Aggregation of beta-1,4-galactosyltransferase on mouse sperm induces the acrosome reaction

beta-1,4-Galactosyltransferase (GalTase) is present on the surface of mouse sperm, where it functions during fertilization by binding to oligosaccharide residues in the egg zona pellucida. The specific oligosaccharide substrates for sperm GalTase reside on the glycoprotein ZP3, which possesses both sperm-binding and acrosome reaction-inducing activity. A variety of reagents that perturb sperm GalTase activity inhibit sperm binding to the zona pellucida, including UDP-galactose, N-acetylglucosamine, alpha-lactalbumin, and anti-GalTase Fab fragments. However, none of these reagents are able to cross-link GalTase within the membrane nor are they able to induce the acrosome reaction. On the other hand, intact anti-GalTase IgG blocks sperm-zona binding as well as induces the acrosome reaction. Anti-GalTase IgG induces the acrosome reaction by aggregating GalTase on the sperm plasma membrane, as shown by the inability of anti-Gal-Tase Fab fragments to induce the acrosome reaction unless cross-linked with goat anti-rabbit IgG. These data suggest that zona pellucida oligosaccharides induce the acrosome reaction by clustering GalTase on the sperm surface.     

Overexpressing sperm surface beta 1,4-galactosyltransferase in transgenic mice affects multiple aspects of sperm-egg interactions

Sperm surface beta 1,4-galactosyltransferase (GalTase) mediates fertilization in mice by binding to specific O-linked oligosaccharide ligands on the egg coat glycoprotein ZP3. Before binding the egg, sperm GalTase is masked by epididymally derived glycosides that are shed from the sperm surface during capacitation. After binding the egg, sperm- bound oligosaccharides on ZP3 induce the acrosome reaction by receptor aggregation, presumably involving GalTase. In this study, we asked how increasing the levels of sperm surface GalTase would affect sperm-egg interactions using transgenic mice that overexpress GalTase under the control of a heterologous promoter. GalTase expression was elevated in many tissues in adult transgenic animals, including testis. Sperm from transgenic males had approximately six times the wild-type level of surface GalTase protein, which was localized appropriately on the sperm head as revealed by indirect immunofluorescence. As expected, sperm from transgenic mice bound more radiolabeled ZP3 than did wild-type sperm. However, sperm from transgenic animals were relatively unable to bind eggs, as compared to sperm from wild-type animals. The mechanistic basis for the reduced egg-binding ability of transgenic sperm was attributed to alterations in two GalTase-dependent events. First, transgenic sperm that overexpress surface GalTase bound more epididymal glycoside substrates than did sperm from wild-type mice, thus masking GalTase and preventing it from interacting with its zona pellucida ligand. Second, those sperm from transgenic mice that were able to bind the zona pellucida were hypersensitive to ZP3, such that they underwent precocious acrosome reactions and bound to eggs more tenuously than did wild-type sperm. These results demonstrate that sperm-egg binding requires an optimal, rather than maximal, level of surface GalTase expression, since increasing this level decreases sperm reproductive efficiency both before and after egg binding. Although sperm GalTase is required for fertilization by serving as a receptor for the egg zona pellucida, excess surface GalTase is counterproductive to successful sperm-egg binding.     

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.     

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.     

Receptor function of mouse sperm surface galactosyltransferase during fertilization

