O-linked oligosaccharides of mouse egg ZP3 account for its sperm receptor activity

Previously, we reported that ZP3, one of three different glycoproteins present in the mouse egg's zona pellucida, serves as a sperm receptor. Furthermore, small glycopeptides derived from egg ZP3 retain full sperm receptor activity, suggesting a role for carbohydrate, rather than polypeptide chain in receptor function. Here, we report that removal of O-linked oligosaccharides from ZP3 destroys its sperm receptor activity, whereas removal of N-linked oligosaccharides has no effect. A specific size class of O-linked oligosaccharides, recovered following mild alkaline hydrolysis and reduction of ZP3, is shown to possess sperm receptor activity and to bind to sperm. The results presented strongly suggest that mouse sperm bind to eggs via O-linked oligosaccharides present on ZP3.     

Carboxy-terminal proteolytic processing at a consensus furin cleavage site is a prerequisite event for quail ZPC secretion

In avian species, a glycoprotein homologous to mammalian ZPC is synthesized in the granulosa cells of developing follicles. We have previously reported that the newly synthesized ZPC (proZPC) in granulosa cells is cleaved at a consensus furin cleavage site to generate mature ZPC prior to secretion. In the present study, we examined the effect of the proteolytic cleavage of proZPC on ZPC secretion by using a specific inhibitor of furin endoprotease and site-directed mutagenesis of the furin cleavage site. Western blot analysis demonstrated that the furin inhibitor efficiently blocked both the proteolytic cleavage of proZPC and the subsequent ZPC secretion. A site-directed mutant that possessed a mutated sequence for furin cleavage was not secreted from the cells. The immunocytochemical observations indicated that proZPC produced in the presence of a furin inhibitor or those produced by the site-directed mutant of the furin cleavage site had accumulated in the endoplasmic reticulum. These results indicate that proZPC is proteolytically cleaved at the consensus furin cleavage site with furin-like protease, and the failure of this cleavage results in its accumulation in the endoplasmic reticulum. Therefore, the C-terminal proteolytic processing of proZPC at the consensus furin cleavage site is a prerequisite event for quail ZPC secretion.     

Cloning of rat sp56,the homologue of mouse sperm ZP3 receptor—sp56

Mouse sp56 is considered as one of the candidates for mouse zona pellucida 3(mZP3)receptor,Up to date,its homologue has only been cloned from guinea pig,namely,AM67.Based on the cDNA sequence of mouse sp56,we designed a pair of primer to amplify its homologue from rat testis cDNA.Using RT-PCR, two tragments of 743 bp and 938 bp were amplified.The PCR products show very high homology to mouse sp56.However,the 743 bp product completely lacks one of the seven Sushi domains of mouse sp56.Using the 743 bp product as the probe to detect the expression profile of sp56 in rat tissues,Northern blot shows that a-2.0kb mRNA expresses specifically in testis.Employed the RACE method,two full cDNA sequences of rat sp56 were obtained.A Mr-42KD band was detected in denatured and non-reducing protein sample of rat testis and sperm with anti-mouse sp56 monoclonal antibody by Western blot method.Rat sp56 was localized on rat sperm head by the indirect immunofluorescence method.Rat sp56 immunoreactivity was detected from the early pachytene spermatocytes and throughout the spermatogenesis.Its cloning will further our understanding of the mechanism of the sperm-egg recognition and binding.     

Cloning, sequencing and site of origin of the rat sperm receptor protein, ZP3

The ZP3 gene encodes for a zona glycoprotein that serves as both a cell-specific binding site for capacitated spermatozoa and an inducer of acrosomal exocytosis during fertilisation. In this study we have determined the nucleotide sequence of rat ZP3 (accession no. Y10823), predicted primary amino acid structure and determined the cellular origin of this molecule within the ovary. Rat ZP3 was found to have an open reading frame of 1272 nucleotides encoding a polypeptide chain of 424 amino acids that was expressed exclusively by the actively growing oocyte population. Rat ZP3 exhibited 91%, 78% and 66% identity with the mouse, hamster and human homologues, respectively. Key features of mouse ZP3, including the number and location of cysteine and proline residues and N-linked glycosylation sites, were also conserved in the rat homologue. The putative O-linked glycosylation sites, a series of serine residues at ZP3(329-334), were also conserved in rat and mouse ZP3, although immediately downstream of this site the amino acid sequences deviated over a short stretch of amino acids. The hydropathicity profile revealed two hydrophobic domains. The first was associated with a putative N-terminal signal sequence which is unusual in the rat in possessing a proline residue at the -1 position relative to the signal cleavage site, a feature it shares with human and marmoset ZP3 but not mouse. The second hydrophobic domain was observed at the C-terminus downstream of a TGF-beta type III receptor domain that appears to be common to all ZP3 sequences examined to date.     

