Immunolocalization of bovine sperm protease BSp120 by light and electron microscopy during capacitation and the acrosome reaction: its role in in vitro fertilization

Mammalian fertilization involves various steps in which the participation of specific enzymes has been demonstrated by numerous studies. Acrosin is one of the most widely acrosomal protease in mammalian spermatozoa studied, including bovine; however, other proteases have also been described. A new trypsin-like serine protease named bovine serine protease of 120 kDa (BSp120) and its pre-cursor BSp66 (66 kDa) were identified in bovine spermatozoa. Cytological and ultrastructural immunolocalization studies on BSp120 were performed in live and fixed cells. Immunoflorescence assays with specific polyclonal antibodies revealed localization of BSp120 on the sperm head, with a signal homogeneously distributed over the acrosome resembling a horseshoe. After the acrosome reaction, sperm showed a patchy pattern in the acrosomal cap. Immune electron microscopy analysis indicated that BSp120 is located over the head plasma membrane of capacitated spermatozoa and acrosome reacting spermatozoa. To assess BSp120 function in sperm-oocyte interaction, in vitro fertilization studies were conducted. Oocytes were incubated with spermatozoa pre-treated with anti-BSp120, anti-guinea pig acrosin, and anti-BSp120 plus anti-guinea pig acrosin. Pre-treatment of bovine spermatozoa with antibodies towards each protein did not significantly modify fertilization rates. However, when both anti-acrosin and anti-BSp120 antibodies were simultaneously added, there was a significant decrease in the fertilization rate, suggesting that both enzymes may be required for fertilization. Altogether, the results from the present study described the localization of BSp120 over the acrosome of bovine sperm, and suggest its involvement in fertilization.     

MMP2 and acrosin are major proteinases associated with the inner acrosomal membrane and may cooperate in sperm penetration of the zona pellucida during fertilization

Sperm-zona pellucida (ZP) penetration during fertilization is a process that most likely involves enzymatic digestion of this extracellular coat by spermatozoa. Since the inner acrosomal membrane (IAM) is the leading edge of spermatozoa during penetration and proteins required for secondary binding of sperm to the zona are present on it, the IAM is the likely location of these enzymes. The objectives of this study were to identify and characterize proteinases present on the IAM, confirm their localization and provide evidence for their role in fertilization. Gelatin zymography of detergent extracts of the IAM revealed bands of enzymatic activity identified as serine and matrix metallo-proteinases (MMPs). Specific inhibitors to MMPs revealed that MMP activity was due to MMP2. Immunoblotting determined that the serine protease activity on the zymogram was due to acrosin and also confirmed the MMP2 activity. Immunogold labeling of spermatozoa at the electron microscope level showed that acrosin and MMP2 were confined to the apical and principal segments of the acrosome in association with the IAM, confirming our IAM isolation technique. Immunohistochemical examination of acrosin and MMP2 during spermiogenesis showed that both proteins originate in the acrosomic granule during the Golgi phase and later redistribute to the acrosomal membrane. Anti-MMP2 antibodies and inhibitors incorporated into in vitro fertilization media significantly decreased fertilization rates. This is the first study to demonstrate that MMP2 and acrosin are associated with the IAM and introduces the possibility of their cooperation in enzymatic digestion of the ZP during penetration.     

