Expression of bonnet monkey (Macaca radiata) zona pellucida-3 (ZP3) in a prokaryotic system and its immunogenicity

An internal fragment (978 bp) corresponding to the bonnet monkey (Macaca radiata) ZP3, excluding the N-terminus signal sequence and the C-terminus transmembrane-like domain, was amplified by PCR from a full-length cDNA clone. The amplified Bam HI and Sacl restricted fragment was cloned in frama downstream of the T5 promoter under lac operator control for expression in the pQE-30 vector. Recombinant ZP3 (r-ZP3) was expressed as a poly-histidine fusion protein in E. coli strains SG13009[pREP4] and BL-21(DE3). Immunoblot with a murine monoclonal antibody, MA-451 (raised against porcine ZP3β—a homologue of bonnet ZP3, and cross-reactive with bonnet zona pellucida) revealed a predominant band of 50 kDa besides degraded fragments. Optimum expression of r-ZP3 was observed at 0.5 mM IPTG. Antisera generated in monkeys against synthetic peptides from the N-(23–45 aa residues) and C-(300–322 and 324–347 aa residues) termini of the deduced bonnet monkey precursor ZP3 sequence reacted with the r-ZP3 protein in ELISA. The r-ZP3 expressed in SG13009[pREP4] was purified on Ni-NTA resin under denaturing conditions and conjugated with diphtheria toxoid (DT). Immunization of a female rabbit and six female bonnet monkeys with the r-ZP3-DT conjugate generated antibodies reactive with r-ZP3 in ELISA. Rabbit r-ZP3 antiserum reacted with porcine ZP3β and bonnet r-ZP3 but failed to react with porcine ZP3α in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on bonnet monkey ovarian sections, showed positive fluorescence with zona pellucida. The availability of r-ZP3 will further help in evaluating its efficacy for fertility regulation and understanding the autoimmune oophoritis associated with ZP3 immunization in nonhuman primates. Mol. Reprod. Dev. 47:140–147, 1997. © 1997 Wiley-Liss, Inc.     

Endocrine response in rabbits immunized with native versus deglycosylated porcine zona pellucida antigens

Previous studies evaluating porcine zona pellucida antigens for immunocontraceptive purposes have in some cases revealed altered ovarian function in association with antibody response. This study was undertaken in an attempt to identify zona immunogens that do not cause adverse endocrine effects. To this end, we investigated the effects of highly purified preparations of native and deglycosylated pig zona pellucida antigens on ovarian function and immune response in the rabbit. Thirty female rabbits were immunized, 5 per group, with 100 micrograms each of either 1) SIZP, solubilized isolated zonae pellucidae; 2) ZP3, a purified porcine zona preparation containing the two principle glycoproteins, ZP3 alpha and ZP3 beta, endo-beta-galactosidase-digested ZP3 glycoproteins (approximately 30% deglycosylated) termed 3) ZP3 alpha/EBGD and 4) ZP3 beta/EBGD; and chemically deglycosylated ZP3 alpha and ZP3 beta (greater than or equal to 92% deglycosylated), termed 5) ZP3 alpha/DG and 6) ZP3 beta/DG. Rabbits injected with saline (n = 2) or Freund's adjuvant alone (n = 3) served as controls. Serum LH, FSH, estradiol, and progesterone were measured at 5-day intervals during seven 20-day cycles of hCG-induced pseudopregnancy over 42 wk. Anti-ZP3 titers, determined by RIA, developed in all treatment groups and correlated directly with carbohydrate content. Animals immunized with SIZP, ZP3, and ZP3 beta/EBGD showed a significant elevation of LH and FSH and a significant decline of peak progesterone levels by the fourth pseudopregnancy cycle. In contrast, animals immunized with ZP3 alpha/EBGD, ZP3 alpha/DG, and ZP3 beta/DG showed no significant elevations of gonadotropins and continued to display cyclic progesterone secretion in response to hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of monoclonal antibodies against porcine zona pellucida-3 and their functional relevance.

