Efficacy of antibodies against Escherichia coli expressed chimeric recombinant protein encompassing multiple epitopes of zona pellucida glycoproteins to inhibit in vitro human sperm-egg binding

To minimize ovarian dysfunction subsequent to immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing the antigenic B cell epitopes as immunogens have been proposed. In this study, attempts have been made to clone and express a recombinant chimeric protein encompassing the epitopes corresponding to bonnet monkey (Macaca radiata) ZP glycoprotein-1 (bmZP1, amino acid residues 132-147), ZP glycoprotein-2 (bmZP2, amino acid residues 86-113), and ZP glycoprotein-3 (bmZP3, amino acid residues 324-347). The above chimeric recombinant protein (r-bmZP123) was expressed as a polyhistidine fusion protein in Escherichia coli. Immunoblot with murine monoclonal antibody, MA-813, generated against recombinant bmZP1 revealed a major band of approximately 10 kDa. The r-bmZP123 was purified on nickel-nitrilotriacetic acid resin under denaturing conditions. The female rabbits immunized with purified r-bmZP123 conjugated to diphtheria toxoid (DT) generated antibodies that reacted with r-bmZP123 and DT in an ELISA. In addition, the immune sera also reacted with E. coli expressed recombinant bmZP1, bmZP2, and bmZP3. In an indirect immunofluorescence assay, the antibodies against r-bmZP123 recognized native ZP of bonnet monkey as well as human. The immune sera also inhibited, in vitro, the binding of human spermatozoa to the human zona in the hemizona assay (HZA). These studies, for the first time, demonstrate the feasibility of assembling multiple epitopes of different ZP glycoproteins as a recombinant protein that elicit antibodies which are reactive with native zona and also inhibit, in vitro, human sperm-oocyte binding.     

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.     

Delineation of a conserved B cell epitope on bonnet monkey (Macaca radiata) and human zona pellucida glycoprotein-B by monoclonal antibodies demonstrating inhibition of sperm-egg binding

To circumvent autoimmune oophoritis after immunization with zona pellucida (ZP) glycoproteins, synthetic peptides encompassing B cell epitope(s) and devoid of oophoritogenic T cell epitopes as immunogens have been proposed. In this study, bonnet monkey (Macaca radiata) ZP glycoprotein-B (bmZPB) was expressed as polyhistidine fusion protein in Escherichia coli. Rabbit polyclonal antibodies against recombinant bmZPB (r-bmZPB) significantly inhibited human sperm-oocyte binding. To map B cell epitopes on ZPB, a panel of 7 murine monoclonal antibodies (mAbs) was generated against r-bmZPB. All 7 mAbs, when tested in an indirect immunofluorescence assay, reacted with bonnet monkey ZP, and only 6 recognized human zonae. Monoclonal antibodies MA-809, -811, -813, and -825 showed significant inhibition in the binding of human spermatozoa to human ZP in a hemizona assay. Epitope-mapping studies using multipin peptide synthesis strategy revealed that these 4 mAbs recognized a common epitope corresponding to amino acids (aa) 136-147 (DAPDTDWCDSIP). Competitive binding studies revealed that the synthetic peptide corresponding to the identified epitope (aa 136-147) inhibited the binding of MA-809, -811, -813, and -825 to r-bmZPB in an ELISA and to bonnet monkey ZP in an indirect immunofluorescence assay. The epitopic domain corresponding to aa 136-147 of bmZPB was completely conserved in human ZPB. These studies will further help in designing ZP-based synthetic peptide immunogens incorporating relevant B cell epitope for fertility regulation in humans.     