The molecular evolution of acrosin in placental mammals

Acrosin is thought to fulfill several different roles in fertilization including that of a serine protease and in secondary zona pellucida (ZP) binding. However, acrosin's importance as a fertilization protein has been questioned. Especially since it was discovered that acrosin knockout mice are fertile. In this study, we explored the sites involved in serine protease activity and secondary binding. We also assessed conservation in functional sites across species and examined whether amino acid changes present in the human population have the potential to affect fertility. In addition, since many mammalian reproduction proteins have been found to evolve rapidly, we tested for positive selection. Sequences from 43 mammals from all 19 placental orders, which included a total of 828 nucleotides from acrosin exons 2, 3, 4, and a portion of exon 5, were obtained. We found that all sites of the serine catalytic triad as well as three other sites linked to catalytic activity were completely conserved. Five of six sites proposed to play a role in secondary binding were 100% conserved as basic residues. These results support an evolutionary conserved role for acrosin as a serine protease and secondary binding protein across placental mammals. We found statistically significant support for positive selection within acrosin, but no single amino acid site reached the significance level of P > 0.95 for inclusion within the category omega > 1. Based upon two amino acid mutation scoring systems, three out of seven human residue changing single nucleotide polymorphisms (SNPs) were found to be potentially protein-altering mutations.     

EARLY STEPS OF SPERM—EGG INTERACTIONS DURING MAMMALIAN FERTILIZATION

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, inin vitrofertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.     

Mammalian zona pellucida glycoproteins: structure and function during fertilization

Zona pellucida (ZP) is a glycoproteinaceous translucent matrix that surrounds the mammalian oocyte and plays a critical role in the accomplishment of fertilization. In humans, it is composed of 4 glycoproteins designated as ZP1, ZP2, ZP3 and ZP4, whereas mouse ZP is composed of ZP1, ZP2 and ZP3 (Zp4 being a pseudogene). In addition to a variable sequence identity of a given zona protein among various species, human ZP1 and ZP4 are paralogs and mature polypeptide chains share an identity of 47%. Employing either affinity purified native or recombinant human zona proteins, it has been demonstrated that ZP1, ZP3 and ZP4 bind to the capacitated human spermatozoa and induce an acrosome reaction, whereas in mice, ZP3 acts as the putative primary sperm receptor. Human ZP2 only binds to acrosome-reacted spermatozoa and thus may be acting as a secondary sperm receptor. In contrast to O-linked glycans of ZP3 in mice, N-linked glycans of human ZP3 and ZP4 are more relevant for induction of the acrosome reaction. Recent studies suggest that Sialyl-Lewis^x sequence present on both N- and O-glycans of human ZP play an important role in human sperm?Cegg binding. There are subtle differences in the downstream signaling events associated with ZP3 versus ZP1/ZP4-mediated induction of the acrosome reaction. For example, ZP3 but not ZP1/ZP4-mediated induction of the acrosome reaction is dependent on the activation of the G_i protein-coupled receptor. Thus, various studies suggest that, in contrast to mice, in humans more than one zona protein binds to spermatozoa and induces an acrosome reaction.     

Phosphoinositide-dependent pathways in mouse sperm are regulated by egg ZP3 and drive the acrosome reaction

Sperm of many animals must complete an exocytotic event, the acrosome reaction, in order to fuse with eggs. In mammals, acrosome reactions are triggered during sperm contact with the egg extracellular matrix, or zona pellucida, by the matrix glycoprotein ZP3. Here, we show that ZP3 stimulates production of phosphatidylinositol-(3,4,5)-triphosphate in sperm membranes. Phosphatidylinositol-3-kinase antagonists that prevent acrosome reactions and fertilization in vitro, while generation of this phosphoinositide in the absence of ZP3 triggered acrosome reactions. Downstream effectors of phosphatidylinositol-(3,4,5)-triphosphate in sperm include the protein kinases, Akt and PKCzeta. These studies outline a signal transduction pathway that plays an essential role in the early events of mammalian fertilization.     