Seven monoclonal antibodies (MAs) against 55 kDa glycoprotein family of porcine zona pellucida (ZP3) reacting with either ZP3 alpha (MA-7, MA-27, MA-28) or ZP3 beta (MA-1, MA-2, MA-11, MA-30) have been described. MA-1, -2, -27, -28 and -30 do not recognize carbohydrate determinants as shown by their reactivity to the deglycosylated (DG) ZP3 alpha and ZP3 beta. Indirect immunoperoxidase studies showed that all MAs reacted with zona pellucida from porcine and monkey ovaries. Only MA-1 and -27 reacted with ZP from rabbit ovary as well, while none of the MAs recognised mouse ZP, MA-7, -11, -27, -28 and -30 inhibited in vitro, the zona lysis by trypsin as well as the binding of ZP3 to sperm membrane vesicle as investigated by ELISA.     

Structural characterization of the Mr = 55,000 antigen (ZP3) of porcine oocyte zona pellucida. Purification and characterization of alpha- and beta-glycoproteins following digestion of lactosaminoglycan with endo-beta-galactosidase

The major macromolecular component of the porcine oocyte zona pellucida is a Mr = 55,000 antigen, termed ZP3, comprised of greater than 25 charge isomers. ZP3 was purified to apparent electrophoretic homogeneity from nonreduced, sodium dodecyl sulfate-treated porcine zonae pellucidae by chromatography on Sephacryl S-400 and hydroxylapatite resins. The carbohydrate moiety of purified ZP3 was comprised of a heterogeneous population of acidic lactosaminoglycans as evidenced by the saccharide composition and size distribution of glycopeptides produced by Pronase digestion of ZP3, as well as by the sensitivity of ZP3 to digestion with Escherichia freundii endo-beta-galactosidase. Endo-beta-galactosidase-digested ZP3 was resolved by gel electrophoresis into two components, termed alpha-glycoprotein and beta-glycoprotein, with Mr values (nonreduced) of 46,000 and 42,000, respectively. Each was comprised of fewer and more neutral charge isomers than ZP3. Following purification by reverse phase high performance liquid chromatography, the alpha- and beta-glycoproteins of endo-beta-galactosidase-digested ZP3 were distinguished on the basis of amino acid and carbohydrate compositions, amino-terminal sequence analyses and peptide mapping experiments, thus suggesting differences in the primary structures of their respective polypeptide moieties. Corresponding dissimilarities in the immunoreactivities of the alpha- and beta-glycoproteins toward polyclonal antisera raised against ZP3, alpha-glycoprotein, and beta-glycoprotein were revealed by competitive binding radioimmunoassays as well as by immunoblotting experiments. Collectively, the data were interpreted to indicate that the Mr = 55,000 antigen of porcine oocyte zona pellucida is in fact comprised of overlapping families of charge isomers corresponding to two structurally and immunologically distinct lactosaminoglycan-containing glycoproteins.     

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.     

Blocking of human sperm-zona interaction by monoclonal antibodies to a glycoprotein family (ZP4) of porcine zona pellucida.

To study zona pellucida antigens involved in human fertilization, five monoclonal antibodies (MAbs)--2A1, 2G3, 4A2, 4E12, and 5H4--were produced to a glycoprotein family (ZP4) isolated from heat-solubilized porcine zonae pellucidae. Each MAb reacted not only with solubilized porcine zona glycoproteins but also with the glycoproteins deglycosylated by trifluoromethanesulfonic acid treatment. They also reacted with intact zonae pellucidae of porcine and human oocytes. Three (4A2, 4E12, and 5H4) of the five MAbs showed a significant blocking effect on human sperm binding and penetration of human zonae pellucidae. The 5H4 MAb showed a strong reaction with ZP4 and ZP1 glycoprotein families of porcine zonae pellucidae, and four other MAbs reacted more strongly with ZP3 than with ZP4. The reactivity of 5H4 with porcine zona glycoproteins was destroyed by chymotrypsin digestion, but the antigen epitope was resistant to proteolysis by trypsin and endoproteinase Lys-C. A peptide fragment reactive to 5H4 was isolated by reverse-phase HPLC from endoproteinase Lys-C-treated ZP4 glycoproteins, and its molecular mass was determined to be 7 kDa by SDS-PAGE. These results suggested that the antigen epitope corresponding to 5H4 is a good candidate for development of a contraceptive vaccine.     

