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A lactosaminoglycan-associated antigen is associated with a carbohydrate moiety of all three zona pellucida (ZP) glycoproteins of pig and rabbit but is absent in the mouse and rat. A monoclonal antibody (PS1) recognizing this determinant was obtained by immunizing mice with a porcine ZP glycoprotein isoform purified by two-dimensional polyacrylamide gel electrophoresis. Conditions known to remove O-linked or sialic acid carbohydrate moieties (alkaline reduction; O-glycanase or neuraminidase enzymatic cleavage) did not remove the carbohydrate epitope. However, treatment with endo-beta-glycosidase, endoglycosidase F, or combinations of neuraminidase plus beta-galactosidase, totally removed the determinant, indicating that it is associated with a poly-N-acetyllactosaminoglycan structure present on an N-linked oligosaccharide. Molecular morphology studies using immunofluorescence and confocal microscopy techniques demonstrate that the PS1 antigen is localized at the surface of the ZP. Confirmation of this localization was obtained through studies that show that this antibody will inhibit homologous sperm binding to the pig ZP. Additional analyses using modular contrast microscopy and immunocytochemistry demonstrate that this carbohydrate-associated antigen is localized in discrete layers throughout the ZP matrix. These studies are the first to demonstrate the presence of a lactosaminoglycan type carbohydrate moiety in all three ZP proteins using a monoclonal antibody that appears to be involved in sperm recognition and structural organization.

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.

Changes in rabbit ovarian hormonal responses and cellular differentiation of ovarian follicles after immunization with porcine zona pellucida (ZP) have been examined. Steroid and peptide hormone levels were monitored after immunization to evaluate ovulation and pseudopregnancy cycles in immunized and control animals. All immunized rabbits developed serum antibodies to specific ZP antigens and failed to form functional corpora lutea in response to hCG administration, as evidenced by the absence of elevated serum progesterone concentrations. This is in contrast to control rabbits, which had elevated progesterone levels 8-9 days after hCG administration. Furthermore, all immunized animals showed greatly increased serum levels of FSH and LH compared to those of control animals. These effects on ovarian function were apparent within 20 weeks of the primary immunization. Follicular development was analyzed by light and electron microscopies. The numbers of primary, secondary, and tertiary follicles in ovaries of immunized animals were markedly reduced within 7 weeks compared with control values. By 23 weeks, few if any growing follicles were present. Although numerous distinct clusters of cells with ultrastructural properties that resemble those of normal follicular cells were present in immunized animals, they contained no oocytes. These studies suggest that antibodies to ZP glycoprotein alter ovarian function by interfering with cells during the stage of follicle differentiation at which the ZP proteins are being synthesized and secreted. This system should provide an excellent model with which to study the early events associated with ovarian follicular cell differentiation and subsequent hormonal responsiveness.