Transitional states of acrosomal exocytosis and proteolytic processing of the acrosomal matrix in guinea pig sperm

In this study, we adapted a FluoSphere bead-binding assay to study the exposure and release of guinea pig sperm acrosomal components during the course of capacitation and acrosomal exocytosis. Prior to capacitation or the initiation of exocytosis, acrosomal proteins were not accessible to FluoSpheres coated with antibodies against two acrosomal matrix (AM) proteins, AM67 and AM50; during the course of capacitation and ionophore-induced acrosomal exocytosis, however, we detected the transient exposure of the solid-phase AM proteins on the surface of guinea pig sperm using the antibody-coated fluorescent beads. Several different transitional stages leading to complete acrosomal exocytosis were classified, and we propose these represent true, functional intermediates since some of the AM proteins are orthologues of mouse proteins that bind the zona pellucida (ZP) of unfertilized eggs. In addition, we present evidence that implicates acrosin in the proteolytic processing of AM50 during AM disassembly. Thus, we propose that the transitional states of acrosomal exocytosis involve early binding of AM proteins to the ZP (by what visually appear to be "acrosome-intact" sperm), maintenance of ZP binding that coincides with the progressive exposure of AM proteins, and gradual proteolytic disassembly of the AM to allow sperm movement through the ZP. We feel this "transitional states" model provides a more refined view of acrosomal function that supports a move away from the widely held, overly simplistic, and binary "acrosome-reaction" model, and embraces a more dynamic view of acrosomal exocytosis that involves intermediate stages of the secretory process in ZP binding and penetration.     

Molecular basis of fertilization in the mouse

Recent studies of mouse fertilization have identified two complementary gamete receptors that mediate sperm-egg binding. Sperm surface β1,4-galactosyltransferase (GalTase) binds to specific oligosaccharides of the egg coat (zona pellucida) glycoprotein ZP3. Evidence suggests that these same molecules may stimulate the acrosome reaction in sperm. After the acrosome reaction, it is thought that sperm remain adherent to the zona by binding another glycoprotein, ZP2. The acrosome-reacted sperm releases hydrolytic enzymes, including acrosin and N-acetylglucosaminidase, enabling it to penetrate the zona pellucida. After the penetrating sperm binds to the egg membrane and activates development, N-acetylglucosaminidase is exocytosed from egg cortical granules and, as part of the zona block to polyspermy, globally removes the sperm GalTase binding site from ZP3 oligosaccharides.     

Interference with the 19S proteasomal regulatory complex subunit PSMD4 on the sperm surface inhibits sperm-zona pellucida penetration during porcine fertilization

Proteolysis of ubiquitinated sperm and oocyte proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis and sperm-zona pellucida (ZP) penetration. The present study examined the role of PSMD4, an essential non-ATPase subunit of the proteasomal 19S regulatory complex responsible for proteasome-substrate recognition, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Porcine sperm-ZP penetration, but not sperm-ZP binding, was blocked in the presence of a monoclonal anti-PSMD4 antibody during IVF. Inclusion in the fertilization medium of mutant ubiquitins (Ub+1 and Ub5+1), which are refractory to processing by the 19S regulatory complex and associated with Alzheimer’s disease, also inhibited fertilization. This observation suggested that subunit PSMD4 is exposed on the sperm acrosomal surface, a notion that was further supported by the binding of non-cell permeant, biotinylated proteasomal inhibitor ZL3VS to the sperm acrosome. Immunofluorescence localized PSMD4 in the sperm acrosome. Immunoprecipitation and proteomic analysis revealed that PSMD4 co-precipitated with porcine sperm-associated acrosin inhibitor (AI). Ubiquitinated species of AI were isolated from boar sperm extracts by affinity purification of ubiquitinated proteins using the recombinant UBA domain of p62 protein. Some proteasomes appeared to be anchored to the sperm head inner acrosomal membrane, as documented by co-fractionation studies. In conclusion, the 19S regulatory complex subunit PSMD4 is involved in the sperm-ZP penetration during fertilization. The recognition of substrates on the ZP by the 19S proteasomal regulatory complex is essential for the success of porcine/mammalian fertilization in vitro.     