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

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

Mapping of dominant B-cell epitopes of a human zona pellucida protein (ZP1)

Zona pellucida (ZP) glycoproteins contain numerous antigenic determinants including carbohydrate, protein, and conformational epitopes; and the immunogenicity of these complex glycoproteins varies in different mammalian hosts. Studies have now shown that antibodies from primates immunized with a cDNA-expressed recombinant rabbit ZP protein (the homologue of the human ZP1 [hZP1]) inhibit sperm binding to the ZP without altering ovarian function, unlike immunization with ZP3 and ZP2 protein families. The ZP1 protein or peptides derived from it (recombinant or synthetic) are therefore primary candidates for use in designing safe and reversible human and animal contraceptive vaccines. In order to define peptide epitope(s) that may be critical for eliciting an immune response sufficient to effect immunological contraception without causing any adverse effects on ovarian physiology, studies have been carried out to identify immunodominant B-cell epitopes of the ZP1 protein. The amino acid sequence of the hZP1 was used to design a set of 94 (15-mer) biotinylated peptides having an overlap of 9 amino acids. Using these peptides in a modified enzyme-linked immunoassay, antibodies in sera from rabbits or baboons immunized with native porcine ZP protein were screened for ZP1 peptide recognition. These studies demonstrate that there are a limited number of peptides recognized by primate antibodies but that the overlapping peptides sharing the sequence GPLTLELQI are recognized by both rabbit and baboon antibodies regardless of the adjuvant system used to induce the immune response. This peptide is 100% conserved in amino acid sequence between the human and pig, although the rabbit protein has two conserved amino acid substitutions (100% similar, 77% identical). Because this peptide is immunogenic as well as antigenic in primates, it could play a major role in the development of human contraceptive vaccines.     

Glycosylation of zona pellucida glycoprotein-3 is required for inducing acrosomal exocytosis in the bonnet monkey.

To investigate the role of polypeptide backbone vis-à-vis glycosylation of the putative primary sperm receptor, the bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 (bmZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was expressed as polyhistidine fusion protein either in Escherichia coli orusing baculovirus expression system. The recombinant bmZP3 (r-bmZP3) was purified using nickel-nitrilotriacetic acid resin and subsequently refolded in the presence of oxidized and reduced glutathione. SDS-PAGE and Western blot analysis revealed approximately 43 and approximately 52 kDa bands corresponding to E. coli and baculovirus expressed r-bmZP3, respectively. The r-bmZP3 purified from both E. coli and baculovirus binds to the principal segment of the acrosomal cap of the capacitated bonnet monkey spermatozoa as evaluated by indirect immunofluoresence assay. In a competitive inhibition assay, the binding of biotinylated baculovirus expressed r-bmZP3 to capacitated spermatozoa was inhibited not only by cold baculovirus expressed r-bmZP3 but also by E. coli expressed r-bmZP3 and vice-versa. Lectin binding studies revealed that the baculovirus r-bmZP3 has N-linked glycosylation with galactose and mannose residues. Capacitated spermatozoa, in the presence of baculovirus expressed r-bZP3, undergoes a significant increase (p < 0.01) in the acrosomal exocytosis as compared to control whereas E. coli expressed r-bmZP3 failed to have a significant effect. These results suggest that though the polypeptide backbone of ZP3 is sufficient for its binding to capacitated spermatozoa, yet glycosylation is required for induction of acrosomal exocytosis.     

Molecular cloning and expression in Escherichia coli of cDNA encoding bonnet monkey (Macaca radiata) zona pellucida glycoprotein

Zona pellucida (ZP) glycoproteins have been proposed as candidate antigens for an immunocontraceptive vaccine. The efficacy of such a vaccine has to be evaluated in nonhuman primates, thus necessitating the characterization of their ZP glycoproteins. A bonnet monkey (Macaca radiata) ovarian cDNA λgt11 library was screened for ZP2 (bZP2) using full-length human ZP2 cDNA as a probe. Two identical full-length clones with an open reading frame of 2235 nt encoding a polypeptide of 745 aa residues were isolated. The deduced aa sequence of bZP2 revealed high sequence identity (94.2%) with human ZP2. The bZP2 cDNA (115–1914 nt, 1.8 kb), excluding sequences coding for N-terminal signal sequence and C-terminal transmembranelike domain, was PCR amplified and Sac1-Sal1 restricted fragment cloned in frame downstream of the T5 promoter under the lac operator control in a pQE-30 vector. Recombinant bZP2 (r-bZP2) was expressed as a polyhistidine fusion protein in Escherichia coli strain M15 [pREP4]. Immunoblot with rabbit polyclonal antibodies against bZP2 synthetic peptide (corresponding to aa residues 429–444; K₄₃₄ replaced by R and I₄₃₆ by V) revealed a major band of 68 kDa. Immunization of male rabbits with the r-bZP2 protein purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with r-bZP2 in ELISA as well as with native protein as revealed by positive fluorescence of ZP of bonnet monkey ovary. The availability of r-bZP2 and its aa sequence will help in the development and evaluation of a contraceptive vaccine based on ZP2. Mol. Reprod. Dev. 50:229–239, 1998. © 1998 Wiley-Liss, Inc.     