Past studies from this laboratory have suggested that mouse sperm binding to the egg zona pellucida is mediated by a sperm galactosyltransferase (GalTase), which recognizes and binds to terminal N-acetylglucosamine (GlcNAc) residues in the zona pellucida (Shur, B. D., and N. G. Hall, 1982, J. Cell Biol. 95:567-573; 95:574-579). We now present evidence that directly supports this mechanism for gamete binding. GalTase was purified to homogeneity by sequential affinity-chromatography on GlcNAc-agarose and alpha-lactalbumin-agarose columns. The purified enzyme produced a dose-dependent inhibition of sperm binding to the zona pellucida, relative to controls. To inhibit sperm/zona binding, GalTase had to retain its native conformation, since neither heat-inactivated nor Mn++-deficient GalTase inhibited sperm binding. GalTase inhibition of sperm/zona binding was not due to steric blocking of an adjacent sperm receptor on the zona, since GalTase could be released from the zona pellucida by forced galactosylation with UDPGal, and the resulting galactosylated zona was still incapable of binding sperm. In control experiments, when UDPGal was replaced with the inappropriate sugar nucleotide, UDPglucose, sperm binding to the zona pellucida remained normal after the adsorbed GalTase was washed away. The addition of UDPGal produced a dose-dependent inhibition of sperm/zona binding, and also dissociated preformed sperm/zona adhesions by catalyzing the release of the sperm GalTase from its GlcNAc substrate in the zona pellucida. Under identical conditions, UDP-glucose had no effect on sperm binding to the zona pellucida. The ability of UDPGal to dissociate sperm/zona adhesions was both time- and temperature-dependent. UDPGal produced nearly total inhibition of sperm/zona binding when the zonae pellucidae were first galactosylated to reduce the number of GalTase binding sites. Finally, monospecific anti-GalTase IgG and its Fab fragments produced a dose-dependent inhibition of sperm/zona binding and concomitantly blocked sperm GalTase catalytic activity. Preimmune IgG or anti-mouse brain IgG, which also binds to the sperm surface, had no effect. The sperm GalTase was localized by indirect immunofluorescence to a discrete plasma membrane domain on the dorsal surface of the anterior head overlying the intact acrosome. These results, along with earlier studies, show clearly that sperm GalTase serves as a principal gamete receptor during fertilization.     

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.     

Zonadhesin Is Essential for Species Specificity of Sperm Adhesion to the Egg Zona Pellucida

Interaction of rapidly evolving molecules imparts species specificity to sperm-egg recognition in marine invertebrates, but it is unclear whether comparable interactions occur during fertilization in any vertebrate species. In mammals, the sperm acrosomal protein zonadhesin is a rapidly evolving molecule with species-specific binding activity for the egg zona pellucida (ZP). Here we show using null mice produced by targeted disruption of Zan that zonadhesin confers species specificity to sperm-ZP adhesion. Sperm capacitation selectively exposed a partial von Willebrand D domain of mouse zonadhesin on the surface of living, motile cells. Antibodies to the exposed domain inhibited adhesion of wild-type spermatozoa to the mouse ZP but did not inhibit adhesion of spermatozoa lacking zonadhesin. Zan^−/− males were fertile, and their spermatozoa readily fertilized mouse eggs in vitro. Remarkably, however, loss of zonadhesin increased adhesion of mouse spermatozoa to pig, cow, and rabbit ZP but not mouse ZP. We conclude that zonadhesin mediates species-specific ZP adhesion, and Zan^−/− males are fertile because their spermatozoa retain adhesion capability that is not species-specific. Mammalian sperm-ZP adhesion is therefore molecularly robust, and species-specific egg recognition by a protein in the sperm acrosome is conserved between invertebrates and vertebrates, even though the adhesion molecules themselves are unrelated.     

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.     

Isolation of antiSLIP1-reactive boar sperm P68/62 and its binding to mammalian zona pellucida

Single-step purification of boar sperm P68/62 that is cross-reactive with a polyclonal antibody against sulfolipidimmobilizing protein 1 (SLIP1) was achieved by chromatofocusing. This method is useful for obtaining P68/62 in quantity. The two proteins, P68 and P62, were antigenically related, since the antibody generated specifically against the 68-kDa band reacted with both the 68- and 62-kDa bands. Like rat testis SLIP1, purified boar sperm P68/62 bound to sulfogalactosylglycerolipid (SGG) and inhibited sperm-egg binding in a dose-dependent manner when added exogenously to sperm-egg coincubates. This inhibitory effect occurred at the level of the zona pellucida (ZP), and further studies showed that biotinylated boar sperm P68/62 bound to the ZP of unfertilized mouse eggs. Furthermore, biotinylated boar sperm P68/62 bound to isolated ZP of unfertilized eggs from other species, including pig, rat, cat, dog, and human, as well as to ZP of intact fertilized mouse eggs and preimplantation embryos of various developmental stages, although the degree of its binding to the ZP of intact eight-cell embryos, morulae, and blastocysts was much lower than that of fertilized eggs and two-cell embryos. These results suggest that P68/62 of capacitated sperm must act together with other sperm surface proteins/molecules that regulate zona binding specificity within homologous species and in unfertilized eggs. Together with our previous findings, we suggest that rather than being a true ZP receptor, sperm P68/62 may be involved in the initial step of sperm-ZP binding that is adhesive in nature. Mol. Reprod. Dev. 49:203–216, 1998 © 1998 Wiley-Liss, Inc.     

