bovine spermatozoa are characterized by a lack of homogeneity in the acrosomal membrane. Therefore, it is difficult to visualize the acrosome to assess morphology. Synthetic proteinase inhibitors were tested on thawed bovine semen for their effect on the integrity of acrosomal membranes. The proteinase inhibitors 4-nitrophenyl-4-guanidinobenzoate (NPGB) and N-L-p-tosyl-L-lysine-chloromethylketone (TLCK) were added to a medium containing spermatozoa separated on a percoll gradient. After incubation for 30 min at 38 degrees C in 5% CO(2), 95% air (final concentration 1 mM), the action of these inhibitors was controlled by measuring the activity of acrosome proteinases. The acrosomal membrane was evaluated by means of a dual stain procedure (trypan blue, Giemsa). In contrast to spermatozoa that had been incubated with proteinase inhibitor-free solution, samples that had been incubated with TLCK showed homogeneity in 90% of the acrosomal membranes and excellent visualization of the acrosome itself; in the NPGB-treated samples, homogeneous staining was observed in 83% of spermatozoa (P < 0.0005). It is concluded that alteration of the acrosomal membrane in thawed semen is not directly caused by freezing-thawing, but may be due to activation of acrosomal proteinases, which is increased during staining procedures. The addition of proteinase inhibitors before staining offers a new possibility for improved assessment of the acrosome in bovine spermatozoa.     

Evaluation of the human sperm acrosome reaction using a monoclonal antibody, GB24, and fluorescence-activated cell sorter

GB24 is a mouse monoclonal antibody raised against human trophoblast microvilli, which recognizes an antigenic determinant on the acrosomal region of the human sperm head. By indirect immunofluorescence, reactivity of GB24 could not be detected on freshly ejaculated spermatozoa but was strongly positive after sperm permeabilization with acetone. On viable, motile spermatozoa, reactivity appeared after induction of the acrosomal reaction with the calcium ionophore A23187. These results suggest that the antigen recognized by GB24 is present on the inner acrosomal membrane. A quantitative evaluation assay of the acrosome reaction on viable spermatozoa by flow cytometry using GB24 and indirect immunofluorescence is proposed.     

Production,characterization, and use of ionophore-induced,calcium-dependent acrosome reaction in ram spermatozoa

A system has been developed for inducing a calcium-dependent acrosome reaction in ram spermatozoa in vitro using the calcium ionophore A23187. The resultant reaction is accompanied by release of the acrosomal enzymes hyaluronidase and acrosin, but there is no release of the cytoplasmic enzyme glucose 6-phosphate isomerase. In any given cell, the visible acrosome reaction apparently takes place rapidly, but there is a variable delay before the reaction occurs. Under optimum conditions, about 90% of treated spermatozoa show an acrosome reaction within one hour. Preincubation of the spermatozoa with the proteinase inhibitors p-amino-benzamidine or p-nitrophenylguanidinobenzoate allows two stages of the reaction to be distinguished ultrastructurally, a membrane fusion stage followed by a dispersal of the acrosomal matrix. In the presence of the inhibitors, the first stage is delayed but is completed within 1 hour, whereas the second remains largely incomplete. In the presence of calcium, ionophore concentrations which induce an acrosome reaction abolish sperm motility rapidly and completely. However, by adding serum albumin shortly after addition of ionophore, motility can be preserved while the acrosome reaction occurs as usual; the motility pattern observed under these conditions is of the “whip-lash” or “activated” type. Although the motile ionophore-treated spermatozoa were unsuccessful at penetrating normal mature sheep oocytes in vitro, they were able to penetrate zona-free oocytes, after which swelling and decondensation of the sperm head took place.     

Influence of a capacitation period on human sperm acrosome loss.

The present study investigates whether a 5 hour capacitation period modifies the ability of human spermatozoa to undergo induced acrosomal loss. Human sperm acrosomal loss was induced by treatment with either the calcium ionophore A23187, low concentrations of the phospholipid dilauroylphosphatidylcholine (PC12), or 2 hours incubation in conditioned medium prepared from human cumulus cells (CM/CC). The use of a dual staining method (FITC-ConA and Hoechst 33258) for simultaneous assessment of acrosomal status and viability demonstrated that induction of acrosomal loss with calcium ionophore was not dependent on a capacitation period. A short (5 hour) incubation period was not sufficient to induce acrosomal loss with CM/CC above spontaneous acrosome reaction rates in medium alone. A significant capacitation-dependent increase (P < 0.05) in acrosomal loss was observed when human spermatozoa were incubated with PC12. Induction of acrosomal loss of capacitated human spermatozoa with PC12 therefore provides a simple assay for the simultaneous assessment of human sperm capacitation and the acrosome reaction in vitro.     

Assessment of the human sperm acrosome reaction using concanavalin A lectin

A method for assessment of the human sperm acrosome reaction is reported using fluorescein isothiocyanate (FITC)-conjugated Concanavalin A (ConA). The technique involved labelling prefixed spermatozoa, where only those spermatozoa that showed a complete loss of the acrosome bound FITC-ConA to the acrosomal region. Competitive sugar binding studies demonstrated that binding of ConA lectin to the acrosomal area of human spermatozoa was inhibited in the presence of 0.2 M D-mannose. Staining with the supravital stain Hoechst 33258 (H258) concomitantly with FITC-ConA allowed determination of only those spermatozoa that had undergone a true and not degenerative acrosomal loss. Incubation of human spermatozoa with 0, 1, 5, and 25 microM calcium ionophore, A23187, for 60 min demonstrated that changes in acrosomal status due to the different treatment protocols may be determined by the dual-staining method. Electron microscopy studies revealed that gold-conjugated ConA bound specifically to the surface of the inner acrosomal membrane of acrosome-reacted spermatozoa. A significant correlation (r = +.97) between transmission electron microscopy (TEM) and FITC-ConA labelling methods of acrosomal status assessment was achieved. The simple ConA labelling procedure reported here therefore provides a reliable method for quantitation of the physiological acrosome reaction of a population of human spermatozoa.     