Structure and function of the zona pellucida: Identification and characterization of the proteins of the mouse oocyte's zona pellucida

The zona pellucida is an acellular coat which surrounds the plasma membrane of fully grown mammalian oocytes and which performs a variety of important functions during oogenesis, fertilization, and preimplantation development. In this investigation the proteins of the mouse oocyte's zona pellucida have been identified and characterized by using zonae pellucidae isolated individually from fully grown oocytes with mouth-operated micropipets. Various morphological and biochemical criteria were employed to assess the purity of the isolated zonae pellucidae and, in most cases, they were found to be virtually free of contamination by other oocyte proteins. It was determined that each zona pellucida contains 4.8 ng of protein, which represents 80% or more of the dry weight of the zona pellucida and about 17% of the oocyte's total protein. Electrophoretic analyses of as few as five isolated zonae pellucidae treated with diazotized [¹²⁵I]iodosulfanilic acid revealed the presence of only three radiolabeled proteins, designated ZP1, ZP2, and ZP3. The same three proteins were identified by Coomassie blue staining when large numbers of isolated zonae pellucidae (approximately 750) were subjected to SDS-polyacrylamide gel electrophoresis. These three proteins migrate as broad bands on SDS-polyacrylamide gels, consistent with their being glycoproteins, with apparent molecular weights of 200,000 (ZP1), 120,000 (ZP2), and 83,000 (ZP3). The same proteins were radiolabeled when intact oocytes were treated with diazotized [¹²⁵I]iodosulfanilic acid, a reagent which does not penetrate the oocyte's plasma membrane, or when isolated zonae pellucidae were treated with ³H-labeled 1-dimethylaminonaphthalene-5-sulfonyl chloride. Results of amino acid analysis and high-resolution two-dimensional electrophoresis of the individual proteins suggest that each protein represents a unique polypeptide chain. The proteins ZP1, ZP2, and ZP3 represent about 36, 47, and 17%, respectively, of the total protein of the zona pellucida. In the presence of reducing agents which cause dissolution of the zona pellucida, ZP1 is converted into a species which migrates with an apparent molecular weight of 130,000, suggesting that it exists as an oligomer, stabilized by disulfide bonds, in the unreduced state. The results of these experiments are discussed in terms of the properties of the zona pellucida before and after fertilization and are compared with results obtained using vitelline envelopes of eggs from nonmammalian animal species.     

Incorporation of mouse zona pellucida proteins into the envelope of Xenopus laevis oocytes

 All vertebrate eggs have extracellular matrices, referred to as the zona pellucida in Mus musculus and the vitelline envelope in Xenopus laevis. The mouse zona, composed of three sulfated glycoproteins (ZP1, ZP2, ZP3), is critical for fertilization and early development, and mice lacking a zona pellucida produce no live offspring. The primary structures of mouse ZP1 (623 amino acids), ZP2 (713 amino acids) and ZP3 (424 amino acids) have been deduced from full-length cDNAs, but posttranslational modifications result in mature zona proteins with molecular masses of 200–180 kDa, 140–120 kDa, and 83 kDa, respectively. The vitelline envelope forms a similar structure around Xenopus eggs and contains three glycoproteins that are structurally related (39–48% amino acid similarity) to the three mouse zona proteins. To investigate whether the structural semblances are sufficient to allow incorporation of the mouse zona proteins into the Xenopus vitelline envelope, capped synthetic mRNAs encoding ZP1, ZP2, and ZP3 proteins were injected into the cytoplasm of stage VI Xenopus oocytes. After 20 h of incubation the oocytes were harvested, and posttranslationally modified zona proteins were detected with monoclonal antibodies specific to mouse ZP1, ZP2, and ZP3. The oocytes were imaged with confocal microscopy to detect individual zona proteins in the extracellular matrix of the oocytes, and this localization was confirmed biochemically. Thus the mouse zona proteins appear to have been sufficiently conserved through 350 million years of evolution to be incorporated into the extracellular envelope surrounding Xenopus eggs. Received: 5 January 1999 / Accepted: 12 February 1999     

