Recombinant porcine zona pellucida glycoproteins expressed in Sf9 cells bind to bovine sperm but not to porcine sperm

The zona pellucida, which surrounds the mammalian oocyte, consists of the ZPA, ZPB, and ZPC glycoproteins and plays roles in species-selective sperm-egg interactions via its carbohydrate moieties. In the pig, this activity is conferred by tri- and tetraantennary complex type chains; in cattle, it is conferred by a chain of 5 mannose residues. In this study, porcine zona glycoproteins were expressed as secreted forms, using the baculovirus-Sf9 insect cell system. The sperm binding activities of the recombinant proteins were examined in three different assays. The assays clearly demonstrated that recombinant ZPB bound bovine sperm weakly but did not bind porcine sperm; when recombinant ZPC was also present, bovine sperm binding activity was greatly increased, but porcine sperm still was not bound. The major sugar chains of ZPB were pauci and high mannose type chains that were similar in structure to the major neutral N-linked chain of the bovine zona. In fact, the nonreducing terminal alpha-mannose residues were necessary for the sperm binding activity. These results show that the carbohydrate moieties of zona glycoproteins, but not the polypeptide moieties, play an essential role in species-selective recognition of porcine and bovine sperm. Moreover, Asn to Asp mutations at either of two of the N-glycosylation sites of ZPB, residue 203 or 220, significantly reduced the sperm binding activity of the ZPB/ZPC mixture, whereas a similar mutation at the third N-glycosylation site, Asn-333, had no effect on binding. These results suggest that the N-glycans located in the N-terminal half of the ZP domain of porcine ZPB are involved in sperm-zona binding.     

Heterocomplex formation and cell-surface accumulation of hen's serum zona pellucida B1 (ZPB1)with ZPC expressed by a mammalian cell line (COS-7): a possible initiating step of egg-envelope matrix construction

The egg envelope, referred to as zona pellucida (ZP) in mammalian eggs, is a fibrous and noncollagenous extracellular matrix surrounding vertebrate eggs, and composed of three to four homologous glycoproteins with a common ZP domain. In birds, a liver-derived ZP glycoprotein (ZP1/ZPB1) is transported through the bloodstream to ovarian follicles and joins the egg-envelope matrix construction together with the other ZP glycoproteins, such as ZPC and ZPD/ZPX2, both secreted from follicular granulosa cells. We report here that, through its ZP domain, ZPB1 specifically associates with ZPC, which might lead to the construction of egg-envelope matrix. The ZPB1 in laying hen's serum specifically bound to ZPC, but not to ZPX2, separated by SDS-PAGE and blotted on a membrane. Hemagglutinin (HA)-tagged ZPC expressed in a mammalian cell line (COS-7) cells was processed and secreted as a mature-form into the culture medium. From the culture supernatant of ZPC-expressing transfectants cultured in the presence of ZPB1, both ZPB1 and ZPC were recovered as heterocomplexes by immunoprecipitation using either anti-HA or anti-ZPB1 antibody. Interestingly, a monoclonal antibody, 8E1, which immunoprecipitated free ZPB1, did not immunoprecipitate the ZPB1-ZPC heterocomplexes. An 8E1 epitope was mapped on a C-terminal region of the ZP domain in a ZPB1 molecule by identifying an 8E1-positive peptide using mass spectroscopy. Furthermore, by laser scanning confocal microscopy, ZPB1 and ZPC were observed to colocalize on the surface of ZPC-expressing transfectants cultured in the presence of ZPB1, whereas almost no ZPC was detected on the surface of the transfectants cultured in the absence of ZPB1. Taken together, these results suggest that ZPB1 transported into ovarian follicles encounters and associates with ZPC secreted from granulosa cells, resulting in the formation of heterocomplexes around an oocyte. In addition, it appears that such ZPB1-ZPC complexes accumulated on the oocyte surface act as a scaffold for subsequent matrix construction events including ZPX2 association.     

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.     

