Porcine zona pellucida ZP3α glycoprotein mediates binding of the biotin-labeled Mr 55,000 family (ZP3) to boar sperm membrane vesicles

The two M_r 55,000 glycoproteins, ZP3α and ZP3b?, of porcine zona pellucida copurify as a preparation designated ZP3. Gamete binding assays have implicated ZP3α, but not ZP3b?, as participating in sperm-zona recognition events. We now report that boar sperm contain membrane-associated binding sites with specificity for ZP3α. Biotin-labeled (b-) preparations of ZP3 bind to intact boar sperm in a saturable manner, with localization on the anterior head region. Membrane vesicles obtained from capacitated sperm by nitrogen cavitation retain b-ZP3 binding sites as determined by an enzyme-linked method employing alkaline phosphatase-conjugated strepavidin. In competitive binding assays using b-ZP3 (0.1μg/ml) as probe, heat-solubilized zonae and ZP3 were effective competitors, whereas the nonzona molecules fetuin and fucoidin were not. Digestion of ZP3 with endo-b?-galactosidase, an enzyme that trims polylactosamines, enhanced its affinity for membrane receptors. In contrast treatments such as chemical deglycosylation, pronase digestion, or disruption of disulfide bonds abolished the ligand activity of ZP3. Finally, purified ZP3α was an at least 100-fold better antagonist than purified ZP3b?. The results demonstrate that binding of b-ZP3 to isolated boar sperm membranes is mediated by sperm receptors with specificity for the ZP3α macromolecular component and reveal a complex contribution of both carbohydrate and protein moieties toward the ligand activity of this sperm adhesive zona molecule. © 1993 Wiley-Liss, Inc.     

Lewis X-containing glycans are specific and potent competitive inhibitors of the binding of ZP3 to complementary sites on capacitated, acrosome-intact mouse sperm

Mammalian fertilization requires a cascade of interactions between sperm and the egg's zona pellucida (ZP). O-linked glycans on mouse glycoprotein ZP3 have been implicated in mediating one step of the fertilization process, the firm adhesion of acrosome-intact sperm to the ZP. Experiments to identify structural requirements of a sperm-binding glycan have demonstrated that a Lewis X (Le(x))-containing glycan (Gal beta 4[Fuc alpha 3]GlcNAc-R) was a potent, competitive inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273: 1888-1895). However, those experiments did not define the particular step in the fertilization pathway that was blocked. The experiments described herein test the hypothesis that Le(x)-containing glycans are specific, competitive inhibitors of the binding of Alexa Fluor 568 fluorochrome (Alexa(568))-labeled ZP3 to sperm and, thus, bind the same sperm surface sites as ZP3. Dose-response analyses demonstrated that these glycans are potent inhibitors (IC(50) approximately 180 nM), which at saturation, reduced Alexa(568)-ZP3 binding by approximately 70%. A Lewis A (Le(a))-capped glycan (Gal beta 3[Fuc alpha 4]GlcNAc) was also a potent inhibitor (IC(50) approximately 150-200 nM), but at saturation, it reduced Alexa(568)-ZP3 binding by only 30%. In contrast, nonfucosylated glycans with nonreducing GlcNAc beta 4 or Gal beta 4 residues did not compete; neither did sialyl-Le(x) (Neu5Ac alpha 3Gal beta 4[Fuc alpha 3]GlcNAc-Lewis X) nor sulfo-Le(x) (3'-O-SO(3)-Lewis X). However, at saturation, Gal alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc reduced Alexa(568)-ZP3 binding by approximately 70% but with moderate apparent affinity (IC(50) approximately 3000 nM). Fluorescence microscopy revealed that Alexa(568)-labeled Le(x)-Lac-BSA, Le(a)-Lac-BSA, and ZP3 bound to the same sperm surface domains. However, Le(a)-Lac did not inhibit binding of Alexa(568)-Le(x)-Lac-BSA, and Le(x)-Lac did not inhibit binding of Alexa(568)-Le(a)-Lac-BSA. Finally, Le(x)-Lac and Le(a)-Lac had an additive inhibitory effect on Alexa(568)-ZP3 binding. Thus, Le(x) is a ligand for a major class of ZP3 binding sites on mouse sperm, whereas Le(a) binding defines a different but less-abundant class of sites.     

