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.     

Complexin I is required for mammalian sperm acrosomal exocytosis

Regulated exocytosis in many cells is controlled by the SNARE complex, whose core includes three proteins that promote membrane fusion. Complexins I and II are highly related cytosolic proteins that bind tightly to the assembled SNARE complex and regulate neuronal exocytosis. Like somatic cells, sperm undergo regulated exocytosis; however, sperm release a single large vesicle, the acrosome, whose release has different characteristics than neuronal exocytosis. Acrosomal release is triggered upon sperm adhesion to the mammalian egg extracellular matrix (zona pellucida) to allow penetration of the egg coat. Membrane fusion occurs at multiple points within the acrosome but how fusion is activated and the formation and progression of fusion points is synchronized is unclear. We show that complexins I and II are found in acrosome-intact mature sperm, bind to SNARE complex proteins, and are not detected in sperm after acrosomal exocytosis (acrosome reaction). Although complexin-I-deficient sperm acrosome-react in response to calcium ionophore, they do not acrosome-react in response to egg zona pellucida proteins and have reduced fertilizing ability, in vitro. Complexin II is present in the complexin-I-deficient sperm and its expression is increased in complexin-I-deficient testes. Therefore, complexin I functions in exocytosis in two related but morphologically distinct secretory processes. Sperm are unusual because they express both complexins I and II but have a unique and specific requirement for complexin I.     

Zonadhesin assembly into the hamster sperm acrosomal matrix occurs by distinct targeting strategies during spermiogenesis and maturation in the epididymis

Zonadhesin is the only sperm protein known to bind in a species-specific manner to the zona pellucida. The zonadhesin precursor is a mosaic protein with a predicted transmembrane segment and large extracellular region composed of cell adhesion, mucin, and tandem von Willebrand D domains. Because the precursor possesses a predicted transmembrane segment and localizes to the anterior head, the mature protein was presumed to be a sperm surface zona pellucida-binding protein. In this study of hamster spermatozoa, we demonstrate that zonadhesin does not localize to the sperm surface but is instead a constituent of the acrosomal matrix. Immunoelectron microscopy revealed that distinct targeting pathways during spermiogenesis and sperm maturation in the epididymis result in trafficking of zonadhesin to the acrosomal matrix. In round spermatids, zonadhesin localized specifically to the acrosomal membrane, where it appeared to be evenly distributed between the outer and inner membrane domains. Subsequent redistribution of zonadhesin resulted in its elimination from the inner acrosomal membrane and restriction to the outer acrosomal membrane of the apical and principal segments and the contents of the posterior acrosome. During sperm maturation in the epididymis, zonadhesin dissociated from the outer acrosomal membrane and became incorporated into the forming acrosomal matrix. These data suggest an important structural role for zonadhesin in assembly of the acrosomal matrix and further support the view that the species specificity of zona pellucida adhesion is mediated by egg-binding proteins contained within the acrosome rather than on the periacrosomal plasma membrane.     

The acrosomal vesicle of mouse sperm is a calcium store

Subsequent to binding to the zona pellucida, mammalian sperm undergo a regulated sequence of events that ultimately lead to acrosomal exocytosis. Like most regulated exocytotic processes, a rise in intracellular calcium is sufficient to trigger this event although the precise mechanism of how this is achieved is still unclear. Numerous studies on mouse sperm have indicated that a voltage-operated Ca2+ channel plays some immediate role following sperm binding to the zona pellucida glycoprotein ZP3. However, there is also evidence that the mammalian sperm acrosome contains a high density of IP3 receptors, suggesting that the exocytotic event involves the release of Ca2+ from the acrosome. The release of Ca2+ from the acrosome may directly trigger exocytosis or may activate store-operated Ca2+ channels in the plasma membrane. To test the hypothesis that the acrosome is an intracellular store we loaded mammalian sperm with the membrane permeant forms of three Ca2+-sensitive fluorescent indicator dyes: fura-2, indo-1, and Calcium Green-5N. Fluorescence microscopy revealed that the sperm were labeled in all intracellular compartments. When fura-2 labeled sperm were treated with 150 microM MnCl2 to quench all fluorescence in the cytosol, or when the sperm were labeled with the low affinity dye Calcium Green-5N, there was a large Ca2+ signal in the acrosome. Consistent with the acrosome serving as an intracellular Ca2+ reservoir, the addition of 20 microM thapsigargin, a potent inhibitor of the smooth endoplasmic reticular Ca2+-ATPase (SERCA), to populations of capacitated sperm resulted in nearly 100% acrosomal exocytosis within 60 min (tau1/2 approximately 10 min), in the absence of extracellular Ca2+. Additionally, treatment of sperm with 100 microM thimerosal, an IP3 receptor agonist, also resulted in acrosomal exocytosis. Taken together, these data suggest that the mouse sperm acrosome is a Ca2+ store that regulates its own exocytosis through an IP3 Ca2+ mobilization pathway.     