Structural conservation of mouse and rat zona pellucida glycoproteins. Probing the native rat zona pellucida proteome by mass spectrometry

The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mouse zonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2, ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report, we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a "minimum structure(s)" to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents. These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome (defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstrating that the rat zonae glycoproteins share a high degree of conservation of structural features with respect to their mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA. Each zona protein undergoes extensive co- and post-translational modification prior to its secretion and incorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that is cleaved off once protein translation begins and an anchoring C-terminal transmembrane domain from which the mature protein is released. Mass spectrometric analysis with a limited amount of native material allowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized by cyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation. The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue that is part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was not determined. Each zona protein contains a "zona domain" with eight conserved cysteine residues that is thought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfide bond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identical to those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites are occupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and four or five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59-83 Ser/Thr residues), but only two regions were observed to carry O-glycans in rat ZP3.     

Equine zona protein synthesis and ZP structure during folliculogenesis, oocyte maturation, and embryogenesis

In the equine, the zona pellucida (ZP) is the major barrier to successful in vitro fertilization. Therefore the aim of our studies was to analyze species-specific features of the equine ZP in regard to structure and glycoprotein ZPB and ZPC expression sites during oocyte development and embryogenesis. The equine ZP revealed high immunological cross-reactivity to porcine ZPB and ZPC. In the ovary, the distribution of ZPB and ZPC was co-localized and correlated with the developmental stage of the follicle. ZPB and ZPC expression started in the oocyte of the late primordial and primary follicle. In the secondary follicle, both the oocyte and the cumulus cells contributed to ZPB and ZPC synthesis. After in vivo maturation the oocyte stopped ZPB and ZPC production whereas the cumulus cells continued synthesis. Contrary, in vitro matured (IVM) cumulus-oocyte-complexes (COCs) revealed a reverse expression pattern. This was correlated to alterations in the distribution, number, and size of pores in the ZP. In the zona, N-acetylglucosamine residues were co-localized with ZPC. The acellular glycoprotein capsule surrounding early equine embryos was negative for ZPB and ZPC. Our results imply that in the horse ZPB and ZPC glycoprotein expression is differentially regulated during folliculogenesis, oocyte maturation, and embryogenesis. Contrary to the bovine and porcine, zona protein synthesis during in vivo maturation is completely overtaken by the cumulus cells implying that in the horse these cells are crucial for zona integrity. During IVM, the cumulus cells lose their ability to synthesize glycoproteins leading to alterations in the zona structure.     

Localization of N-linked carbohydrate chains in glycoprotein ZPA of the bovine egg zona pellucida.

The zona pellucida, a transparent envelope surrounding the mammalian oocyte, consists of three glycoproteins, ZPA, ZPB and ZPC, and plays a role in sperm-egg interactions. In bovines, these glycoproteins cannot be separated unless the acidic N-acetyllactosamine regions of the carbohydrate chains are removed by endo-beta-Galactosidase digestion. Endo-beta-Galactosidase-digested ZPB retains stronger sperm-binding activity than ZPC. It is still unclear whether ZPA possesses significant activity. Recently, we reported that bovine sperm binds to Man5GlcNAc2, the neutral N-linked chain in the cow zona proteins. In this study, we investigated the localization of the sperm-ligand active high-mannose-type chain and the acidic complex-type chains in bovine ZPA. Three N-glycopeptides of ZPA, containing an N-glycosylation site at Asn83, Asn191 and Asn527, respectively, were obtained from endo-beta-Galactosidase-digested ZPA. Of these glycosylation sites, only Asn527 is present in the ZP domain common to all the zona proteins. The carbohydrate structures of the N-linked chains obtained from each N-glycopeptide were characterized by two-dimensional sugar mapping analysis, while considering the structures of the N-linked chains of the zona protein mixture reported previously. Acidic complex-type chains were found at all three N-glycosylation sites, while Man5GlcNAc2 was found at Asn83 and Asn191, but there was very little of this sperm-ligand active chain at Asn527 in the ZP domain of ZPA.     

A hatching enzyme substrate in the Xenopus laevis egg envelope is a high molecular weight ZPA homolog

The Xenopus laevis egg envelope is composed of six or more glycoproteins, three of which have been cloned and identified as the mammalian homologs ZPA (ZP2), ZPB (ZP1) and ZPC (ZP3). The remaining glycoproteins are a triplet of high molecular weight components that are selectively hydrolyzed by the hatching enzyme. We have isolated one of these proteins and cloned its cDNA. The mRNA for the protein was found to be expressed only in early stage oocytes, as are other envelope components. From the deduced amino acid sequence, it was indicated to be a secreted glycoprotein with a characteristic ZP domain in the C-terminal half of the molecule. The N-terminal half was unrelated to any known glycoprotein. Comparative sequence analysis of the ZP domain indicated that it was derived from an ancestor of ZPA and ZPB, with the greatest identity to ZPA. This envelope component has been designated ZPAX.     