Plasma membrane association, purification, and partial characterization of mouse sperm beta 1,4-galactosyltransferase

Gamete recognition in the mouse is mediated, in part, by the binding of sperm surface galactosyltransferase (GalTase) to appropriate substrates in the egg zona pellucida. In this paper, sperm GalTase is shown to be an externally oriented, integral plasma membrane component. GalTase is not peripherally adsorbed to the cell surface, nor is it bound to cell surface glycoside substrates. GalTase can be released from the surface of intact sperm by either mild proteolysis or by detergent under conditions in which the sperm membranes remain intact as judged by double-label indirect immunofluorescence. Detergent-solubilized sperm GalTase has been purified to apparent homogeneity by affinity chromatography and characterized as a beta 1,4-GlcNAc:GalTase by substrate and kinetic analyses. Purified and membrane-bound GalTase both show an unusual thermal inactivation above 39-40 degrees C, whereas other sperm enzyme activities as well as GalTase activity from other cell types are temperature-dependent. Purified sperm GalTase inhibits sperm binding to the egg zona pellucida, consistent with its proposed role during gamete recognition.     

Porcine sperm surface beta1,4galactosyltransferase binds to the zona pellucida but is not necessary or sufficient to mediate sperm-zona pellucida binding.

The binding of sperm to the zona pellucida is an integral part of the mammalian fertilization process, investigated most extensively in the mouse. Several sperm receptors for the murine zona pellucida have been studied (Snell WJ, White JM. 1996. Cell 85:629-637; Wassarman PM. 1999. Cell 96:175-183), but the most compelling evidence exists for beta-1,4-galactosyltransferase (GalTase). Considering that GalTase is present on the surface of porcine sperm (Larson JL, Miller DJ. 1997. Biol Reprod 57:442-453), we investigated the role of GalTase in porcine sperm-zona binding. Sperm surface GalTase catalyzed the addition of uridine diphosphate-[(3)H]galactose to the 55 kDa group of the porcine zona pellucida proteins implicated in sperm binding, demonstrating that GalTase binds the porcine zona. The functional importance of GalTase-zona pellucida binding was tested. Addition of uridine diphosphate galactose, a substrate that completes the GalTase enzymatic reaction and disrupts GalTase mediated adhesion, had no effect on binding of sperm to porcine oocytes. Furthermore, removal of the GalTase zona ligand by incubation of oocytes with N-acetylglucosaminidase had no effect on binding of sperm to oocytes. These results suggest that GalTase is not necessary for sperm to bind to the zona pellucida. Digestion of isolated porcine zona proteins with N-acetylglucosaminidase did not affect the biological activity of soluble porcine zona proteins in competitive sperm-zona binding assays, suggesting that GalTase alone is not sufficient to mediate sperm-zona attachment. From these results, it appears that, although GalTase is able to bind porcine zona proteins, its function in porcine sperm-zona binding is not necessary or sufficient for sperm-zona binding. This supports the contention that porcine sperm-zona binding requires redundant gamete receptors.     