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

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

Ultrastructural localization of proacrosin and acrosin in ram spermatozoa

Proacrosin and acrosin were localized immunocytochemically at the electron microscope level in ram spermatozoa undergoing an ionophore-induced acrosome reaction. Antigenicity was preserved after fixation with 0.5% w/v ethyl-(dimethylaminopropyl)-carbodimide, and an antibody preparation was used that reacted with all major forms of ram acrosin. All stages of the acrosome reaction could be observed in a single preparation. At the earliest stage, labeling was observed throughout the acrosomal contents, which were just beginning to disperse. As dispersal proceeded, labeling diminished, being associated only with visible remnants of the acrosomal matrix. By the time the acrosome had emptied, almost no labeling could be detected on the inner acrosomal membrane. The relationship between matrix dispersal and proacrosin activation was studied in isolated ram sperm heads. While proacrosin was prevented from activating, the acrosomal matrix remained compact; but as activation proceeded, the matrix decondensed and dispersed in close parallel. By the time proacrosin activation was complete, the acrosomal contents had almost entirely disappeared. We conclude that proacrosin is distributed throughout the acrosomal contents as an intrinsic constituent of the acrosomal matrix. During the acrosome reaction, proacrosin activation occurs, resulting directly in decondensation of the matrix. All the contents of the acrosome including acrosin disperse and, by the time the acrosome is empty and the acrosomal cap is lost, only occasional traces of acrosin remain on the inner acrosomal membrane. Since the acrosomal cap is normally lost during the earliest stages of zona penetration, acrosin's role in fertilization is unclear: it does not appear to be a zona lysin bound to the inner acrosomal membrane.     

Sperm epidermal growth factor receptor (EGFR) mediates α7 acetylcholine receptor (AChR) activation to promote fertilization

To attain fertilization the spermatozoon binds to the egg zona pellucida (ZP) via sperm receptor(s) and undergoes an acrosome reaction (AR). Several sperm receptors have been described in the literature; however, the identity of this receptor is not yet certain. In this study, we suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might be a sperm receptor activated by ZP to induce epidermal growth factor receptor (EGFR)-mediated AR. We found that isolated ZP or α7 agonists induced the AR in sperm from WT but not α7-null spermatozoa, and the induced AR was inhibited by α7 or EGFR antagonists. Moreover, α7-null sperm showed very little binding to the egg, and microfluidic affinity in vitro assay clearly showed that α7nAChR, as well as EGFR, interacted with ZP3. Induction of EGFR activation and the AR by an α7 agonist was inhibited by a Src family kinase (SFK) inhibitor. In conclusion we suggest that activation of α7 by ZP leads to SFK-dependent EGFR activation, Ca(2+) influx, and the acrosome reaction.     

Sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis

At fertilization, spermatozoa bind to the zona pellucida (ZP1, ZP2, ZP3) surrounding ovulated mouse eggs, undergo acrosome exocytosis and penetrate the zona matrix before gamete fusion. Following fertilization, ZP2 is proteolytically cleaved and sperm no longer bind to embryos. We assessed Acr3-EGFP sperm binding to wild-type and huZP2 rescue eggs in which human ZP2 replaces mouse ZP2 but remains uncleaved after fertilization. The observed de novo binding of Acr3-EGFP sperm to embryos derived from huZP2 rescue mice supports a ;zona scaffold' model of sperm-egg recognition in which intact ZP2 dictates a three-dimensional structure supportive of sperm binding, independent of fertilization and cortical granule exocytosis. Surprisingly, the acrosomes of the bound sperm remain intact for at least 24 hours in the presence of uncleaved human ZP2 regardless of whether sperm are added before or after fertilization. The persistence of intact acrosomes indicates that sperm binding to the zona pellucida is not sufficient to induce acrosome exocytosis. A filter penetration assay suggests an alternative mechanism in which penetration into the zona matrix initiates a mechanosensory signal transduction necessary to trigger the acrosome reaction.     

