Structural and functional relationships between mouse and hamster zona pellucida glycoproteins

The hamster egg's extracellular coat, or zona pellucida, consists of three glycoproteins, designated hZP1, hZP2, and hZP3, that exhibit extensive heterogeneity on SDS-PAGE. hZP1 is a relatively minor component of hamster zonae pellucidae, as compared with hZP2 and hZP3. In the presence of reducing agents, hZP1, 200,000 apparent Mr, migrates on SDS-PAGE with an apparent Mr of 103,000. This suggests that hZP1, like mouse ZP1, is composed of two polypeptides held together by intermolecular disulfides. When purified hamster ZP glycoproteins were tested at relatively low concentrations in an in vitro competition assay, employing either hamster or mouse gametes, only hZP3 (56,000 apparent Mr) exhibited sperm receptor activity (i.e., inhibited binding of sperm to eggs). Thus, apparently hZP3 is the hamster counterpart of mouse ZP3, the mouse egg receptor for sperm. Furthermore, at relatively high concentrations, solubilized hamster egg ZP preparations induced both hamster and mouse sperm to undergo the acrosome reaction in vitro. hZP3 is encoded by a relatively abundant ovarian mRNA that is detected by a mouse ZP3 cDNA probe and is the same size, about 1.5 kb, as mRNA encoding the mouse sperm receptor, ZP3 (83,000 apparent Mr). Like mouse ZP2, hZP2 undergoes limited proteolysis following artificial activation of hamster eggs in vitro. Results of in vitro assays employing intact eggs and isolated zonae pellucidae demonstrate that hamster eggs possess a ZP2-proteinase which has a substrate specificity similar to that of the mouse enzyme. These observations are discussed in terms of structural and functional relationships that may exist between hamster and mouse zona pellucida glycoproteins.     

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.     

Transgenic mouse eggs with functional hamster sperm receptors in their zona pellucida.

Sperm receptors are located in the mammalian egg extracellular coat, or zona pellucida. Mouse and hamster sperm receptor glycoproteins, mZP3 (83 x 10(3) M(r)) and hZP3 (56 x 10(3) M(r)), respectively, have very similar polypeptides (44 x 10(3) M(r); 81% identical) that are glycosylated to different extents. Purified mZP3 and hZP3 can bind to mouse sperm, prevent them from binding to eggs and induce them to undergo exocytosis, the acrosome reaction, in vitro. A DNA construct that placed the hZP3 gene under the control of mZP3 gene 5'-flanking sequence was used in this report to produce two mouse lines that harbored the foreign sperm receptor transgene. In both lines, the transgene was expressed only by growing oocytes, at a level comparable to that of the endogenous mZP3 gene, and the developmental pattern of transgene expression resembled that of the mZP3 gene. In addition to mZP3, transgenic mouse oocytes synthesized and secreted a glycoprotein indistinguishable from hZP3, and incorporated both glycoproteins into a mosaic zona pellucida. Importantly, hZP3 purified from such zonae pellucidae exhibited both sperm receptor and acrosome reaction-inducing activities in vitro and, following fertilization of transgenic mouse eggs, was inactivated. These results demonstrate that a biologically active foreign sperm receptor can be synthesized and secreted by transgenic mouse oocytes, assembled into a mosaic zona pellucida, and inactivated following fertilization as part of the secondary block to polyspermy.     

How the egg regulates sperm function during gamete interaction: facts and fantasies