A contraceptive peptide vaccine targeting sulfated glycoprotein ZP2 of the mouse zona pellucida

In this study, we have mapped and characterized a B cell epitope of sulfated glycoprotein ZP2 (ZP2) as a step toward the development of a multi-epitope zona pellucida (ZP) vaccine. Recombinant polypeptides expressed by random deoxyribonuclease-digested fragments of ZP2 cDNA were screened for binding to IE-3, a monoclonal antibody to murine ZP2. Positive clones contained cDNA inserts encoding polypeptide corresponding to ZP2(103-134). When normal or ovariectomized female mice were immunized with three overlapping peptides that span this region of ZP2 (101-120, 111-130, 121-140), only ZP2(121-140) elicited IgG antibodies that reacted with mouse ovarian ZP, indicative of the presence of native B epitope and helper T cell epitope in ZP2(121-140). To more finely map the ZP2 B cell epitope, a random peptide display library was screened with the IE-3 antibody, and a consensus tetramer sequence VxYK that matched the ZP2(123-126) sequence VRYK was located. Competitive immunofluorescence analysis with single alanine-substituted VxYK peptides ranked the relative contribution of the three critical B cell epitope residues as Y > V > K. A chimeric peptide was constructed that contained the YRYK motif of ZP2 and a bovine RNase T cell epitope. Although (C57BL/6xA/J) F1 (B6AF1) female mice immunized with the chimeric peptide developed ZP antibody response, this peptide elicited antibody only in mice of the histocompatibility complex (MHC) H-2(k or b) haplotype. In contrast, ZP2(121-140) peptide elicited antibody in inbred mice with three additional mouse MHC haplotypes. Moreover, although ZP2(121-140) contained a T cell epitope, no oophoritis was observed after immunization of B6AF1 mice with ZP2(121-140) in complete Freund's adjuvant (CFA). In a preliminary trial, female B6AF1 mice immunized with ZP2(121-140) in CFA had reduced litter sizes as compared with mice injected with CFA alone.     

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.     

Cytochemical demonstration of modification of carbohydrates in the mouse zona pellucida during folliculogenesis

In the present study, lectin-gold cytochemistry and antibodies against ZP2 and ZP3 glycoproteins were used to investigate the oligosaccharide content of mouse ovarian zona pellucida (ZP) during follicular development. The entire thickness of the ZP and several organelles of the oocyte (cortical granules, Golgi apparatus, and vesicular aggregates) were reactive to RCA-I, DSA, AAA, WGA, MAA, and LFA throughout follicular development. HPA labeling was not detected at the earliest stages of follicular folliculogenesis. HPA reactivity was first observed in the ZP, Golgi apparatus, and the vesicles of oocytes at the trilaminar primary follicle stage. HPA labeling in the ZP was always restricted to the inner region of the zona matrix. After neuraminidase treatment, HPA reacted with the entire ZP in ovarian follicles at different stages of development. Immunolabeling with specific antibodies showed that, although ZP2 and ZP3 glycoproteins were uniformly distributed in the zona matrix of ovarian oocytes, there was a progressive increase in thickness of the ZP in parallel with the proliferation of follicular cells. ZP3 glycoprotein was also localized to the Golgi apparatus and vesicular aggregate. The present results suggest: (1) a difference in composition of carbohydrate content between the inner and outer region of the fully developed ZP generated probably by a modification in the biosynthetic pathway of oligosaccharides in the oocyte during folliculogenesis, (2) that newly synthesized ZP glycoproteins displace previously synthesized ZP components in a direction toward the follicular cells and, therefore, no redistribution of the ZP matrix occurs during folliculogenesis, and (3) that the vesicular aggregates in the ooplasm constitute an intermediate step in the secretory pathway of ZP glycoproteins. Accepted: 10 January 2000     

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.     