Effects of a phorbol ester on mouse eggs: dissociation of sperm receptor activity from acrosome reaction-inducing activity of the mouse zona pellucida protein, ZP3

Biologically active phorbol esters or a diacylglycerol induce mouse eggs to modify the zona pellucida such that sperm receptor activity is retained but the ability of bound sperm to undergo a complete acrosome reaction is lost (Y. Endo, R.M. Schultz, and G.S. Kopf. 1987. Dev. Biol. 119, 199-209). We now show that purified ZP3 from 12-O-tetradecanoyl phorbol-13-acetate (TPA)-treated eggs possesses full sperm receptor activity but has lost its ability to induce a complete acrosome reaction. The modification of the acrosome reaction-inducing activity of ZP3 from these TPA-treated eggs differs from ZP3 isolated from two-cell embryos, which cannot initiate the acrosome reaction. These results demonstrate the dissociation of the two biological activities of ZP3 and may provide a system to assess the components of ZP3 involved in the acrosome reaction.     

Characterization of zona pellucida glycoprotein 3 (ZP3) and ZP2 binding sites on acrosome-intact mouse sperm

There is considerable evidence that mouse fertilization requires the binding of sperm to two of the three glycoproteins that form the zona pellucida (ZP), ZP3 and ZP2. Despite the biologic importance of this binding, no one has demonstrated that sperm express separate, saturable, and specific binding sites for ZP3 and for ZP2. Such a demonstration is a prerequisite for defining the distribution, numbers, affinities, and regulation of function of ZP3 and ZP2 binding sites on sperm. The experiments reported herein used fluorochrome-labeled ZP3 and ZP2 and quantitative image analysis to characterize the saturable binding of ZP3 and ZP2 to distinct sites on living, capacitated, acrosome-intact mouse sperm. Approximately 20% of the ZP3 binding sites were found over the acrosomal cap, and the remaining sites were located over the postacrosomal region of the head. In contrast, ZP2 binding sites were detected only over the postacrosomal region. Saturation analysis estimated numbers and affinities of the binding sites for ZP3 (B(max) approximately 185 000 sites per sperm; K(d) approximately 67 nM) and ZP2 (B(max) approximately 500 000 sites per sperm; K(d) approximately 200 nM). Use of unlabeled ZP3, ZP2, and ZP1 as competitive inhibitors of the binding of fluorochrome-labeled ZP3 and ZP2 demonstrated that ZP3 and ZP2 bound specifically to their respective sites on sperm. Finally, we demonstrate that extracellular calcium as well as capacitation and maturation of sperm are required for these sites to bind their respective ligands.     

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.     

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.     

Embryonal carcinoma cells transfected with ZP3 genes differentially glycosylate similar polypeptides and secrete active mouse sperm receptor

Mouse and hamster sperm receptors, called mZP3 (approximately 83,000 Mr) and hZP3 (approximately 56,000 Mr), respectively, are glycoproteins located in the ovulated egg zona pellucida. Certain of the glycoprotein O-linked oligosaccharides are essential for sperm receptor activity. Here, we transfected mouse embryonal carcinoma (EC) cells with mZP3 and hZP3 genes placed under control of a constitutive promoter. Transfected cells synthesized and secreted large amounts of the glycoproteins, called EC-mZP3 and EC-hZP3. Although the primary structures of mZP3 and hZP3 polypeptides (44,000 Mr) are very similar to one another, EC-mZP3 (approximately 83,000 Mr) and EC-hZP3 (approximately 49,000 Mr) were glycosylated to very different extents, such that they resembled their egg counterparts. Like egg mZP3, EC-mZP3 inhibited binding of sperm to ovulated eggs and induced sperm to acrosome-react in vitro. In addition, large numbers of sperm bound to aggregates of mZP3-transfected EC cells in vitro. On the other hand, unlike egg hZP3, EC-hZP3 did not exhibit either sperm receptor or acrosome reaction-inducing activity, and sperm failed to bind to aggregates of hZP3-transfected EC cells. Thus, transfected EC cells not only express sperm receptor genes, but also discriminate between very similar polypeptides with respect to glycosylation and, in the case of mZP3, add specific oligosaccharides essential for biological activity. In addition, the results demonstrate that EC cells can serve as a source for large amounts of functional mouse sperm receptor.     

Conserved furin cleavage site not essential for secretion and integration of ZP3 into the extracellular egg coat of transgenic mice

The extracellular zona pellucida surrounding mammalian eggs is formed by interactions of the ZP1, ZP2, and ZP3 glycoproteins. Female mice lacking ZP2 or ZP3 do not form a stable zona matrix and are sterile. The three zona proteins are synthesized in growing oocytes and secreted prior to incorporation into the zona pellucida. A well-conserved furin site upstream of a transmembrane domain near the carboxyl terminus of each has been implicated in the release of the zona ectodomains from oocytes. However, mutation of the furin site (RNRR --> ANAA) does not affect the intracellular trafficking or secretion of an enhanced green fluorescent protein (EGFP)-ZP3 fusion protein in heterologous somatic cells. After transient expression in growing oocytes, normal EGFP-ZP3 and mutant EGFP-ZP3 associate with the inner aspect of the zona pellucida, which is distinct from the plasma membrane. These in vitro results are confirmed in transgenic mice expressing EGFP-ZP3 with or without the mutant furin site. In each case, EGFP-ZP3 is incorporated throughout the width of the zona pellucida and the transgenic mice are fertile. These results indicate that the zona matrix accrues from the inside out and, unexpectedly, suggest that cleavage at the furin site is not required for formation of the extracellular zona pellucida surrounding mouse eggs.     