Porcine zona pellucida glycoprotein ZP4 is responsible for the sperm-binding activity of the ZP3/ZP4 complex

Summary The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm-egg interactions. Porcine and bovine ZPs consist of glycoproteins ZP2, ZP3, and ZP4. In both pig and bovine a heterocomplex consisting of ZP3 and ZP4 binds to sperm, however it is not clarified whether ZP3 or ZP4 in the complex is responsible for the sperm binding. Previously, we have established a baculovirus-Sf9 cell expression system for porcine ZP glycoproteins. A mixture of recombinant ZP3 (rZP3) and rZP4 displayed sperm-binding activity toward bovine sperm but not porcine sperm, probably due to differences in carbohydrate structure between the native and recombinant ZP glycoproteins. In this study, a mixture of porcine rZP3 and native ZP4 (nZP4) inhibited the binding of porcine sperm to the ZP. In contrast, a mixture of porcine nZP3 and rZP4 did not inhibit the binding of porcine sperm, although the mixture inhibited the binding of bovine sperm. The porcine rZP3/nZP4 mixture bound to the acrosomal region of porcine sperm, in a manner similar to that of the nZP3/nZP4 mixture. nZP3 was precipitated with rZP4, and nZP4 was precipitated with rZP3 by utilising the N-terminal tags on the recombinant proteins. These results indicated that nZP4, but not rZP4, is necessary for binding activity of porcine ZP3/ZP4 complex towards porcine sperm and further suggested that the carbohydrate structures of ZP4 in the porcine ZP3/ZP4 complex are responsible for porcine sperm-binding activity of the complex.     

Egg's ZP3 structure speaks volumes

Binding of mammalian sperm to eggs depends in part on ZP3, a glycoprotein in the egg's extracellular coat, the zona pellucida. In this issue, Han et?al. (2010) describe the structure of an avian ZP3 homolog, providing insights into ZP3 processing and polymerization and the roles of the ZP3 polypeptide and its carbohydrate in sperm binding.     

Lewis X-containing neoglycoproteins mimic the intrinsic ability of zona pellucida glycoprotein ZP3 to induce the acrosome reaction in capacitated mouse sperm

The binding of zona pellucida (ZP) glycoprotein ZP3 to mouse sperm surface receptors is mediated by protein-carbohydrate interactions. Subsequently, ZP3 induces sperm to undergo the acrosome reaction, an obligatory step in fertilization. We have previously identified Lewis X (Le(x); Gal beta 4[Fuc alpha 3]GlcNAc) as a potent inhibitor of in vitro sperm-ZP binding (Johnston et al. J Biol Chem 1998; 273:1888-1895). This glycan is recognized by approximately 70% of the ZP3 binding sites on capacitated, acrosome-intact mouse sperm, whereas Lewis A (Le(a); Gal beta 3[Fuc alpha 4]GlcNAc) is recognized by most of the remaining sites (Kerr et al. Biol Reprod 2004; 71:770-777). Herein, we test the hypothesis that Le(x)- and Le(a)-containing glycans, when clustered on a neoglycoprotein, bind ZP3 receptors on sperm and induce sperm to undergo the acrosome reaction via the same signaling pathways as ZP3. Results show that a Le(x)-containing neoglycoprotein induced the acrosome reaction in a dose-dependent and capacitation-dependent manner. A Le(a)-containing neoglycoprotein also induced sperm to undergo the acrosome reaction but was less potent than Le(x)-containing neoglycoproteins. In contrast, neoglycoproteins containing beta4-lactosamine (Gal beta 4GlcNAc), Lewis B (Fuc alpha 2Gal beta 3[Fuc alpha 4]GlcNAc), and sialyl-Le(x) glycans were inactive, as were four other neoglycoproteins with different nonfucosylated glycans. Consistent with these results, unconjugated Le(x)- and Le(a)-capped glycans were dose-dependent inhibitors, which at saturation, reduced the ZP-induced acrosome reaction by about 60% and 30%, respectively. Experiments utilizing pharmacological inhibitors suggest that induction of the acrosome reaction by solubilized ZP and Le(x)- and Le(a)-containing neoglycoproteins require the same calcium-dependent pathway. However, only the ZP-induced acrosome reaction requires a functional G(i) protein. Thus, Le(x)-containing neoglycoproteins bind to a major class of ZP3 receptors on capacitated sperm. A Le(a)-containing neoglycoprotein binds a second ZP3 receptor but is a less-potent inducer of the acrosome reaction.     