The regulation of acrosomal exocytosis. I. Sperm capacitation is required for the induction of acrosome reactions by the bovine zona pellucida in vitro

The regulation of acrosomal exocytosis in capacitated bovine spermatozoa by soluble extracts of zonae pellucidae was examined. Kinetic studies demonstrated that zonae pellucidae stimulated synchronous acrosome reactions. The t1/2 of this process was 5-10 min and response was maximal at 20 min. The apparent initial rate of exocytosis in sperm populations was dependent upon the concentration of zona pellucida protein, with an ED50 and a maximally effective dosage of 20 and 50 ng protein/microliter, respectively. Zonae pellucidae caused up to a 48-fold increase in the apparent initial rate and a 3- to 4-fold stimulation in the net occurrence of exocytosis. In contrast, solubilized zonae pellucidae did not induce acrosome reactions in uncapacitated sperm. The development of a capacitated state, as assayed by the ability of sperm to fertilize eggs in vitro, was compared to the expression of zona pellucida-regulated acrosome reactions in a series of kinetic experiments. Both activities were manifest with similar kinetics and displayed identical dependencies toward stimulatory and inhibitory agents in vitro. It is concluded that capacitation is an essential prerequisite for the induction of acrosomal exocytosis in bovine sperm by the zona pellucida.     

The role of the acrosomal matrix in fertilization

Mammalian sperm must have properly formed acrosomes to be fully functional in the process of binding and penetrating the zona pellucida (ZP), the extracellular matrix surrounding the egg. There is much evidence to raise doubts about the old "bag of enzymes" paradigm of acrosomal function, although this is the model that seems to prevail. We concur with other scientists that acrosomal exocytosis is not an all or none event where the acrosome is either "intact" or "reacted". As determined by transmission electron microscopy of human sperm undergoing acrosomal exocytosis, six stages can be identified, with the intermediate ones involving loss of acrosomal matrix material. In the mouse, there is a temporal relationship among four stages of acrosomal exocytosis. Numerous evidences suggest a more complex role for the acrosome in fertilization in which the acrosomal matrix is a scaffold for sperm-ZP interactions that self-regulates by a controlled disassembly mechanism.     

PH‐20 but not acrosin is involved in sperm penetration of the macaque zona pellucida

In this study, we investigated the functions of PH‐20 and acrosin during the interaction of macaque sperm with the zona pellucida. Both of these sperm enzymes have been reported to be present on the inner acrosomal membrane of acrosome reacted sperm, and have been suggested to play a role during secondary sperm‐zona binding in other species. Anti‐macaque PH‐20 IgG, anti‐pig acrosin IgG and soybean trypsin inhibitor (SBTI) were used as probes for immunolocalization of the two proteins at the ultrastructural level, and as reagents for blocking sperm penetration of the macaque zona pellucida in vitro. As a control, we performed similar studies with antibodies to CD‐46, which is also located on the inner acrosomal membrane, but has no known function in sperm‐zona pellucida interaction. After labeling with anti‐acrosin IgG, gold label was not present on the sperm surface before the acrosome reaction, but was detected over the entire head of sperm that were induced to acrosome react with calcium ionophore A23187. In contrast, when sperm were induced to acrosome react by binding to intact zona pellucida, acrosin was present in the acrosomal shroud but not on the inner acrosomal membrane. Similar results were obtained when SBTI was used as a probe for enzyme localization. PH‐20 and CD‐46 were demonstrated on the inner acrosomal membrane of sperm induced to acrosome react by ionophore treatment and by zona binding. Neither anti‐acrosin IgG nor anti‐CD‐46 IgG affected sperm penetration of the zona at concentrations up to 300 μg/ml, but zona penetration was blocked completely when anti‐PH‐20 IgG (100 μg/ml) was present during sperm‐oocyte interaction. Ultrastructural observations of oocytes incubated with anti‐PH‐20 IgG showed that acrosomal shrouds were present on the zona surface but no sperm had begun to penetrate into the zona substance. We conclude that anti‐PH‐20 IgG prevented sperm penetration of the macaque zona pellucida by interference with secondary sperm‐zona binding, rather than primary sperm‐zona binding or the zona‐induced acrosome reaction. Acrosin was not detected on the inner acrosomal membrane of sperm that are induced to acrosome react after zona binding, and acrosin does not appear to be critical for sperm penetration of the macaque zona pellucida. Mol. Reprod. Dev. 53:350–362, 1999. © 1999 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.     

Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction.

Mammalian fertilization involves interactions of sperm surface receptors with ligands of the zona pellucida, an extracellular matrix surrounding the ovulated egg. In mouse, the zona is composed of three glycoproteins. One of them, ZP3, participates in primary sperm binding and in the subsequent triggering of the sperm's acrosome reaction. Considerable evidence suggests that carbohydrate determinants of ZP3 are responsible for binding to sperm and may be important for acrosomal exocytosis. A full-length cDNA encoding mouse ZP3 was assembled and cloned into expression vectors that contained either a cytomegalovirus (CMV) or a vaccinia (P11) promoter. Mouse L-929 cells were stably transformed with the pZP3-CMV constructs, and green monkey CV-1 cells were infected with a recombinant vaccinia virus containing ZP3. rZP3 was affinity purified from culture media and detected on Western blots as a single 60- to 70-kDa band, which differed in molecular weight from native ZP3 (mean, 83 kDa). Nevertheless, rZP3 is biologically active. rZP3 decreases sperm-zona binding with a potency equivalent to that of native zona pellucida and, like native ZP3, rZP3 triggers acrosomal exocytosis in capacitated mouse sperm. Thus, rZP3 isolated from both rodent and primate cells appears to contain those carbohydrate and protein structures necessary for ZP3's dual role in fertilization.     

Cell surface beta-1,4-galactosyltransferase-I activates G protein-dependent exocytotic signaling

ZP3 is a protein in the mammalian egg coat (zona pellucida) that binds sperm and stimulates acrosomal exocytosis, enabling sperm to penetrate the zona pellucida. The nature of the ZP3 receptor/s on sperm is a matter of considerable debate, but most evidence suggests that ZP3 binds to beta-1,4-galactosyltransferase-I (GalTase) on the sperm surface. It has been suggested that ZP3 induces the acrosome reaction by crosslinking GalTase, activating a heterotrimeric G protein. In this regard, acrosomal exocytosis is sensitive to pertussis toxin and the GalTase cytoplasmic domain can precipitate G(i) from sperm lysates. Sperm from mice that overexpress GalTase bind more soluble ZP3 and show accelerated G protein activation, whereas sperm from mice with a targeted deletion in GalTase have markedly less ability to bind soluble ZP3, undergo the ZP3-induced acrosome reaction, and penetrate the zona pellucida. We have examined the ability of GalTase to function as a ZP3 receptor and to activate heterotrimeric G proteins using Xenopus laevis oocytes as a heterologous expression system. Oocytes that express GalTase bound ZP3 but did not bind other zona pellucida glycoproteins. After oocyte maturation, ZP3 or GalTase antibodies were able to trigger cortical granule exocytosis and activation of GalTase-expressing eggs. Pertussis toxin inhibited GalTase-induced egg activation. Consistent with G protein activation, both ZP3 and anti-GalTase antibodies increased GTP-gamma[(35)S] binding as well as GTPase activity in membranes from eggs expressing GalTase. Finally, mutagenesis of a putative G protein activation motif within the GalTase cytoplasmic domain eliminated G protein activation in response to ZP3 or anti-GalTase antibodies. These results demonstrate directly that GalTase functions as a ZP3 receptor and following aggregation, is capable of activating pertussis toxin-sensitive G proteins leading to exocytosis.     

Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O- linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and - reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.     