Sperm-egg recognition in the mouse: characterization of sp56, a sperm protein having specific affinity for ZP3

Recognition between mammalian gametes occurs when the plasma membrane of the sperm head binds to the zona pellucida (ZP), an extracellular coat surrounding eggs. ZP3, one of three glycoproteins in the ZP, is the egg protein recognized by sperm. A mouse sperm surface protein, sp56 (M(r) = 56,000), has been identified on the basis of its specific affinity for ZP3 (Bleil, J. D., and P. M. Wassarman. 1990. Proc. Natl. Acad. Sci. USA. 87:5563-5567). Studies presented here were designed to characterize mouse sperm sp56 and to further test whether or not this protein specifically recognizes ZP3. sp56 was purified by both ZP3 affinity chromatography and by ion exchange chromatography followed by size-exclusion chromatography. The purified native protein eluted from size-exclusion columns as a homomultimer (M(r) approximately 110,000). Each monomer of the protein contains intramolecular disulfide bonds, consistent with its extracellular location. Immunohistochemical and immunoblotting studies, using monoclonal antibodies, demonstrated that sp56 is a peripheral membrane protein located on the outer surface of the sperm head plasma membrane, precisely where sperm bind ZP3. Results of crosslinking experiments demonstrated that the ZP3 oligosaccharide recognized by sperm has specific affinity for sp56. Collectively, these results suggest that sp56 may be the sperm protein responsible for sperm-egg recognition in the mouse.     

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.     

Relationship between the M42 antigen of mouse sperm and the acrosome reaction induced by ZP3

A murine monoclonal antibody, M42 mAb, directed against 200/220 Kd protein of mouse sperm, has been employed to study the molecular events of gamete interaction. We have reported previously that M42 mAb blocks mouse fertilization in a zona-dependent manner; the reagent specifically inhibits physiologically induced (zonae), but not pharmacologically induced (A23187), acrosome reactions in mouse sperm. Using solubilized mouse zonae pellucidae and purified ZP3, we demonstrate that M42 mAb inhibits acrosome reactions (ARs) induced by ZP3 to the same extent as those induced by total zonae. We have also studied AR inhibition using the fluorescent antibiotic chlortetracycline (CTC), which permits visualization of three different acrosomal patterns during the AR. In the presence of M42 IgG, greater than 70% of capacitated sperm treated with zonae are arrested in the acrosome-intact state (B-pattern), in contrast to the majority of sperm (60-70%) in the absence of M42 IgG, which progress through the intermediate phase (S-pattern) to the fully acrosome-reacted (AR-pattern) state. Incubation of sperm with zona proteins modified by incubating eggs with phorbol esters arrests sperm in the S-pattern (Y. Endo, R.M. Schultz, and G.S. Kopf, 1987, Dev. Biol. 119, 199-209). We show that once sperm have reached such a state, M42 mAb no longer exerts an inhibitory effect. The addition of unmodified ZP to S-pattern sperm permits the completion of the acrosome reaction. These results indicate that M42 mAb blocks an early step in the AR cascade and that M42 mAb is unable to prevent subsequent events of this cascade once it has been initiated.     

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.     

Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3

In many cells, receptor activation initiates sustained Ca2+ entry which is critical in signal transduction. Mammalian transient receptor potential (Trp) proteins, which are homologous to the Drosophila photoreceptor-cell Trp protein, have emerged as candidate subunits of the ion channels that mediate this influx. As a consequence of overexpression, these proteins produce cation currents that open either after depletion of internal Ca2+ stores or through receptor activation. However, determining the role of endogenous Trp proteins in signal transduction is complicated by the absence of selective antagonists. Here we examine Trp function during sperm-egg interaction. The sperm acrosome reaction is a Ca2+-dependent secretory event that must be completed before fertilization. In mammals, exocytosis is triggered during gamete contact by ZP3, a glycoprotein constituent of the egg's extracellular matrix, or zona pellucida (ZP). ZP3 activates trimeric G proteins and phospholipase C and causes a transient Ca2+ influx into sperm through T-type Ca2+ channels. These early responses promote a second Ca2+-entry pathway, thereby producing sustained increases in intracellular Ca2+ concentration ([Ca2+]i) that drive acrosome reactions. Our results show that Trp2 is essential for the activation of sustained Ca2+ influx into sperm by ZP3.