Evidence that aggregation of mouse sperm receptors by ZP3 triggers the acrosome reaction

In the mouse, considerable evidence indicates that initial sperm binding to the zona pellucida (ZP) is mediated by ZP3. In addition, this same glycoprotein is also responsible for inducing the acrosome reaction (AR). Whereas the O-linked oligosaccharides of ZP3 appear to mediate sperm-ZP binding, the portion of ZP3 bearing AR activity has not been defined. To try to understand the bifunctional role of ZP3 (binding and AR inducing activities), we have examined the hypothesis that ZP3 aggregates sperm receptor molecules. By analogy with findings in a variety of other extracellular signal transducing systems, including receptors for growth factors and insulin, this aggregation event could initiate the cascade resulting in the AR. To test this hypothesis, we have generated monospecific polyclonal antibodies against ZP2 and against ZP3, and examined the effects of these probes on capacitated sperm incubated in the absence or presence of various ZP protein preparations. For some experiments, we have used proteolytic fragments of ZP3, a preparation known to retain specific binding, but not AR-inducing, activity. We show here that capacitated mouse sperm, incubated with ZP glycopeptides, displayed ARs when incubated subsequently with anti-ZP3 IgG; ARs did not occur when parallel sperm samples were incubated with anti-ZP2 IgG or with anti-ZP3 Fab fragments. When capacitated sperm were treated successively, with (a) ZP3 glycopeptides, (b) anti-ZP3 Fab fragments, and (c) goat anti-rabbit IgG, ARs occurred in the majority of sperm. An alternative approach to examine this hypothesis used ZP proteins obtained from tubal eggs treated previously with bioactive phorbol diester (12-O-tetradecanoyl phorbol-13-acetate [TPA]). This preparation arrests capacitated sperm in an intermediate state of the AR. We demonstrate here that these sperm can be induced to undergo a complete AR by subsequent treatment with anti-ZP3 IgG. Together, these findings are consistent with the hypothesis under examination, and suggest that the aggregation of sperm molecules recognized by ZP3 glycopeptides or by TPA-treated ZP is sufficient to trigger the events that occur during acrosomal exocytosis.     

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.     

猪精子中与卵透明带糖蛋白ZP3结合的蛋白质

依次经ＰＳＬ－Ｓｅｐｈａｒｏｓｅ亲和层析柱和纤维素ＣＭ－５２离子交换层析柱,从猪精子的ＣＨＡＰＳ抽提液分离得４个蛋白质组分。用固相透明带精蛋白结合试验（ＩＺＰＧＢＡ）检测;表明精子蛋白ＳＰ１和ＳＰ２具有结合透明带糖蛋白ＺＰ３的活性,ＳＰ２并显示凝集血球的活性。精子蛋白ＳＰ１与卵预温育明显抑制精卵结合,抑制活性与加入的精子蛋白的浓度呈正相关。用生物素标记的ＺＰ３和蛋白质印迹技术,证明ＳＰ１中的６８ｋＤ精子蛋白与ＺＰ３结合,提示６８ｋＤ精子蛋白参与精卵结合。     

Characterization of two zebrafish cDNA clones encoding egg envelope proteins ZP2 and ZP3.