Identification and characterization of RING-finger ubiquitin ligase UBR7 in mammalian spermatozoa

The ubiquitin-proteasome system (UPS) controls intracellular protein turnover in a substrate-specific manner via E3-type ubiquitin ligases. Mammalian fertilization and particularly sperm penetration through the oocyte vitelline coat, the zona pellucida (ZP), is regulated by UPS. We use an extrinsic substrate of the proteasome-dependent ubiquitin-fusion degradation pathway, the mutant ubiquitin UBB⁺¹, to provide evidence that an E3-type ligase activity exists in sperm-acrosomal fractions. Protein electrophoresis gels from such de novo ubiquitination experiments contained a unique protein band identified by tandem mass spectrometry as being similar to ubiquitin ligase UBR7 (alternative name: C14ORF130). Corresponding mRNA was amplified from boar testis and several variants of the UBR7 protein were detected in boar, mouse and human sperm extracts by Western blotting. Genomic analysis indicated a high degree of evolutionary conservation, remarkably constant purifying selection and conserved testis expression of the UBR7 gene. By immunofluorescence, UBR7 was localized to the spermatid acrosomal cap and sperm acrosome, in addition to hotspots of proteasomal activity in spermatids, such as the cytoplasmic lobe, caudal manchette, nucleus and centrosome. During fertilization, UBR7 remained with the ZP-bound acrosomal shroud following acrosomal exocytosis. Thus, UBR7 is present in the acrosomal cap of round spermatids and within the acrosomal matrix of mature boar spermatozoa. These data provide the first evidence of ubiquitin ligase activity in mammalian spermatozoa and indicate UBR7 involvement in spermiogenesis.     

A synthetic decapeptide from a conserved ZP3 protein domain induces the G protein-regulated acrosome reaction in bovine spermatozoa

In some animal species, the zona pellucida protein 3 (ZP3) plays a central role during fertilization, functioning as a specific receptor for sperm and as an inducer of the acrosome reaction. On the other hand, the zona pellucida protein 2 (ZP2) acts as a secondary receptor, binding to acrosome-reacted sperm. The objective of these studies was to identify ZP2 and ZP3 domains that may be of importance for the induction of the acrosome reaction. For this purpose, we synthesized a number of ZP2 and ZP3 peptides that were either conserved among species or that were species-specific according to their respective primary structures. We identified a defined, conserved ZP3 decapeptide (ZP3-6 peptide) that bound to the surface of the acrosomal region and induced the acrosome reaction in a concentration-dependent manner in capacitated bovine sperm; this effect was significant in the nanomolar range. Pertussis toxin inhibited the ZP3-6 peptide-induced acrosome reaction but had no effect on the progesterone-induced exocytotic event. Our data are in accordance with previous studies showing that progesterone induces acrosomal exocytosis via a different pathway than ZP3 and strengthen the hypothesis that the effect of ZP3-6 peptide upon acrosomal exocytosis is G protein regulated. Despite the commonly accepted idea that glycosylation of ZP proteins is required for successful sperm-oocyte interaction, we found that acrosomal exocytosis can be induced by a synthetic ZP3 peptide that is not glycosylated. The results presented in this study may be useful for the investigation of the molecular mechanisms of sperm-egg interaction in bovine and other species.     

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.     

Status of the rat acrosome during sperm-zona pellucida interactions

Over the past 40 years evidence from many sources has indicated that the mammalian acrosome reaction occurs within or near the cumulus oophorus. Recently, however, workers investigating in vitro fertilization in the mouse have concluded that in this system the acrosome reaction takes place on the surface of the zona pellucida. We have investigated the interaction of rat spermatozoa and the zona pellucida by using the scanning electron microscope (SEM) and two monoclonal antibodies which are directed to antigens of the rat sperm acrosome. When in vitro inseminated eggs from which the cumulus has been removed are viewed with the SEM some sperm heads on the surface of the zona pellucida appear unaltered whereas others appear to be undergoing changes. In vivo, all displayed altered head morphology. Using immunogold labeling we found that the two antibodies employed, 2C4 and 5B1, were directed to acrosomal content and vesiculating acrosomal membranes. Immunofluoresence staining of zonae pellucidae in in vitro fertilization studies revealed numerous small positive regions. These were presumably acrosomal content and membranes which had been left on the zona surface by spermatozoa which had been associated with the zona surface. Our results suggest that the rat acrosome interacts with the zona pellucida. During this interaction some acrosomal content and membranes detach from the spermatozoon and remain on the surface of the zona pellucida.     