Molecular cloning of bovine zona pellucida glycoproteins ZPA and ZPB and analysis for sperm-binding component of the zona.

The zona pellucida, a transparent envelope surrounding the mammalian oocyte, comprises three glycoproteins, ZPA, ZPB and ZPC, and plays important roles in fertilization. We have previously reported that apparent relative molecular masses of bovine zona glycoproteins on SDS/PAGE under nonreducing conditions after removal of poly N-acetyllactosamine at the nonreducing portion of sugar chains with endo-beta-galactosidase are 72 000, 58 000 and 45 000 [Noguchi, S., Yonezawa, N., Katsumata, T., Hashizume, K.,Kuwayama, M., Hamano, S., Watanabe, S. & Nakano, M. (1994) Biochim. Biophys. Acta 1201, 7-14]. The N-terminal amino-acid sequences and crossreactivity to antibodies specific to each porcine zona component show that the bovine components correspond to porcine ZPA, ZPB and ZPC, respectively. In this study, we deduced amino-acid sequences of bovine ZPA and ZPB by cDNA cloning and sequencing. Identities in amino-acid sequences between bovine and porcine counterparts were 77% for ZPA and 75% for ZPB, whereas between bovine and murine counterparts identities were 57% for ZPA and 37% for ZPB. The positions of Cys were completely conserved in bovine ZPA and ZPB compared with counterparts of other mammalian species. Bovine ZPA was processed between Ala and Asp on fertilization, suggesting that the consensus motif for the processing is Ala-Asp-Asp/Glu. We purified bovine zona components and examined their sperm-binding activity with an in vitro competition assay and sperm-bead-binding assay. As a result, ZPB showed the strongest sperm-binding activity among the components. ZPC also showed sperm-binding activity and the activity per molecule was about one-sixth that of ZPB according to the result of the sperm-bead-binding assay. We could not determine if ZPA has significant sperm-binding activity, but the activity may be much lower than that of ZPB even if ZPA has significant activity. Thus, ZPB may play a major role in sperm binding in bovine zona.     

Phylogenetic analysis and identification of pseudogenes reveal a progressive loss of zona pellucida genes during evolution of vertebrates

Vertebrate eggs are surrounded by an extracellular matrix with similar functions and conserved individual components: the zona pellucida (ZP) glycoproteins. In mammals, chickens, frogs, and some fish species, we established an updated list of the ZP genes, studied the relationships within the ZP gene family using phylogenetic analysis, and identified ZP pseudogenes. Our study confirmed the classification of ZP genes in six subfamilies: ZPA/ZP2, ZPB/ZP4, ZPC/ZP3, ZP1, ZPAX, and ZPD. The identification of a Zpb pseudogene in the mouse genome, Zp1 pseudogenes in the dog and bovine genomes, and Zpax pseudogenes in the human, chimpanzee, macaque, and bovine genomes showed that the evolution of ZP genes mainly occurs by death of genes. Our study revealed that the extracellular matrix surrounding vertebrate eggs contains three to at least six ZP glycoproteins. Mammals can be classified in three categories. In the mouse, the ZP is composed of three ZP proteins (ZPA/ZP2, ZPC/ZP3, and ZP1). In dog, cattle and, putatively, pig, cat, and rabbit, the zona is composed of three ZP proteins (ZPA/ZP2, ZPB/ZP4, and ZPC/ZP3). In human, chimpanzee, macaque, and rat, the ZP is composed of four ZP proteins (ZPA/ZP2, ZPB/ZP4, ZPC/ZP3, and ZP1). Our review provides new directions to investigate the molecular basis of sperm-egg recognition, a mechanism which is not yet elucidated.     