Two zebrafish cDNA clones encoding homologs of mammalian zona pellucida proteins ZP2 and ZP3 were isolated from a whole adult cDNA library. The ZP2 clone encodes a protein of 428 amino acids. Unlike other teleost ZP2s that contain an N-terminal repetitive domain enriched with prolines and glutamines, the zebrafish ZP2 has no such repetitive domain. In the C-terminal non-repetitive domain, the zebrafish ZP2 shares 55-76% sequence identity with other teleost ZP2s. The ZP3 cDNA clone encodes a protein of 431 amino acids, which shares 61% sequence identity with a carp ZP3. Similar to mammalian ZP proteins, both zebrafish ZP2 and ZP3 contain several potential phosphorylation sites. However, unlike mammalian ZP proteins, both zebrafish ZP proteins contain almost no glycosylation site, which has been proposed to be important for interaction with sperm; thus, the ZP proteins may behave differently in mammals and teleosts. Northern blot analysis indicated that both zebrafish ZP2 and ZP3 mRNAs were expressed exclusively in the ovary and hence the ovary is likely the only site for ZP2 and ZP3 biosynthesis.     

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.     

95 kd sperm proteins bind ZP3 and serve as tyrosine kinase substrates in response to zona binding

In the mouse, the zona pellucida (ZP) glycoprotein ZP3 both binds intact sperm and induces acrosomal exocytosis. The subsequent signaling pathway(s) is still uncertain, but Gi-like proteins have been implicated. By analogy with other signal transduction mechanisms, we examined anti-phosphotyrosine antibody reactivity in mouse sperm. Antibodies reacted with three proteins of 52, 75, and 95 kd. Indirect immunofluorescence localized reactivity to the acrosomal region of the sperm head. The 52 kd and 75 kd phosphoproteins are detected only in capacitated sperm, whereas the 95 kd protein is detected in both fresh and capacitated sperm. For the 95 kd protein, the level of immunoreactivity is not related to sperm motility but is enhanced by both capacitation and sperm interaction with solubilized ZP proteins. In addition, binding of radiolabeled whole ZP or purified ZP3 to blots of separated sperm proteins identified two ZP binding proteins of 95 kd and 42 kd. 95 kd sperm proteins that bind to ZP3 also react with anti-phosphotyrosine antibodies (in a ZP concentration-dependent manner), supporting the idea that the same 95 kd sperm protein serves as a ZP3 receptor and as a tyrosine kinase substrate. These findings and our evidence on acrosome reaction triggering via sperm receptor aggregation suggest that a 95 kd protein in the sperm plasma membrane is aggregated by ZP3, which stimulates tyrosine kinase activity leading to acrosomal exocytosis.     

Evidence for the presence of high-mannose/hybrid oligosaccharide chain(s) on the mouse ZP2 and ZP3.

Previous studies from this laboratory have identified a novel alpha-D-mannosidase on plasma membranes of rat, mouse, hamster, and human spermatozoa [Tulsiani et al. J Cell Biol 1989; 109:1257; Biol Reprod 1990; 42:843]. Inhibition of the mouse sperm surface alpha-D-mannosidase inhibits sperm-egg binding in vitro, suggesting that the sperm enzyme may have a receptor-like role in binding to the complementary molecules (presumably mannose-containing oligosaccharide [OS] chains) on the mouse zona pellucida (ZP) glycoconjugates [Cornwall et al. Biol Reprod 1991; 44:913]. In the studies reported here, we demonstrate the presence of high-mannose/hybrid-type OS on mouse zona components. Zona-intact eggs, prepared from superovulated mice, were radioiodinated, and the individual zona components (ZP1, ZP2, and ZP3) were isolated by electrophoresis followed by electroelution. The purified ZP components, when resolved by immobilized concanavalin A column chromatography, showed the following results: 1) Nearly all of the ZP1 applied to the immobilized lectin eluted in the column flow-through (effluent) fractions, and no radioactivity eluted with alpha-methyl mannoside, suggesting that ZP1 may not contain high-mannose/hybrid OS. 2) A significant amount of both ZP2 and ZP3 bound to the immobilized lectin, and nearly 16% and 8% of the two components, respectively, were repeatedly eluted with alpha-methyl mannoside.(ABSTRACT TRUNCATED AT 250 WORDS)     