Antibodies generated in response to plasmid DNA encoding zona pellucida glycoprotein-B inhibit in vitro human sperm-egg binding

To investigate the immunogenicity of plasmid DNA encoding bonnet monkey (Macaca radiata) zona pellucida (ZP) glycoprotein-B (bmZPB), the cDNA corresponding to bmZPB, excluding the N-terminal signal sequence and C-terminus transmembrane-like domain, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRbmZPB). In vitro transfection of COS-1, COS-7, CHO, HEK-293, and UM-449 mammalian cells with VRbmZPB plasmid DNA led to the expression of bmZPB. Expression of bmZPB in transfected cells was cytosolic. Flow cytometry analysis of COS-1 cells transfected with VRbmZPB revealed that approximately 15% cells expressed bmZPB. The expressed bmZPB has an apparent molecular weight of 57 kDa. Immunization of male BALB/cJ mice with VRbmZPB plasmid DNA in saline as compared to VR1020 immunized group, elicited significant antibodies against E. coli expressed recombinant bmZPB as evaluated in ELISA. The antibodies generated by VRbmZPB plasmid DNA recognized bonnet monkey as well as human ZP. The immune sera obtained from mice immunized with VRbmZPB plasmid DNA also inhibited, in vitro, the binding of spermatozoa to the ZP in the hemizona assay. These studies, for the first time, demonstrate the feasibility of DNA vaccine to generate antibodies against ZP that recognize native protein and inhibit human sperm-oocyte binding.     

Expression in Escherichia coli and immunological characterization of three zona pellucida proteins (ZP1, ZP2, and ZP3) from a marsupial,the brushtail possum (Trichosurus vulpecula)

The brushtail possum (Trichosurus vulpecula) zona pellucida (ZP) is composed of three major glycoproteins, designated ZP1, ZP2, and ZP3 based on their size and homology with eutherian ZP proteins. These proteins are candidate antigens for the development of an immunocontraceptive vaccine to control the fertility of the brushtail possum in New Zealand, where it is an introduced pest. In order to further their immunological and functional characterization, recombinant possum ZP proteins were produced in Escherichia coli (E. coli) strain JM109, M15, SG13009, or BL21 codon plus. Each of the proteins produced possessed a N-terminal six histidine tag (His)(6) to facilitate purification and consisted of amino acid (aa) residues 18-471 of possum ZP1, aa residues 40-311 of ZP2 (ZP2-N), aa residues 305-634 of ZP2 (ZP2-C), and aa residues 23-342 of ZP3. Immunoblot using anti-RGS(His)(4) antibodies and polyclonal rabbit anti-porcine ZP antibodies detected major bands at 54 kDa for ZP1, 32 kDa for ZP2-N, 39 kDa for ZP2-C, and 40 kDa for ZP3. Immunization of male and female rabbits with ZP2-N, ZP2-C, and ZP3 purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with recombinant ZP proteins on Western blot and with native ZP proteins in possum ovarian sections using immunofluorescence. Antibodies generated against ZP1 in the same way were reactive with recombinant ZP proteins on Western blot only. The recombinant possum ZP proteins and specific antibodies produced in this study give an indication of the antigenic relationship of the possum ZP proteins and are vital tools for future studies of sperm-ZP binding in marsupials and for the evaluation of ZP-based contraceptive vaccines in possums and other marsupials.     

Ovaries remain functional in squirrel monkeys (Saimiri sciureus) immunized with porcine zona pellucida 55,000 macromolecule

Fifty female squirrel monkeys were each immunized with 200 micrograms of a purified preparation of the 55,000 macromolecule (ZP3) from porcine zona pellucida. The fertility status of these immunized monkeys, as well as the effect of ZP3 antibodies on ovarian function, was monitored. High anti-ZP3 titers were achieved (greater than 75% binding levels as determined by radioimmunoassay) and remained high (approximately 67% binding level) for the duration of this study. Hormonal evaluations indicated initial disturbances in normal ovarian steroid secretion and function that were confirmed by laparoscopic observation and oocyte production data. Histological examination of ovaries at 6-7 mo post-injection suggested an interference in folliculogenesis. No pregnancies were observed in the immunized monkeys during this period. By 10-15 mo post-immunization, hormonal and laparoscopic data indicated that ovarian function was recovering in injected monkeys despite the continued presence of high titers to ZP3. Collectively, these results demonstrate that although immunization with ZP3 initially produces disturbances in normal ovarian function that inhibit fertility, these effects are reversible. Such findings encourage the continued intensive investigation of purified porcine zona macromolecules for immunocontraceptive purposes.     