Analysis of N-linked glycans of porcine zona pellucida glycoprotein ZPA by MALDI-TOF MS: a contribution to understanding zona pellucida structure

The mammalian oocyte is encased by a transparent extracellular matrix, the zona pellucida (ZP), which consists of three glycoproteins, ZPA, ZPB, and ZPC. The glycan structures of the porcine ZP and the complete N-glycosylation pattern of the ZPB/ZPC oligomer has been recently described. Here we report the N-glycan pattern and N-glycosylation sites of the porcine ZP glycoprotein ZPA of an immature oocyte population as determined by a mass spectrometric approach. In-gel deglycosylation of the electrophoretically separated ZPA protein and comparison of the pattern obtained from the native, the desialylated and the endo-beta-galactosidase-treated glycoprotein allowed the assignment of the glycan structures by MALDI-TOF MS by considering the reported oligosaccharide structures. The major N-glycans are neutral biantennary complex structures containing one or two terminal galactose residues. Complex N-glycans carrying N-acetyllactosamine repeats are minor components and are mostly sialylated. A significant signal corresponding to a high-mannose type chain appeared in the three glycan maps. MS/MS analysis confirmed its identity as a pentamannosyl N-glycan. By the combination of tryptic digestion of the endo-beta-galactosidase-treated ZP glycoprotein mixture and in-gel digestion of ZPA with lectin affinity chromatography and reverse-phase HPLC, five of six N-glycosylation sites at Asn(84/93), Asn268, Asn316, Asn323, and Asn530 were identified by MS. Only one site was found to be glycosylated in the N-terminal tryptic glycopeptide with Asn(84/93.) N-glycosidase F treatment of the isolated glycopeptides and MS analysis resulted in the identification of the corresponding deglycosylated peptides.     

Participation of the nonreducing terminal beta-galactosyl residues of the neutral N-linked carbohydrate chains of porcine zona pellucida glycoproteins in sperm-egg binding

The zona pellucida (ZP) surrounding the mammalian oocyte is composed of three glycoprotein components (ZPA, ZPB, and ZPC). Mammalian sperm bind to carbohydrate chains of a ZP glycoprotein in the initial phase of fertilization. Sperm-ligand carbohydrate chains have been characterized in mouse, cow, and pig. In pigs, triantennary/tetraantennary neutral complex-type chains from ZPB/ZPC mixture possess stronger sperm-binding activity than those of biantennary chains (Kudo et al., 1998: Eur J Biochem 252:492-499). Most of these oligosaccharides have beta-galactosyl residues at the nonreducing ends. This study used two in vitro competition assays to investigate the participation of the nonreducing terminal beta-galactosyl residues of the ligand active chains in porcine sperm binding. The removal of the nonreducing terminal beta-galactosyl residues from either the ligand active carbohydrate chains or endo-beta-galactosidase-digested glycoproteins significantly reduced their inhibition of sperm-egg binding, indicating that the beta-galactosyl residues at the nonreducing ends are involved in porcine sperm-egg binding. A correlation between the sperm-binding activity and in vitro fertilization rate is also presented.     

Identification of the true human orthologue of the mouse Zp1 gene: evidence for greater complexity in the mammalian zona pellucida?

The mammalian zona pellucida is a mixture of glycoproteins, believed to be encoded by three distinct genes, ZP1/ZPB, ZP2/ZPA, and ZP3/ZPC. We have now determined that the true human orthologue of the mouse Zp1 gene is not ZPB, but that there is a distinct human ZP1 gene. Comparison of the human ZP1 and murine Zp1 genes indicates significant conservation of nucleotide and amino acid sequences, of intron-exon size and organisation, and of regulatory sequences. In addition, the mouse and human ZP1 genes are in a region of conserved synteny between human chromosome 11 and mouse chromosome 19.     

Disulfide linkage patterns of pig zona pellucida glycoproteins ZP3 and ZP4

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

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.     

Secretion and assembly of zona pellucida glycoproteins by growing mouse oocytes microinjected with epitope-tagged cDNAs for mZP2 and mZP3