Although details of the molecular mechanism are not yet clear, considerable evidence suggests that the egg-specific extracellular matrix component ZP3 regulates an essential event of sperm function, the acrosome reaction. Spatial control of this exocytotic event appears to be exerted by immobilization of the triggering ligand, ZP3, in the zona pellucida matrix surrounding the egg. Our data suggest that the signal transduction pathway in sperm activated by this ligand involves highly conserved components that are involved in many other eukaryotic signalling events. Recent experiments indicate that the murine zona pellucida glycoprotein ZP3 regulates acrosomal exocytosis by aggregating its corresponding receptors (ZP3-Rs) located in the mouse sperm plasma membrane. In other experiments, we have identified a putative ZP3-R of mouse sperm with Mr 95,000. Indirect immunofluorescence localizes this ZP3-R, termed p95, to the acrosomal region of the mouse sperm head, which is the anticipated location for ZP3-Rs. Membrane fractionation studies indicate that p95 cofractionates with a plasma membrane-enriched preparation from sperm that contains zona pellucida-receptor activity. In addition to its role as a ZP3-R, p95 also serves as a substrate for a tyrosine kinase in response to zona pellucida binding. On the basis of the data presented here, and borrowing heavily from findings for other signalling systems, we have formulated two testable hypotheses that are compatible with the available data: either p95 is itself a protein tyrosine kinase receptor, or p95 serves as a ZP3 receptor and is separate from a protein tyrosine kinase that is activated during gamete interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polypeptide encoded by mouse ZP3 exon-7 is necessary and sufficient for binding of mouse sperm in vitro

Fertilization in mice is initiated by species-specific binding of sperm to mZP3, one of three mouse zona pellucida (ZP) glycoproteins. At nanomolar concentrations, purified egg mZP3 binds to acrosome-intact sperm heads and inhibits binding of sperm to eggs in vitro. Although several reports suggest that sperm recognize and bind to a region of mZP3 encoded by mZP3 exon-7 (so-called, sperm combining-site), this issue remains controversial. Here, exon-swapping and an IgG(Fc) fusion construct were used to further evaluate whether mZP3 exon-7 is essential for binding of sperm to mZP3. In one set of experiments, hamster ZP3 (hZP3) exon-6, -7, and -8 were individually replaced with the corresponding exon of mZP3. Stably transfected embryonal carcinoma (EC) cell lines carrying the recombinant genes were produced and secreted recombinant glycoprotein was purified and assayed for the ability to inhibit binding of sperm to eggs. While EC-hZP3, a recombinant form of hZP3 made by EC cells, is unable to inhibit binding of mouse sperm to eggs in vitro, the results suggest that substitution of mZP3 exon-7 for hZP3 exon-7, but not mZP3 exon-6 or -8, can impart inhibitory activity to EC-hZP3. In this context, a fusion construct consisting of human IgG(Fc) and mZP3 exon-7 and -8 was prepared, an EC cell line carrying the recombinant gene was produced, and secreted chimeric glycoprotein, called EC-huIgG(Fc)/mZP3(7), was purified and assayed. It was found that the chimeric glycoprotein binds specifically to plasma membrane overlying sperm heads to a similar extent as egg mZP3 and, at nanomolar concentrations, inhibits binding of mouse sperm to eggs in vitro. Collectively, these observations provide new evidence that sperm recognize and bind to a region of mZP3 polypeptide immediately downstream of its ZP domain that is encoded by mZP3 exon-7. The implications of these findings are discussed.     

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.     

Porcine zona pellucida: association of sperm receptor activity with the alpha-glycoprotein component of the Mr = 55,000 family

The immunogenicity and sperm receptor activity of five preparations of the major porcine zona pellucida glycoprotein family ZP3 (Mr = 55,000) were investigated. These included (1) ZP3, a chromatographically purified preparation of the 55,000 family; (2) ZP3 alpha, and (3) ZP3 beta, the two-component glycoproteins of the ZP3 family; (4) ZP3-EBGD, a partially deglycosylated preparation of ZP3 obtained by enzymatic treatment; and (5) ZP3-DG, a chemically deglycosylated preparation of ZP3. Titer studies using mouse and rabbit antisera prepared against each preparation yielded the following order of immunogenicity: ZP3 and ZP3 beta greater than ZP3-EBGD and ZP3 alpha greater than ZP3-DG, indicating that ZP3 becomes less immunogenic as more carbohydrate is removed. Pretreatment of intact zona with the various antisera prior to zona exposure to sperm resulted in an inhibition of sperm attachment to those zona treated with antibodies to ZP3, ZP3-EBGD, and ZP3 alpha. Pretreatment of zona with antibodies to ZP3 beta and ZP3-DG had no effect on sperm attachment. Studies involving pretreatment of boar sperm with the various ZP3 preparations prior to their use in a sperm-zona attachment assay and investigations involving displacement of the radiolabeled ZP3 preparations from sperm by unlabeled ZP3 preparations also yielded findings similar to the antibody studies. Collectively, these data indicate that ZP3 alpha probably functions as a zona receptor for boar sperm and that carbohydrate has an important role in maintaining the functional integrity of the ZP3 alpha glycoprotein.     