N-glycosylation of zona glycoproteins during meiotic maturation is involved in sperm-zona pellucida interactions of porcine oocytes

The objective was to determine whether N-glycosylation of zona pellucida (ZP) glycoproteins occurred during meiotic maturation of porcine oocytes, and whether this had a role in fertilization. In the first of three experiments, carbohydrate residues in the ZP of in vitro matured porcine oocytes were blocked with various lectins and the influence of such blocking on sperm-ZP interactions was studied. The second experiment used a lectin-binding assay to determine whether the number of GlcNAc residues in ZP was changed by N-glycosylation during in vitro maturation (IVM) of porcine oocytes. The last experiment determined the effects of tunicamycin, a specific N-glycosylation inhibitor, for various intervals during IVM, on sperm-ZP interactions in porcine oocytes. The primary findings were that: 1) N-glycosylation of GlcNAc residues in porcine ZP occurred during the first 24 h of IVM; and 2) such glycosylation was indispensible for sperm-ZP interactions, e.g., number of sperm bound to ZP, acrosome-reacted sperm, sperm penetration rate, and level of polyspermy (P < 0.05). However, blocking N-glycosylation by tunicamycin treatment during IVM did not adversely influence the progression of oocytes to meiotic metaphase II and male pronucleus formation, indicating that this glycosylation was involved only in the initial stages of fertilization. We inferred that the increase in terminal GlcNAc residues in ZP glycoprotein through new N-glycosylation during the first 24 h of meiotic maturation played a critical role in porcine ZP acquiring the capacity to accept sperm.     

Characterization of a novel ZP3-independent sperm-binding ligand that facilitates sperm adhesion to the egg coat

During mammalian fertilization, sperm adhere to the extracellular coat of the egg, or zona pellucida, in a species-specific manner. In mouse, evidence suggests that sperm recognize and bind to specific oligosaccharide ligands within the zona pellucida glycoprotein, ZP3, via beta1,4-galactosyltransferase I (GalT I), a lectin-like receptor on the sperm surface. Although in vitro experiments using isolated gametes lend support to this model, recent in vivo studies of genetically altered mice question whether ZP3 and/or GalT I are solely responsible for sperm-egg binding. In this regard, sperm from GalT I-null mice bind poorly to ZP3 and fail to undergo a zona-induced acrosome reaction; however, they still bind to the ovulated egg coat in vitro. In this report, we characterize a novel ZP3- and GalT I-independent mechanism for sperm adhesion to the egg coat. Results show that the ovulated zona pellucida contains at least two distinct ligands for sperm binding: a ZP3-independent ligand that is peripherally associated with the egg coat and facilitates gamete adhesion; and a ZP3-dependent ligand that is present in the insoluble zona matrix and is recognized by sperm GalT I to facilitate acrosomal exocytosis. The ZP3-independent ligand is not a result of contamination by egg cortical granules, nor is it the mouse homolog of oviduct-specific glycoprotein. It behaves as a 250 kDa, WGA-reactive glycoprotein with a basic isoelectric point, distinguishing it from the acidic glycoproteins that form the insoluble matrix of the egg coat. When eluted from isoelectric focusing gels, the acidic matrix glycoproteins possess sperm-binding activity for wild-type sperm, but not for GalT I-null sperm, whereas the basic glycoprotein retains sperm-binding activity for both wild-type and GalT I-null sperm. Thus, GalT I-null sperm are able to resolve gamete recognition into at least two distinct binding events, leading to the characterization of a novel, peripherally associated, sperm-binding ligand on the ovulated zona pellucida.     

Binding of caput epididymal mouse sperm to the zona pellucida

Immature sperm from the caput epididymis are immotile and infertile. It is thought that caput epididymal sperm are infertile due to their immotility, as well as to an inability to bind to the zona pellucida, suggesting the absence of a functional receptor for the zona. However, the sperm receptor for the zona pellucida has been identified previously as the enzyme galactosyltransferase (GalTase) (L. C. Lopez et al. (1985) J. Cell Biol. 101, 1501-1510) and is present on the surface of caput as well as cauda epididymal sperm (N. F. Scully et al., (1987) Dev. Biol. 124, 111-124.). In this paper we examine this apparent conflict and show that immotile caput epididymal sperm are able to bind to the zona pellucida if they are first washed free of caput epididymal secretions, which contain factors that inhibit sperm-zona binding. Consistent with this finding are results that show that caput epididymal fluid is capable of inhibiting the binding of mature, cauda epididymal sperm to the zona pellucida. Caput epididymal fluid contains, among many other components, a soluble GalTase and an alpha-lactalbumin-like protein, both of which are capable of inhibiting mouse sperm-zona binding. Thus, caput epididymal sperm have the appropriate receptor, i.e., GalTase, for the zona pellucida, to which they can bind if removed from the inhibitory factors that mask their zona-binding ability.     