A role for the human sperm glycine receptor/Cl(-) channel in the acrosome reaction initiated by recombinant ZP3.

Previously, we have demonstrated an essential role for the neuronal glycine receptor (GlyR) in the acrosome reaction (AR) of mouse and porcine sperm initiated by the egg zona pellucida (ZP). In the present study, we have demonstrated presence of the GlyR in human sperm by immunoprecipitation and Western blot analysis, investigated the potential of a recombinant human ZP3 (rhZP3) preparation as an alternative research tool to solubilized human ZP, and shown that the human sperm GlyR is essential to the human AR initiated by rhZP3. Additionally, we have been able to demonstrate that rhZP3 possesses biological activity, because it is able to rapidly stimulate the AR in capacitated human sperm and its action is blocked by the addition of pertussis toxin. Moreover, spectrofluorometric studies using fura-2-loaded human sperm have shown that rhZP3 triggers a peak-and-plateau rise in intracellular Ca(2+) levels similar to that seen with solubilized mammalian ZP. These results suggest that the actions of rhZP3 and solubilized ZP are elicited via the same signal transduction pathways. Furthermore, incubation of human sperm with an antibody directed against the alpha1 subunit of the human spinal cord GlyR or with 50 nM strychnine caused significant inhibition in the rhZP3-initated AR. Finally, studies using fura-2-loaded human sperm showed that 50 nM strychnine was also able to inhibit the Ca(2+) influx associated with addition of rhZP3. These results further support the view that rhZP3 and the ZP work through the same mechanisms, show that the GlyR is involved in rhZP3-initiated AR, and suggest that the GlyR may also play a role in the early signal transduction cascades associated with ZP-initiated AR in vivo.     

Adaptive evolution of fertilization proteins within a genus: variation in ZP2 and ZP3 in deer mice (Peromyscus)

Rapid evolution of reproductive proteins has been documented in a wide variety of taxa. In internally fertilized species, knowledge about the evolutionary dynamics of these proteins between closely related taxa is primarily limited to accessory gland proteins in the semen of Drosophila. Investigation of additional taxa and functional classes of proteins is necessary in order to determine if there is a general pattern of adaptive evolution of reproductive proteins between recently diverged species. We performed an evolutionary analysis of 2 egg coat proteins, ZP2 and ZP3, in 15 species of deer mice (genus Peromyscus). Both of these proteins are involved in egg-sperm binding, a critical step in maintaining species-specific fertilization. Here, we show that Zp2 and Zp3 gene trees are not consistent with trees based on nonreproductive genes, Mc1r and Lcat, where species formed monophyletic clades. In fact, for both of the reproductive genes, intraspecific amino acid variation was extensive and alleles were sometimes shared across species. We document positive selection acting on ZP2 and ZP3 and identify specific amino acid sites that are likely targets of selection using both maximum likelihood approaches and patterns of parallel amino acid change. In ZP3, positively selected sites are clustered in and around the region implicated in sperm binding in Mus, suggesting changes may impact egg-sperm binding and fertilization potential. Finally, we identify lineages with significantly elevated rates of amino acid substitution using a Bayesian mapping approach. These findings demonstrate that the pattern of adaptive reproductive protein evolution found at higher taxonomic levels can be documented between closely related mammalian species, where reproductive isolation has evolved recently.     