重组人卵透明带蛋白(rhZP3)的生物活性研究

为了研究毕赤酵母表达的重组人卵透明带蛋白(rhZP3)的生物活性,分别用空白培养液,含孕酮或rhZP3的培养液对人精子进行顶体诱发实验,用考马斯亮蓝染色法对顶体状态进行评价;用不同浓度的rhZP3以及空白培养液分别处理精子,然后再与卵子进行结合实验,观察经过不同处理的精子在精卵结合中的情况;用抗rhZP3抗血清与阴性血清分别处理卵子,再与精子进行结合实验,观察经过不同处理后的卵子在精卵结合中的情况。rhZP3诱发顶体反应实验结果显示,rhZP3处理组与空白对照组之间差异显著(P<0.01);精卵结合实验结果显示,各实验组和对照组之间存在显著性差异(P<0.01),rhZP3、抗rhZP3抗体均能抑制精卵结合。实验结果表明,rhZP3具有天然人卵透明带蛋白相似的活性。     

Proteolytic processing of human zona pellucida proteins.

Formation of the egg's extracellular matrix, the zona pellucida, is critical for fertilization and development of growing embryos. Zona pellucida glycoproteins, ZP1, ZP2, and ZP3, are secreted to form an insoluble extracellular matrix surrounding mammalian eggs. All cloned mammalian zona pellucida sequences contain a furin consensus cleavage site, RX(K)/(R)R, upstream of a putative transmembrane domain, which suggests processing by an endoprotease of the furin-proprotein-convertase family. Recombinant expression of human (h) ZP1, ZP2, and ZP3 produces glycoproteins that are secreted and have migration patterns in SDS-PAGE identical to those of native human zona pellucida proteins. Because a C-terminal epitope tag that is present in the cell-associated zona proteins is, however, absent from the secreted zona proteins, secreted recombinant zona pellucida proteins lack their C-terminal regions. Three different strategies were used to explore processing events in the C-terminal region: site-directed mutagenesis of the furin cleavage site, treatment with a competitive inhibitor of all furin family members, and interference with Golgi modifications by Brefeldin A. All treatments altered the SDS-PAGE migration of recombinant hZP3, concordant with cleavage by a furin family member and Golgi glycosylation of secreted hZP3. Furthermore, cleavage of cell-associated hZP3 by exogenous furin converts the migration of cell-associated hZP3 to that of secreted hZP3. To determine whether a similar cleavage pattern exists in zona pellucida proteins that are assembled in the zona matrix, "hZP3 rescue" mouse zonae pellucidae were employed. Immunoblotting experiments revealed that hZP3, assembled and functional in the "hZP3 rescue" mouse zona pellucida, lacks the furin cleavage site, supporting the hypothesis that formation of the zona pellucida matrix involves regulated proteolysis by a member of the furin convertase family.     

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.     