Zona pellucida protein binding ability of porcine sperm during epididymal maturation and the acrosome reaction

In many mammals, the first interaction between gametes during fertilization occurs when sperm contact the zona pellucida surrounding the egg. Although porcine sperm first contact the zona pellucida via their plasma membrane, the regions of the sperm surface that display zona receptors have not been determined. We have used the Alexa 488 fluorophore conjugated to solubilized porcine zona pellucida proteins to observe zona receptors on live boar sperm. Zona proteins bound live, acrosome-intact sperm on the anterior portion of the sperm head, concentrated in a thin band over the acrosomal ridge. When sperm membranes were permeabilized by fixation or acrosome reactions induced by the ionophore A23187, zona binding was extended to a broad area covering the entire acrosomal region. Zona binding proteins were present in the acrosomes of sperm from all regions of the epididymis. In contrast, zona binding sites were found on the plasma membrane of most sperm from the corpus and cauda epididymis, but on only 6% of caput epididymal sperm. In conclusion, acrosome-intact boar sperm exhibit concentrated zona protein binding over the acrosomal ridge and acquire this binding in the corpus region of the epididymis, correlating with the developmental stage at which sperm gain the ability to fertilize oocytes.     

Function of the acrosomal matrix: zona pellucida 3 receptor (ZP3R/sp56) is not essential for mouse fertilization

In mammalian fertilization, sperm-zona pellucida binding is considered to be a critical aspect of gamete interaction. In this study, we examine the mouse sperm acrosomal matrix protein zona pellucida 3 receptor (ZP3R; formerly called sp56) because of our interest in defining the function of the acrosomal matrix, the particulate compartment within the sperm secretory acrosome. Using targeted deletion of the Zp3r gene by homologous recombination, we examined the fertility of nullizygous animals. Our experiments showed that males and females homozygous for the affected gene exhibited no differences in litter sizes compared to wild-type and heterozygous animals. Testis weights of nullizygous males were equivalent to those of wild-type and heterozygous males, and no differences in the number of sperm produced by mice of three genotypes were found. In vitro fertilization rates using cumulus-intact and cumulus-free oocytes were also equivalent. Examination of sperm-binding zonae of unfertilized eggs and the ability of the sperm to undergo acrosomal exocytosis in response to calcium ionophore A23187 displayed no differences between wild-type, heterozygous, and nullizygous mouse sperm. These results provide further evidence that either ZP3R is not involved in sperm-zona pellucida binding or this process might be functionally redundant, involving multiple proteins for gamete interactions.     

Acrosomal exocytosis of mouse sperm progresses in a consistent direction in response to zona pellucida

Sperm acrosomal exocytosis is essential for successful fertilization, and the zona pellucida (ZP) has been classically considered as the primary initiator in vivo. At present, following what is referred to as primary binding of the sperm to the ZP, the acrosome reaction paradigm posits that the outer acrosomal membrane and plasma membrane fuse at random points, releasing the contents of the acrosome. It is then assumed that the inner acrosomal membrane mediates secondary binding of the sperm to the ZP. In the present work we used a live fluorescence imaging system and mouse sperm containing enhanced green fluorescent protein (EGFP) in their acrosomes. We compared the processes of acrosomal exocytosis stimulated by the calcium ionophore ionomycin or by solubilized ZP. As monitored by the loss of EGFP from the sperm, acrosomal exocytosis driven by these two agents occurred differently. When ionomycin was used, exocytosis started randomly (no preference for the anterior, middle or posterior acrosomal regions). In contrast, following treatment with solubilized ZP, the loss of acrosomal components always started at the posterior zone of the acrosome and progressed in an anterograde direction. The exocytosis was slower when stimulated with ZP and on the order of 10 sec, which is in accordance with other reports. These results demonstrate that ZP stimulates acrosomal exocytosis in an orderly manner and suggest that a receptor‐mediated event controls this process of membrane fusion and release of acrosomal components. These findings are incorporated into a model. J. Cell. Physiol. 220: 611–620, 2009. © 2009 Wiley‐Liss, Inc.