Recombinant mouse sperm ZP3-binding protein (ZP3R/sp56) forms a high order oligomer that binds eggs and inhibits mouse fertilization in vitro

Many candidates have been proposed as zona pellucida-binding proteins. Without precluding a role for any of those candidates, we focused on mouse sperm protein ZP3R/sp56, which is localized in the acrosomal matrix. The objective of this study was to analyze the role of ZP3R/sp56 in mouse fertilization. We expressed recombinant ZP3R/sp56 as a secreted protein in HEK293 cells and purified it from serum-free, conditioned medium. In the presence of reducing agents, the recombinant ZP3R/sp56 exhibited a molecular weight similar to that observed for the native ZP3R/sp56. Reminiscent of the native protein, recombinant ZP3R/sp56 formed a high molecular weight, disulfide cross-linked oligomer consisting of six or more monomers under non-reducing conditions. Recombinant ZP3R/sp56 bound to the zona pellucida of unfertilized eggs but not to 2-cell embryos, indicating that the changes that take place in the zona pellucida at fertilization affected the interaction of this protein with the zona pellucida. The extent of in vitro fertilization was reduced in a dose-dependent manner when unfertilized eggs were preincubated with recombinant ZP3R/sp56 (74% drop at the maximum concentrations assayed). Eggs incubated with the recombinant protein showed an absence of or very few sperm in the perivitelline space, suggesting that the reduction in the fertilization rate is caused by the inhibition of sperm binding and/or penetration through the zona pellucida. These results indicate that sperm ZP3R/sp56 is important for sperm-zona interactions during fertilization and support the concept that the acrosomal matrix plays an essential role in mediating the binding of sperm to the zona pellucida.     

Insights into egg coat assembly and egg-sperm interaction from the X-ray structure of full-length ZP3

ZP3, a major component of the zona pellucida (ZP) matrix coating mammalian eggs, is essential for fertilization by acting as sperm receptor. By retaining a propeptide that contains a polymerization-blocking external hydrophobic patch (EHP), we determined the crystal structure of an avian homolog of ZP3 at 2.0 ? resolution. The structure unveils the fold of a complete ZP domain module in a homodimeric arrangement required for secretion and reveals how EHP prevents premature incorporation of ZP3 into the ZP. This suggests mechanisms underlying polymerization and how local structural differences, reflected by alternative disulfide patterns, control the specificity of ZP subunit interaction. Close relative positioning of a conserved O-glycan important for sperm binding and the hypervariable, positively selected C-terminal region of ZP3 suggests a concerted role in the regulation of species-restricted gamete recognition. Alternative conformations of the area around the O-glycan indicate how sperm binding could trigger downstream events via intramolecular signaling.     

Primary structure and developmental expression of Dp ZP2, a vitelline envelope glycoprotein homolog of mouse ZP2, in Discoglossus pictus, one of the oldest living Anuran species

A glycoprotein of the Xenopus vitelline envelope, gp 69/64, which mediates sperm binding, is closely related to the components of ZPA family, such as the mouse zona pellucida ZP2. To test the generality of these findings, we studied Discoglossus pictus, a species evolutionary distant from Xenopus and identified as a protein of 63 kDa in the vitelline envelope. Preliminary studies suggest that this protein may bind sperm at fertilization. We found that the 63-kDa protein is glycosylated and contains both N- and O-linked chains. We have cloned the cDNA encoding the Discoglossus protein of 63 kDa (Dp ZP2) by screening a Discoglossus cDNA library using Xenopus gp 69/64 cDNA as a probe. Analysis of the deduced sequence of Discoglossus protein revealed 48% identity with Xenopus gp 69/64 and 37-40% identity with mouse ZP2. The sequence conservation included a ZP domain, a potential furin cleavage site and a putative transmembrane domain. The N-terminus region of Dp ZP2 was 40% identical to the corresponding region of Xenopus gp 69/64 which has been shown to be essential for sperm binding to the VE. Although, as of yet, there is no evidence for sperm binding at the Dp ZP2 N-terminus, it is interesting that in this region three potential O-glycosylation sites are conserved in both species, in contrast to N-glycosylation sites. It was found that the Dp ZP2 mRNA is expressed in stage 1 oocytes and in the follicle cells surrounding the oocyte. Similarly, in Xenopus oocytes, the gp 69/64m RNA, was found in the oocytes, as well as in the somatic cells. Mol. Reprod. Dev. 59:133-143, 2001.     