Polypeptide backbone derived from carboxyl terminal of mouse ZP3 inhibits sperm-zona binding

For mammalian organism, fertilization begins with species-specific recognition between sperm and egg, a process depending upon egg zona pellucida glycoproteins and putative sperm interacting protein(s). In mouse, zona pellucida glycoprotein ZP3 is believed to be the primary receptor for sperm and inducer of sperm acrosomal reaction, and its function has been attributed to the specific O-linked oligosaccharides attached to polypeptide backbone. While lots of reports have focused on the role of ZP3's oligosaccharides in fertilization, there are few concerning its polypeptide backbone. To investigate whether mZP3 polypeptide backbone is involved in sperm-egg recognition, three partially overlapping cDNA fragments, together covering entire mouse ZP3, were cloned, expressed and purified under denaturing condition. Although all three refolded proteins possess native conformation, only one derived from the carboxyl terminal showed inhibitory effect to the sperm-zona binding during in vitro fertilization. This phenomenon could not be explained by enhanced acrosomal exocytosis rate, in that the acrosomal reaction assay demonstrated its inability to induce the acrosomal reaction. Our results suggest that the carboxyl terminal of mZP3 polypeptide backbone interacts with sperm and such interaction plays a significant role in sperm-zona binding, ultimately successful fertilization.     

A structural view of egg coat architecture and function in fertilization

Species-restricted interaction between gametes at the beginning of fertilization is mediated by the extracellular coat of the egg, a matrix of cross-linked glycoprotein filaments called the zona pellucida (ZP) in mammals and the vitelline envelope in nonmammals. All egg coat subunits contain a conserved protein-protein interaction module-the "ZP domain"-that allows them to polymerize upon dissociation of a C-terminal propeptide containing an external hydrophobic patch (EHP). Recently, the first crystal structures of a ZP domain protein, sperm receptor ZP subunit zona pellucida glycoprotein 3 (ZP3), have been reported, giving a glimpse of the structural organization of the ZP at the atomic level and the molecular basis of gamete recognition in vertebrates. The ZP module is divided in two related immunoglobulin-like domains, ZP-N and ZP-C, that contain characteristic disulfide bond patterns and, in the case of ZP-C, also incorporate the EHP. This segment lies at the interface between the two domains, which are connected by a long loop carrying a conserved O-glycan important for binding to sperm in vitro. The structures explain several apparently contradictory observations by reconciling the variable disulfide bond patterns found in different homologues of ZP3 as well as the multiple ZP3 determinants alternatively involved in gamete interaction. These findings have implications for our understanding of ZP subunit biogenesis; egg coat assembly, architecture, and interaction with sperm; structural rearrangements leading to postfertilization hardening of the ZP and the block to sperm binding; and the evolutionary origin of egg coats.     

Zona pellucida from fertilised human oocytes induces a voltage-dependent calcium influx and the acrosome reaction in spermatozoa, but cannot be penetrated by sperm

Background  

The functions of three zona glycoproteins, ZP1, ZP2 and ZP3 during the sperm-zona pellucida (ZP) interaction are now well established in mice. The expression of an additional zona glycoprotein, ZPB/4, in humans, led us to reconsider the classical mouse model of gamete interaction. We investigated the various functions of human ZP (hZP) during the interaction of spermatozoa with fertilised and unfertilised oocytes.     

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.     

Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs

During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head to a glycoprotein, called ZP3, present in the egg extracellular coat or zona pellucida. Bound sperm then undergo the acrosome reaction, which results in exposure of inner acrosomal membrane, penetrate through the zona pellucida, and fuse with egg plasma membrane. Thus, in the normal course of events, acrosome-reacted sperm must remain bound to eggs, despite loss of plasma membrane from the anterior region of the head and exposure of inner acrosomal membrane. Here, we examined maintenance of binding of sperm to the zona pellucida following the acrosome reaction. We found that polyclonal antisera and monoclonal antibodies directed against ZP2, another zona pellucida glycoprotein, did not affect initial binding of sperm to eggs, but inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. On the other hand, polyclonal antisera and monoclonal antibodies directed against ZP3 did not affect either initial binding of acrosome-intact sperm to eggs or maintenance of binding following the acrosome reaction. We also found that soybean trypsin inhibitor, a protein reported to prevent binding of mouse sperm to eggs, did not affect initial binding of sperm to eggs, but, like antibodies directed against ZP2, inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. These and other observations suggest that ZP2 serves as a secondary receptor for sperm during the fertilization process in mice and that maintenance of binding of acrosome-reacted sperm to eggs may involve a sperm, trypsin-like proteinase.     