Fluorescent localization of thiols and disulfides in marsupial spermatozoa by bromobimane labelling

The acrosome of marsupial spermatozoa is a robust structure which, unlike its placental counterpart, resists disruption by detergent or freeze/thawing and does not undergo a calcium ionophore induced acrosome reaction. In this study specific fluorescent thiol labels, bromobimanes, were used to detect reactive thiols in the intact marsupial spermatozoon and examine whether disulfides play a role in the stability of the acrosome. Ejaculated brushtail possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) spermatozoa were washed by swim up and incubated with or without dithiothreitol (DTT) in order to reduce disulfides to reactive thiols. Spermatozoa were then washed by centrifugation and treated with monobromobimane (mBBr), a membranepermeable bromobimane, or with monobromotrimethylammoniobimane (qBBr), a membrane-impermeable bromobimane. Labelled spermatozoa were examined by fluorescence microscopy and sperm proteins (whole sperm proteins and basic nuclear proteins) were analysed by gel electrophoresis. The membrane-permeable agent mBBr lightly labelled the perimeter of the acrosome of non-DTT-treated possum and wallaby spermatozoa, indicating the presence of peri-acrosomal thiol groups. After reduction of sperm disulfides by DTT, mBBr labelled the entire acrosome of both species. The membrane-impermeable agent qBBr did not label any part of the acrosome in non-DTT or DTT-treated wallaby or possum spermatozoa. Thiols and disulfides are thus associated with the marsupial acrosome. They are not found on the overlying plasma membrane but are either in the acrosomal membranes and/or matrix. The sperm midpiece and tail were labelled by mBBr, with increased fluorescence observed in DTT-treated spermatozoa. The nucleus was not labelled in non-DTT or DTT-treated spermatozoa. Electrophoretic analysis confirmed the microscopic observations: Basic nuclear protein (protamines) lacked thiols or disulfide groups. Based on these findings, the stability of the marsupial acrosome may be due in part to disulfide stabilization of the acrosomal membranes and/or acrosomal matrix. In common with placental mammals, thiol and disulfide containing proteins appear to play a role in the stability of sperm tail structures. It was confirmed that the fragile marsupial sperm nucleus lacked thiols and disulfides. © 1994 Wiley-Liss, Inc.     

Localization of a syntaxin isoform, syntaxin 2, to the acrosomal region of rodent spermatozoa

The acrosome reaction includes a membrane fusion event that is a prerequisite for sperm penetration through the zona pellucida and subsequent fertilization. Since SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have been shown to be key players in membrane fusion during regulated exocytosis in nerve terminals and secretory cells, and since the acrosome reaction has some features in common with regulated exocytosis, we hypothesized that SNARE proteins might also regulate acrosomal exocytosis. RT-PCR analysis demonstrated the expression of SNARE proteins, three isoforms of syntaxin 2 (2A, 2B, and 2C) and syntaxin 4A, in rat testes. Immunoblot analysis with anti-syntaxin 2 antibody showed that the protein was expressed in rodent spermatozoa, and that it was associated with membrane components of spermatozoa prepared by sucrose density gradient centrifugation. Confocal laser scanning microscopy with double immunolabeling revealed that syntaxin 2 was colocalized with acrin 1, a 90 kDa acrosomal protein, over the acrosomal region of spermatozoa but was not associated with the posterior half of head or tail. Localization of syntaxin 2 over the acrosomal region was supported by the finding that it was shed from sperm heads during an acrosome reaction induced by calcium ionophore A23187 in vitro. In view of the putative role of syntaxin proteins in other membrane fusion systems, these data suggest that syntaxin 2 may be involved in regulating the acrosomal reaction in rodent spermatozoa.     

Signaling pathways in sperm capacitation and acrosome reaction.

The binding to the egg's zona pellucida stimulates the spermatozoon to undergo acrosome reaction, a process which enables the sperm to penetrate the egg. Prior to this binding, the spermatozoa underago in the female reproductive tract a series of biochemical transformations, collectively called capacitation. The first event in capacitation is cholesterol efflux leading to the elevation of intracellular calcium and bicarbonate to activate adenylyl cyclase (AC) to produce cyclic-AMP, which activates protein kinase A (PKA) to indirectly phosphorylate certain proteins on tyrosine. During capacitation, there is also an increase in protein tyrosine phosphorylation dependent actin polymerization and in the membrane-bound phospholipase C (PLC). Sperm binding to zona-pellucida causes further activation of cAMP/PKA and protein kinase C (PKC), respectively. PKC opens a calcium channel in the plasma membrane. PKA together with inositol-trisphosphate activate calcium channels in the outer acrosomal membrane, which leads to an increase in cytosolic calcium. The depletion of calcium in the acrosome will activate a store-operated calcium entry mechanism in the plasma membrane, leading to a higher increase in cytosolic calcium, resulting in F-actin dispersion which enable the outer acrosomal and the plasma membrane to come into contact and fuse completing the acrosomal reaction.