Relevance of glycosylation of human zona pellucida glycoproteins for their binding to capacitated human spermatozoa and subsequent induction of acrosomal exocytosis

To delineate the functional aspects of zona pellucida (ZP) glycoproteins during fertilization in human, in the present study, fluorochrome-conjugated Escherichia coli (E. coli)- and baculovirus-expressed recombinant human ZP glycoprotein-2 (ZP2), -3 (ZP3), and -4 (ZP4) were employed. In an immunofluorescence assay, capacitated human sperm exhibited binding of the baculovirus-expressed recombinant ZP3 as well as ZP4 to either acrosomal cap or equatorial region whereas acrosome-reacted sperm failed to show any binding to the acrosomal cap. Using double labeling experiments, simultaneous binding of ZP3 and ZP4 to the acrosomal cap was observed suggesting the possibility of different binding sites of these proteins on the sperm surface. No binding of ZP2 was observed to the capacitated sperm. However, acrosome-reacted sperm (20.00 +/- 1.93%) showed binding of ZP2 that was restricted to only equatorial region. Interestingly, E. coli-expressed recombinant human zona proteins also showed very similar binding profiles. Competitive inhibition studies with unlabeled recombinant human zona proteins revealed the specificity of the above binding characteristics. Binding characteristics have been further validated by an indirect immunofluorescence assay using native human heat solubilized isolated zona pellucida. Employing baculovirus-expressed recombinant ZP3 and ZP4 with reduced N-linked glycosylation and respective E. coli-expressed recombinant proteins, it was observed that glycosylation is required for induction of acrosomal exocytosis but its absence may not compromise on their binding ability. These studies have revealed the binding profile of individual human zona protein to spermatozoa and further strengthened the importance of glycosylation of zona proteins for acrosomal exocytosis in spermatozoa.     

Baculovirus expressed C-terminal fragment of bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 inhibits ZP3-mediated induction of acrosomal exocytosis

Zona pellucida glycoprotein-3 (ZP3) has been postulated as the primary sperm receptor in various mammalian species including bonnet monkey (Macaca radiata). However, information on the domain responsible for its binding to spermatozoa is inadequate. In the present study, bonnet monkey ZP3 (bmZP3), corresponding to amino acid (aa) residues 223-348 [bmZP3(223-348)] has been cloned and expressed using baculovirus expression system. SDS-PAGE and Western blot analysis of the purified renatured recombinant protein revealed it as a closely spaced doublet of approximately 25 kDa. Lectin-binding studies documented the presence of both O- as well as N-linked glycans. The biotinylated r-bmZP3(223-348) binds to the acrosomal region of the capacitated spermatozoa but fails to bind to the acrosome-reacted spermatozoa as investigated by immunofluorescence studies. In ELISA, nonbiotinylated r-bmZP3(223-348) and baculovirus expressed r-bmZP3, devoid of signal sequence and transmembrane-like domain [r-bmZP3(23-348)] competitively inhibit its binding to the capacitated spermatozoa. Interestingly, binding of biotinylated r-bmZP3(23-348) to the capacitated sperm is also inhibited by nonbiotinylated r-bmZP3(223-348). In contrast to r-bmZP3(23-348), r-bmZP3(223-348) failed to induce acrosomal exocytosis in the capacitated sperm. Interestingly, it competitively inhibits the acrosomal exocytosis induced by r-bmZP3(23-348). These studies, for the first time, identify a domain of ZP3 capable of binding to capacitated spermatozoa and inhibiting ZP3-mediated induction of acrosomal exocytosis furthering our understanding of mammalian fertilization.