草原兔尾鼠卵透明带3在Pichia pastoris酵母中的分泌表达

本研究通过酵母Pichia pastoris系统表达草原兔尾鼠卵透明带3(IZP3)蛋白．获得控制害鼠生育研究的抗原物质。根据本实验室克隆获得的IZP3基因序列设计引物．并分别在5’引物和3’引物中引入了EcoR Ⅰ和Xba Ⅰ酶切位点。经PCR扩增,将IZP3克隆至穿梭质粒pSuper Y上,获得的重组穿梭质粒pSuper Y—IZP3经线性化后．采用电激法将重组穿梭质粒转入酵母SMD1168菌株中．Zeocin 筛选阳性克隆,经小瓶发酵后,取上清作SDS—PAGE和Weaterm—blot检测,结果表明IZP3在酵母中得到了成功表达,表达产物的分子量约为55kD．为IZP3免疫不育控制鼠害的研究提供了检测抗原。     

ZP2 and ZP3 traffic independently within oocytes prior to assembly into the extracellular zona pellucida

The extracellular zona pellucida surrounds mammalian eggs and mediates taxon-specific sperm-egg recognition at fertilization. In mice, the zona pellucida is composed of three glycoproteins, but the presence of ZP2 and ZP3 is sufficient to form a biologically functional structure. Each zona pellucida glycoprotein is synthesized in growing oocytes and traffics through the endomembrane system to the cell surface, where it is released from a transmembrane domain and assembled into the insoluble zona pellucida matrix. ZP2 and ZP3 colocalize in the endoplasmic reticulum and in 1- to 5-microm post-Golgi structures comprising multivesicular aggregates (MVA), but a coimmunoprecipitation assay does not detect physical interactions. In addition, ZP2 traffics normally in growing oocytes in the absence of ZP3 or if ZP3 has been mutated to prevent incorporation into the zona pellucida matrix, complementing earlier studies indicating the independence of ZP3 secretion in Zp2 null mice. N glycosylation has been implicated in correct protein folding and intracellular trafficking of secreted proteins. Although ZP3 contain five N-glycans, enhanced green fluorescent protein-tagged ZP3 lacking N glycosylation sites is present in MVA and is incorporated into the zona pellucida matrix of transgenic mice. Thus, ZP2 secretion is seemingly unaffected by ZP3 lacking N-glycans. Taken together, these observations indicate that ZP2 and ZP3 traffic independently through the oocyte prior to assembly into the zona pellucida.     

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.     

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.     

Addition of cholesterol loaded cyclodextrin prior to GV-phase vitrification improves the quality of mature porcine oocytes in vitro

The purpose of this study was to determine whether the mitochondrial membrane potential, pro-apoptotic gene expression, and ubiquitylation status of zona pellucida proteins (ZP1, ZP2, and ZP3) of vitrified GV-stage mature oocytes could be protected by treatment with cholesterol-loaded methyl-β-cyclodextrin (CLC) prior to vitrification. Porcine GV oocytes were treated with CLC prior to the vitrification process, and the effects on the mitochondrial membrane potential and ZP ubiquitylation status were determined by JC-1 single staining and western blot assays. We found that porcine GV-stage oocytes were treated with CLC at different concentrations (0.5, 5, and 10 mg/mL) prior to vitrification improved in vitro maturation of these oocytes (P < 0.05). The mitochondrial membrane potential of matured oocyte without vitrification or treated with 5 mg/mL CLC vitrification treatment was higher than that of the 0 mg/mL CLC group and other treatment groups (vitrified) (P < 0.05). The expression of Caspase 3, Caspase 8, and Caspase 9 genes in the high concentration CLC treatment groups (5 and 10 mg/mL) was significantly lower than that in the 0 (vitrified) mg/mL CLC group (P < 0.05). ZPs protein and ZP3 protein ubiquitylation were also higher in the non-vitrified controls, 5 and 10 mg/mL CLC-treated oocytes than in the 0 (vitrified) and 0.5 mg/mL vitrified groups (P < 0.05). Whereas the sperm–oocyte binding capacity was improved in the CLC treatment groups (P < 0.05) but the embryonic development rate was not improved. In conclusion, pretreatment with CLC can improve the survival rate and maturation rate of oocytes and protect their mitochondria and zona pellucida of porcine oocytes from cryodamage during the vitrification process.     