Differential expression of glycoside residues in the mammalian zona pellucida

The mammalian zona pellucida is an extracellular matrix surrounding the oocyte, and is composed of three major glycoproteins, ZP1, ZP2, and ZP3. Previous studies have suggested that the sperm receptor activity of the zona pellucida resides in specific oligosaccharide chains on the ZP3 glycoprotein. However, the nature of the terminal monosaccharide(s) on these glycosidic chains to which sperm bind is a matter of active debate. Evidence has been presented to support a role for at least three distinct monosaccharides in sperm binding, alpha-galactose, L-fucose on Lewis X structures, and beta-N-acetylglucosamine. Previous studies have shown that beta-N-acetylglucosamine is uniformly distributed throughout the zona matrix. In this study, we have investigated the expression and distribution of alpha-galactose and fucose moieties during the maturation of the zona pellucida in mouse, rat, and hamster. Interestingly, alpha-galactose residues are expressed only during later stages of zona secretion and, consequently, are confined to the inner portions of the mature zona pellucida in mouse and rat. In hamster, alpha-galactose residues are only detectable in the zona pellucida of ovulated eggs, and are not found in ovarian oocytes. Fucosyl residues linked to Lewis X glycosides are not detectable at any stage of zona maturation in these three species, whereas fucose linked to N-linked core oligosaccharides are present throughout the zona. These studies indicate a previously unappreciated heterogeneity in the composition of zona glycosides. The specific localization of alpha-galactose residues to the inner portions of the zona matrix suggest a role in the later stages of sperm penetration through the zona. Finally, due to their absence from the zona surface, alpha-galactose and Lewis X fucosyl residues are not likely to be mediators of primary sperm binding.     

Reassessing the molecular biology of sperm-egg recognition with mouse genetics

The zona pellucida is an extracellular coat that surrounds mammalian eggs and early embryos. This insoluble matrix separates germ from somatic cells during folliculogenesis and plays critical roles during fertilization and early development. The mouse and human zona pellucida contain three glycoproteins (ZP1 or ZPB, ZP2, ZP3), the primary structures of which have been deduced by molecular cloning. Targeted mutagenesis of endogenous mouse genes and transgenesis with human homologues provide models to investigate the roles of individual zona components. Collectively, the genetic data indicate that no single mouse zona pellucida protein is obligatory for taxon-specific sperm binding and that two human proteins are not sufficient to support human sperm binding. An observed post-fertilization persistence of mouse sperm binding to "humanized" zona pellucida correlates with uncleaved ZP2. These observations are consistent with a model for sperm binding in which the supramolecular structure of the zona pellucida necessary for sperm binding is modulated by the cleavage status of ZP2.     

Localization and functional importance of a conserved zona pellucida 2 protein domain in the human and bovine ovary using monoclonal anti-ZP2 peptide antibodies

In mammals, gamete recognition and sperm binding to the oocyte are mediated by the zona pellucida (ZP), an acellular coat surrounding the plasma membrane of the oocyte that consists of particular ZP proteins. The ZP2 protein mediates secondary sperm binding to the ZP. Its primary structures are highly conserved as revealed by cDNA cloning. In the present study, we investigated the localization of ZP2 in human and bovine ovaries and oocytes and the influence of monoclonal anti-ZP2 peptide antibodies upon bovine sperm-egg interactions. We generated a monoclonal anti-ZP2 synthetic peptide antibody, mAb ZP2-20, against a sequence that is strongly conserved in the mammalian ZP2 amino acid sequence. Specificity of mAb ZP2-20 was determined by ELISA and immunoblotting, respectively. Our results show that mAb ZP2-20 specifically detected the peptide used as an antigen and reacted with its corresponding protein antigen in human and bovine ovaries. In order to elucidate effects of mAb ZP2-20 upon bovine sperm-ZP binding, we used the competitive hemizona assay (cHZA) and found that the antibodies clearly inhibit sperm binding to the ZP. We conclude that (i). monoclonal antibodies against ZP2 peptides react with ZP proteins present in bovine and human ovaries and can be used as a specific marker for ZP2; and that (ii). mAb ZP2-20 detects a ZP2 epitope that is of functional relevance for sperm-ZP interactions.     