Equatorin is not essential for acrosome biogenesis but is required for the acrosome reaction

The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in mammalian fertilization. However, the regulatory mechanisms controlling acrosome biogenesis and acrosome exocytosis during fertilization are largely unknown. Equatorin (Eqtn) is a membrane protein that is specifically localized to the acrosomal membrane. In the present study, the physiological functions of Eqtn were investigated using a gene knockout mouse model. We found that Eqtn^−/− males were subfertile. Only approximately 50% of plugged females were pregnant after mating with Eqtn^−/− males, whereas more than 90% of plugged females were pregnant after mating with control males. Sperm and acrosomes from Eqtn^−/− mice presented normal motility and morphology. However, the fertilization and induced acrosome exocytosis rates of Eqtn-deficient sperm were dramatically reduced. Further studies revealed that the Eqtn protein might interact with Syntaxin1a and SNAP25, but loss of Eqtn did not affect the protein levels of these genes. Therefore, our study demonstrates that Eqtn is not essential for acrosome biogenesis but is required for the acrosome reaction. Eqtn is involved in the fusion of the outer acrosomal membrane and the sperm plasma membrane during the acrosome reaction, most likely via an interaction with the SNARE complex.     

Evidence that P36, a human sperm acrosomal antigen involved in the fertilization process is triosephosphate isomerase

P36 is one of the immunodominant sperm antigens identified by antibodies eluted from the spermatozoa of infertile men. In a previous study, we isolated and characterized this auto-antigen as a glycoprotein with several isoforms. Specific rabbit antibodies were produced to investigate sperm topography and the role of P36 in the fertilization process and we showed that P36 is present on the equatorial segment of acrosome-reacted spermatozoa and is involved in sperm-binding and the penetration of zona-free hamster oocytes. In the present study, we demonstrated, by means of immunofluorescence and electron microscopy, that P36 is present all over the acrosomal membranes of non-reacted spermatozoa. We also investigated the role of P36 in the acrosome reaction and sperm binding to the zona pellucida (ZP). The exposure of capacitated spermatozoa to rabbit anti-P36 antibodies had no effect on primary fixation to the ZP, but inhibited secondary binding to the ZP and the Ca2+ ionophore-induced acrosome reaction. These results suggest that P36, an acrosomal antigen, is involved in several steps of the fertilization process. On two-dimensional Western blots, human anti-sperm antibodies (ASA) and rabbit anti-P36 antibodies recognized five to six isoforms of P36, all 36/37 kDa in size, with a pI between 5.1 and 5.7. Two major spots were identified as human triosephosphate isomerase (TPI) by MALDI-TOF mass spectrometry. Anti-TPI antibodies were shown to react with the isoforms recognized by human and rabbit anti-P36 antibodies. We also demonstrated the presence of TPI in human sperm heads. Further studies are underway to establish whether there is a sperm-specific isoform of TPI and its role in sperm function.     

Proacrosin/acrosin during guinea pig spermatogenesis

Enriched populations of guinea pig spermatogenic cells were isolated by sedimentation velocity at unit gravity. Each cell population was analyzed for the presence of members of the proacrosin/acrosin family by enzymography, immunoblotting, and immunofluorescence. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis in gels containing 0.1% gelatin, protease activities with molecular weights of 55,000 (major) and 50,000 (minor) were detected in round spermatid extracts. Condensing spermatid extracts contained protease activities with molecular weights between 55,000 and 50,000. These major protease activities had molecular weights similar to antigens detected by immunoblotting with a monospecific rabbit antiserum directed against purified boar acrosin. Extracts of guinea pig sperm and the soluble acrosomal components released following the acrosome reaction induced with ionophore A23187 contained three major protease activities (Mr 32,000, 34,000, 47,000) but only the 47,000 Mr protease cross-reacted with the antibody. The spermatid and sperm protease activities were inhibited and activated by classical effectors of acrosin activity from other species. Immunofluorescence demonstrated that proacrosin/acrosin was present as early as the Golgi phase of spermiogenesis. In addition, immunoreactivity was confined to the acrosomes in a manner characteristic of each spermatid stage. These results demonstrate that proacrosin/acrosin can be detected in the earliest spermiogenic stages by electrophoretic and immunological techniques and suggest that changes in the molecular weights of proacrosin/acrosin occur as spermatids mature.     