Subcellular distribution of ZP1, ZP2, and ZP3 glycoproteins during folliculogenesis and demonstration of their topographical disposition within the zona matrix of mouse ovarian oocytes

The zona pellucida (ZP) is an extracellular coat synthesized and secreted by the oocyte during follicular development and surrounding the plasma membrane of mammalian eggs. To date, the mechanism of synthesis and secretion, mode of assembly, and intracellular trafficking of the ZP glycoproteins have not been fully elucidated. Using antibodies against mouse ZP1, ZP2, and ZP3 in conjunction with the protein A-gold technique, we have shown an association of immunolabeling with the Golgi apparatus, secretory granules, and a complex structure called vesicular aggregate, respectively, in mouse ovarian follicles. In contrast, the neighboring granulosa cells were not reactive to any of the three antibodies used. Immunolabeling of ZP1, ZP2, and ZP3 was detected throughout the entire thickness of the ZP, irrespective of the developmental stage of ovarian follicles. Double and triple immunolocalization studies, using antibodies tagged directly to different sizes of gold particles, revealed an asymmetric spatial distribution of the three ZP glycoproteins in the zona matrix at various stages of follicular development. All three glycoproteins were specifically localized over small patches of darkly stained flocculent substance dispersed throughout the zona matrix. Very often, ZP1, ZP2, and ZP3 were found in close association. These results confirm findings from previous studies demonstrating that ovarian oocytes and not granulosa cells are the only source for mouse ZP glycoproteins. In addition, results from our morphological and immunocytochemical experiments suggest that the vesicular aggregates in the ooplasm are likely to serve as an intermediary in the synthesis and secretion of ZP glycoproteins. The stoichiometric disposition of ZP1, ZP2, and ZP3 in the zona matrix as revealed by double and triple immunolocalization studies provide further insight into some of the unanswered questions pertinent to the current model of mouse ZP structure proposed by the Wassarman group.     

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.     

Immunolocalization of zona pellucida antigens in the ovarian follicle of dogs, cats, horses and elephants

A comparative evaluation of the location of immunoreactive porcine zona pellucida (pZP) glycoproteins was performed with polyclonal rabbit anti-pZP antibodies on ovarian sections of the dog, cat, horse, and elephant. For this, formalin (light microscopy) and glutaraldehyde (transmission electron microscopy [TEM]) fixed ovarian sections were incubated with antibodies raised against highly purified pZP. Staining patterns were determined with diaminobenzidine (DAB) at the light level. The dog ZP had a distinct staining distribution that is characterized by intense staining around the periphery of the ZP and the oolemma and less dense staining throughout the width of the ZP. In dog follicles that contained multiple oocytes, there were oocytes of identical and dissimilar stages. Cat ovarian sections showed uniform staining of the ZP. Horse results showed uniform staining of ZP and ooplasm, and granulosa cells (GC). Elephant sections showed staining of the ZP with dense staining at the oolemma, as well as staining of the ooplasm. In all species the staining of the ZP was not evident until GC differentiation. In all cases there was no staining of ovarian tissue with control normal rabbit serum. Specific staining patterns of ZP were evaluated by TEM and immunogold staining. The immunogold-linked anti-pZP antibodies stained the ZP matrix in all species. There was staining of ooplasm organelles suggesting that ZP secretion originates from the oocyte of the dog and cat. In addition, follicular and ZP measurements were taken that allowed accurate characterization of follicle stage. These findings suggest that in all four species the ZP is recognized by anti-pZP antibodies and there is also evidence to suggest the possible origins of ZP glycoproteins.