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.     

Immunological response and ovarian histology of squirrel monkeys (Saimiri sciureus) immunized with porcine zona pellucida ZP3 (Mr = 55,000) macromolecules

ZP3 (M, = 55,000) is the major electrophoretic component of the porcine zona pellucida (ZP). In a continuing assessment of ZP3 as a candidate antigen for contraceptive vaccine development, female squirrel monkeys were immunized with 200 μg ZP3 using either Freund's adjuvant (FA) or muramyl dipeptide (MDP) and the effect of such immunization on ovarian histology examined. Two experimental and three control groups were immunized: Group 1 (n = 4), ZP3 plus FA; Group 2 (n = 4),ZP3 plus MDP; and controls—Group 3 (n = 2), ZP3 alone; Group 4 (n = 4), FA alone; and Group 5 (n = 4), saline. High antibody response to ZP3 was detected in the ZP3/FA and ZP3/MDP groups, and a very low response, in the ZP3-alone group. Immune profiles for the ZP3iFA and ZP3/MDP groups were comparable, but titers in the MDP group were consistently lower and decreased more rapidly after 300 days post-immunization (PI) than in the FA group. At 6 months PI, all ovaries from the ZP3/FA group revealed a deficiency of zona-encased oocytes and a reduction in secondary and tertiary follicles compared to controls. At 18–24 months PI, normal ovarian histology in one ZP3/FA injected monkey and the presence of zona-encased oocytes in a second monkey suggested ovarian recovery. Normal ovarian histology was present in all monkeys in the ZP3/MDP group as well as in all controls. These findings indicate that while immunization with ZP3/FA does initially perturb normal ovarian histology, such adverse effects appear to be reversible. Furthermore, immunization using ZP3 with MDP has no adverse effect on the ovary, indicating the importance of proper adjuvant selection in immunocontraceptive (IC) studies. These data encourage continued investigation of the zona IC approach using well-characterized zona immunogens with non-Freund's adjuvants.     

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

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

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.     

Characterization of a proteinase that cleaves zona pellucida glycoprotein ZP2 following activation of mouse eggs

Here, we describe an in vitro assay that has permitted further characterization of a proteinase (called "ZP2-proteinase") that is released upon activation of ovulated mouse eggs and cleaves ZP2, one of three glycoproteins present in mouse zonae pellucidae. Results presented suggest that ZP2-proteinase readily diffuses through the zona pellucida within 5 min of activation of eggs by ionophore A23187 and carries out limited proteolysis of ZP2. Appearance of ZP2-proteinase is completely dependent upon activation of eggs, consistent with it being present in cortical granule exudate. The proteinase is insensitive to a wide variety of proteinase inhibitors, but is inhibited when either an anti-ZP2 monoclonal antibody or an Fab fragment of the antibody is bound to ZP2. Proteolysis occurs near the amino- or carboxy-terminus of ZP2, producing a 23,000 Mr glycopeptide(s) that remains attached to ZP2 by intramolecular disulfide bonds. HPLC fractionation of activated egg exudate suggests that ZP2-proteinase has an apparent Mr between 21,000 and 34,000. Proteolysis of ZP2 correlates with "hardening" of the zona pellucida following egg activation and, thus, may be responsible for one aspect of the zona reaction.