Profile of a mammalian sperm receptor

Complementary molecules on the surface of eggs and sperm are responsible for species-specific interactions between gametes during fertilization in both plants and animals. In this essay, several aspects of current research on the mouse egg receptor for sperm, a zona pellucida glycoprotein called ZP3, are addressed. These include the structure, synthesis, and functions of the sperm receptor during oogenesis and fertilization in mice. Several conclusions are drawn from available information. These include (I) ZP3 is a member of a unique class of glycoproteins found exclusively in the extracellular coat (zona pellucida) of mammalian eggs. (II) ZP3 gene expression is an example of oocyte-specific and, therefore, sex-specific gene expression during mammalian development. (III) ZP3 is a structural glycoprotein involved in assembly of the egg extracellular coat during mammalian oogenesis. (IV) ZP3 is a sperm receptor involved in carbohydrate-mediated gamete recognition and adhesion during mammalian fertilization. (V) ZP3 is an inducer of sperm exocytosis (acrosome reaction) during mammalian fertilization. (VI) ZP3 participates in the secondary block to polyspermy following fertilization in mammals. (VII) The extracellular coat of other mammalian eggs contains a glycoprotein that is functionally analogous to mouse ZP3. The unique nature, highly restricted expression, and multiple roles of ZP3 during mammalian development make this glycoprotein a particularly attractive subject for investigation at both the cellular and molecular levels.     

Functional interactions between sulphated polysaccharides and proacrosin: implications in sperm binding and digestion of zona pellucida.

Acrosin is a serine protease located within mammalian acrosome as inactive proacrosin. Sulphated polymers bind to proacrosin and acrosin, to a domain different from the active site. Upon binding, these polymers induce proacrosin activation and some of them, such as fucoidan, inhibit sperm binding to the zona pellucida. In this work we have studied the interaction of solubilised zona pellucida glycoproteins (ZPGs), heparin and ARIS (Acrosome Reaction Inducing Substance of Starfish) with boar and human acrosin. We have found that ARIS, solubilised ZPGs and fucoidan, but not heparin, inhibit the binding of the monoclonal antibody against human acrosin C5F10 to boar or human proacrosin. These results suggest that fucoidan, solubilised ZPGs and ARIS bind to a related domain on the proacrosin surface. Moreover, ARIS was able to induce human proacrosin activation. On the other hand, neither ARIS nor heparin from porcine intestinal mucosa or bovine lung induced hamster sperm acrosome reaction or sperm motility. Recent data showed that acrosin is involved in dispersal of the acrosomal matrix after acrosome reaction. Thus, the control of the ZPG glycan chains over proacrosin activation may regulate both sperm penetration rate and limited proteolysis of zona pellucida proteins.     

Disulfide linkage patterns of pig zona pellucida glycoproteins ZP3 and ZP4

Zona pellucida, a transparent envelope surrounding the mammalian oocyte, plays major roles in fertilization and consists of three or four glycoproteins. Primary structures, and especially the positions of cysteine (Cys) residues in the zona glycoproteins, are well conserved among mammals. In this study, we analyzed the disulfide linkages of pig ZP3 and ZP4 purified from ovaries. While disulfide linkage patterns of four Cys residues in the N-terminal halves of the ZP domains of ZP3 and ZP4 were identical to those previously reported for mice, rats, humans, and fish, the disulfide linkage patterns of six Cys residues in the C-terminal half of the ZP domain in ZP4, as well as eight Cys residues in the C-terminal region of the ZP domain and a following region unique to ZP3, were different from those previously reported. Thus, higher-order structures of zona glycoproteins might not be conserved in the C-terminal regions.     

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.