Recombinant bovine zona pellucida glycoproteins ZP3 and ZP4 coexpressed in Sf9 cells form a sperm-binding active hetero-complex

The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm-egg interactions. Porcine and bovine ZPs are composed of the glycoproteins ZP2, ZP3, and ZP4. We previously established an expression system for porcine ZP glycoproteins (ZPGs) using baculovirus in insect Sf9 cells. Here we established a similar method for expression of bovine ZPGs. The recombinant ZPGs were secreted into the medium and purified by metal-chelating column chromatography. A mixture of bovine recombinant ZP3 (rZP3) and rZP4 coexpressed in Sf9 cells exhibited inhibitory activity for bovine sperm-ZP binding similar to that of a native bovine ZPG mixture, whereas neither bovine rZP3 nor rZP4 inhibited binding. An immunoprecipitation assay revealed that the coexpressed rZP3/rZP4 formed a hetero-complex. We examined the functional domain structure of bovine rZP4 by constructing ZP4 mutants lacking the N-terminal domain or lacking both the N-terminal and trefoil domains. When either of these mutant proteins was coexpressed with bovine rZP3, the resulting mixtures exhibited inhibitory activity comparable to that of the bovine rZP3/rZP4 complex. Hetero-complexes of bovine rZP3 and porcine rZP4, or porcine rZP3 and bovine rZP4, also inhibited bovine sperm-ZP binding. Our results demonstrate that the N-terminal and trefoil domains of bovine rZP4 are dispensable for formation of the sperm-binding active bovine rZP3/rZP4 complex and, furthermore, that the molecular interactions between rZP3 and rZP4 are conserved in the bovine and porcine systems.     

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.     

Mammalian sperm-egg interaction: fertilization of mouse eggs triggers modification of the major zona pellucida glycoprotein, ZP2

Sperm-egg interaction in mammals is initiated by binding of sperm to the zona pellucida, an acellular coat completely surrounding the plasma membrane of unfertilized eggs and preimplantation embryos. Fertilization results in transformation of the zona pellucida (“zona reaction”), such that additional sperm are unable to bind to the zona pellucida of fertilized eggs and embryos, and sperm that had partially penetrated the zona pellucida of eggs prior to fertilization are prevented from further penetration after fertilization. The failure of sperm to bind to fertilized mouse eggs and embryos is attributable to modification of the sperm receptor, ZP3, an 83,000-molecular weight glycoprotein present in zonae pellucidae isolated from both eggs and embryos [Bleil, J. D., and Wassarman, P. M. (1980). Cell, 20, 873–882]. In this investigation, ZP2, the major glycoprotein found in mouse zonae pellucidae [Bleil, J. D., and Wassarman, P. M. (1980). Develop. Biol., 76, 185–202] was analyzed by gel electrophoresis under a variety of conditions in order to determine whether or not it undergoes modification as a result of fertilization. Under nonreducing conditions, ZP2 present in solubilized zonae pellucidae that were isolated individually from mouse oocytes, eggs, and embryos migrates on SDS-polyacrylamide gels with an apparent molecular weight of 120,000. However, under reducing conditions, ZP2 from embryos, but not from oocytes or unfertilized eggs, migrates with an apparent molecular weight of 90,000 and has been designated ZP2_f. The evidence presented suggests that modification of ZP2 following fertilization involves proteolysis of the glycoprotein, but that intramolecular disulfide bonds prevent the release of peptide fragments. It is shown that the same change in ZP2 can be generated in vitro by artificial activation of unfertilized mouse eggs with the calcium ionophore A23187, thus eliminating the possibility that a sperm component is responsible for the modification of ZP2 following fertilization. These results suggest that some of the changes in the biochemical and biological properties of zonae pellucidae, observed following fertilization or activation of mouse eggs, result from modification of the major zona pellucida glycoprotein, ZP2.