The zona pellucida (ZP) is a highly organized extracellular coat that surrounds all mammalian eggs. The mouse egg ZP is composed of three glycoproteins, called mZP1-3, that are synthesized, secreted, and assembled into a ZP exclusively by growing oocytes. Here, we microinjected epitope-tagged (Myc and Flag) cDNAs for mZP2 and mZP3 into the germinal vesicle (nucleus) of growing oocytes isolated from juvenile mice. Specific antibodies and laser scanning confocal microscopy were used to follow nascent, recombinant ZP glycoproteins in both permeabilized and nonpermeabilized oocytes. When such cDNAs were injected, epitope-tagged mZP2 (Myc-mZP2) and mZP3 (Flag-mZP3) were synthesized, packaged into large intracellular vesicles, and secreted by the vast majority of oocytes. Secreted glycoproteins were incorporated into only the innermost layer of the thickening ZP, and the amount of nascent glycoprotein in this region increased with increasing time of oocyte culture. Consistent with prior observations, the putative transmembrane domain at the C terminus of mZP2 and mZP3 was missing from nascent glycoprotein incorporated into the ZP. When the consensus furin cleavage site near the C terminus of mZP3 was mutated, such that it should not be cleaved by furin, secretion and assembly of mZP3 was reduced. On the other hand, mZP3 incorporated into the ZP lacked the transmembrane domain downstream of the mutated furin cleavage site, suggesting that some other protease(s) excised the domain. These results strongly suggest that nascent mZP2 and mZP3 are incorporated into only the innermost layer of the ZP and that excision of the C-terminal region of the glycoproteins is required for assembly into the oocyte ZP.     

Immunohistochemical localization of zona pellucida proteins ZPA, ZPB and ZPC in human, cynomolgus monkey and mouse ovaries

The zona pellucida of mammalian oocytes plays an important role in binding and activation of sperm cells during the molecular events leading to fertilization. The genes coding for the three zona pellucida glycoproteins ZPA, ZPB, and ZPC of various species including mouse, dog, and human have been cloned and sequenced by several groups. However, it has remained a matter of debate as to whether the oocytes alone or in conjunction with the surrounding granulosa cells express and deposit these proteins to form the zona pellucida matrix. Addressing this unresolved issue, we assessed the expression and localization of all three zona pellucida proteins in ovaries of human, cynomolgus monkey and mice using immunohistochemical methods. In addition, oocyte-specific expression of ZPC from the primordial stage onward was confirmed by in situ hybridization. In sections of human ovaries, ZPA, ZPB, and ZPC proteins were immunohistochemically detected in the cytoplasms of primordial oocytes and during later stages of folliculogenesis in the zona pellucida matrices of oocytes. In sections fixed with formalin, a clear homogeneous ring was visible around the oocyte and no staining of granulosa cells was observed. In contrast, staining of ZP proteins was also observed between granulosa cells when Bouin's reagent had been used for tissue fixation. Thus, the original zona pellucida architecture was better preserved by formalin fixation. We further demonstrated that dissolution of the zona pellucida of isolated bovine oocytes occurred after they were exposed to Bouin's reagent. In summary, these results demonstrate that in mice, monkeys and humans, zona proteins are expressed and assembled exclusively by the oocyte and not by the granulosa cells. Previously observed results of ZP expression by an involvement of granulosa cells might therefore be the result of an improper fixation of the tissues leading to the disruption of the zona pellucida. Additionally this study highlights the importance of choosing the correct fixative for immunohistochemistry, not only for the usual reason of retaining antigenicity, but rather to retain the entire architectural structure.     

Mutation of a conserved hydrophobic patch prevents incorporation of ZP3 into the zona pellucida surrounding mouse eggs

Three glycoproteins (ZP1, ZP2, and ZP3) are synthesized in growing mouse oocytes and secreted to form an extracellular zona pellucida that mediates sperm binding and fertilization. Each has a signal peptide to direct it into a secretory pathway, a "zona" domain implicated in matrix polymerization and a transmembrane domain from which the ectodomain must be released. Using confocal microscopy and enhanced green fluorescent protein (EGFP), the intracellular trafficking of ZP3 was observed in growing mouse oocytes. Replacement of the zona domain with EGFP did not prevent secretion of ZP3, suggesting the presence of trafficking signals and a cleavage site in the carboxyl terminus. Analysis of linker-scanning mutations of a ZP3-EGFP fusion protein in transient assays and in transgenic mice identified an eight-amino-acid hydrophobic region required for secretion and incorporation into the zona pellucida. The hydrophobic patch is conserved among mouse zona proteins and lies between a potential proprotein convertase (furin) cleavage site and the transmembrane domain. The cleavage site that releases the ectodomain from the transmembrane domain was defined by mass spectrometry of native zonae pellucidae and lies N-terminal to a proprotein convertase site that is distinct from the hydrophobic patch.