Expression of recombinant human zona pellucida protein 2 and its binding capacity to spermatozoa

The human zona pellucida (ZP) is composed of three major glycoproteins: ZP1, ZP2, and ZP3. The aim of this study was to clarify the role of ZP2 by focusing on the polypeptide structure. We produced in Escherichia coli a recombinant human ZP2 protein (rec-hZP2) corresponding to amino acid sequence 1-206 of the mature protein. The final yield of rec-hZP2 protein was 80 microg/ml Luria Broth medium. After 2-h incubation of human spermatozoa with rec-hZP2 in vitro, an immunofluorescent study indicated that rec-hZP2 bound only to acrosome-reacted spermatozoa. The binding site migrated from the acrosome to the midpiece of the spermatozoa. Rabbit and mouse antisera produced against rec-hZP2 stained native human ZP in the immunofluorescent study, and significantly blocked human sperm binding and penetration into human ZP as compared to control values. The N-terminal polypeptide portion of human ZP2 was shown to contain a binding site for acrosome-reacted spermatozoa and to play an important role in secondary sperm binding and penetration into the ZP.     

Enzymatic dissection of the functions of the mouse egg's receptor for sperm

During the course of sperm-egg interaction in mice, zona pellucida glycoprotein ZP3 (approximately equal to 80 kDa) serves as both receptor for sperm (J. D. Bleil and P. M. Wassarman, 1980c, Cell 20, 873-882) and inducer of the acrosome reaction (J. D. Bleil and P. M. Wassarman, 1983, Dev. Biol. 95, 317-324). In this investigation, small ZP3 glycopeptides (approximately equal to 1.5-6 kDa), obtained by extensive digestion of the purified glycoprotein with insoluble Pronase, were assayed for both sperm receptor and acrosome reaction-inducing activities. While ZP3 glycopeptides were virtually as effective as intact ZP3 in inhibiting binding of sperm to eggs in vitro ("receptor activity"), unlike intact ZP3, they failed to induce sperm to undergo the acrosome reaction. The latter was determined by indirect immunofluorescence using a monoclonal antibody directed against the acrosomal cap region of sperm. These results suggest that the sperm receptor activity of ZP3 is dependent only on its carbohydrate components, whereas acrosome reaction-inducing activity is dependent on the polypeptide chain of ZP3 as well.     

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.     

Transgenic mice with reduced numbers of functional sperm receptors on their eggs reproduce normally.

To initiate fertilization in mice, free-swimming sperm bind to mZP3, an approximately 83-kDa glycoprotein present in the ovulated egg zona pellucida (ZP). mZP3 is located periodically along the filaments that constitute the ZP. Sperm recognize and bind to specific oligosaccharides linked to one or more of five Ser residues clustered in the carboxy-terminal one-third of the mZP3 polypeptide. When all five Ser residues are converted to nonhydroxy amino acids by site-directed mutagenesis of the mZP3 gene, an inactive form of mZP3, called mZP3[ser], is secreted by embryonal carcinoma cells stably transfected with the mutated gene. Here, seven independent transgenic mouse lines were established that harbor the mutated mZP3 gene. In all lines, the mutant gene is expressed by growing oocytes and mZP3[ser] is synthesized, secreted, and incorporated into the ZP. Purified mZP3[ser] prepared from ovaries of transgenic mice, like mZP3[ser] from transfected embryonal carcinoma cells, is inactive in sperm binding assays in vitro. On the other hand, the presence of mZP3[ser] in the ZP does not significantly affect either the binding of sperm to ovulated eggs in vitro or the reproduction of the mice, i.e., the transgenic mice are fertile, breed at normal intervals, and produce litters of normal sizes. These results indicate that the number of functional sperm receptors in the ZP can be reduced by more than 50% without adversely affecting fertilization of eggs in vivo.