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Candidate contraceptive vaccines directed against gamete-specific antigens have been the subject of intensive investigations during the last few decades. Antigens of zona pellucida (ZP) represent a unique target for immunological intervention of fertilization process and recent encouraging data emphasize the strong anti-fertility potential of such an approach.
Studies have been conducted to investigate the immunogenicity of native and deglycosylated porcine ZP antigens and the immunocontraceptive efficacy of active and passive immunization with ZP antigens in rabbits and monkeys. Zonae pellucidae were isolated from porcine ovaries using serial sieving procedures. One-dimensional gel electrophoretic techniques failed to adequately resolve ZP antigens. The pattern exhibited considerable microheterogeneity, presumably due to the presence of carbohydrate and sulfate groups in zona proteins. Two-dimensional gel electrophoresis demonstrated that porcine zonae are composed of three major glycoprotein families, each exhibiting marked charge and molecular weight heterogeneity, which is typical of post-translationally modified proteins. The molecular weight ranges of the three glycoproteins, as determined by reference to standards, were 42,000-120,000; 70,000-101,000; and 95,000-118,000 daltons. The highly sensitive silver staining procedures detected an additional low-molecular weight glycoprotein family, within the molecular weight range of 20,000-25,000 daltons. Deglycosylation of porcine ZP glycoproteins by trifluo methanesulfonic acid resulted in effective deletion of the carbohydrates as revealed by considerable reduction of the molecular weights and apparent heterogeneity of the ZP macromolecules. When subjected to one-dimensional gel electrophoresis, the deglycosylated ZP fraction resolved into three discrete polypeptides with molecular weights ranging from 35,000-70,000 daltons.
Groups of rabbits were immunized with native and deglycosylated porcine ZP antigens admixed in Freund's ad juvant. Normal saline and Freund's ad ju-injected animals served as the controls for the study. The effect of immunization with porcine ZP antigens with and without adjuvant was also evaluated in nonhuman primates. Two groups of rhesus monkeys were immunimed with porcine ZP glycoproteins. The first group received the ZP immunogens emulsified in complete Freund's adjuvant and the second group was immunized with ZP glycoprotein without any adjuvant. No booster immunizations were given to the animals of both these groups. Serum antibodies to native, as well as deglycosylated ZP antigens were monitored at regular intervals in all the immunized animals by Immunoelectrophoresis, immunofluorescence and enzyme immunoassays. All animals immunized with porcine zona glycoproteins along with Freund's adjuvant evoked a vigorous antibody response, which was detectable as early as one month after the primary immunization. The anti-bodies were mainly of the IgG class and did not exhibit any preferential reactivity, and distinctly recognized all the three major glycoprotein families of porcine zona on immunoblots prepared from 2D-PAGE separated ZP antigens. The antibody levels remained high and showed a marginal decline at the end of the observation period, thus indicating that ZP glycoproteins are potent heteroimmunogens. While the monkeys immunized with a single dose of porcine ZP antigens admixed in Freund's adjuvant elicited high-titre anti-ZP antibodies and were rendered amenorrhoic, the monkeys immunized with porcine zonae without adjuvant failed to generate any detectable anti-ZP antibody and had normal menstrual cycles. Zona glycoproteins, thus do not generate effective antibody response, unless administered along with a powerful adjuvant. Although, deglycosylated ZP antigens exhibited significant immunogenicity, the antibody response to these antigens was relatively attenuated, thereby demonstrating that deglycosylation of ZP glycoproteins results in alterations in the immunological properties of the macromolecule.
Contraceptive efficacy of active immunizations with the native and deglycosylated forms of ZP antigens was evaluated both in vitro as well as in vivo employing zona dissolution assay, sperm-egg binding assay and mating experiments. Antisera to both native and deglycosylated ZP antigens were equally effective in preventing the digestion of ZP matrices by proteolytic enzymes and completely abolished in vitro sperm-egg interactions. Heteroimmunization with native ZP glycoproteins also led to complete interception of pregnancies as none of the treated animals delivered any offspring throughout the period of observation. Animals immunized with deglycosylated ZP antigens also revealed significant reduction in fertilities, although, the contraceptive effect had a delayed onset and did not appear to be as drastic as in case of immunizations with native zona antigens.
Passive immunization of rabbits with 30 mg of chromatographically purified gamma globulins to porcine ZP glycoproteins resulted in a complete inhibition of fertilities for a minimum of 68 days and the animals did not deliver any offspring during this period. The induced infertility in the passively immunized animals was completely reversible, since normal fertility was restored to all the treated animals after the anti-ZP antibody levels had receded.
The findings of the study confirm the immunogenicity and contraceptive efficacy of deglycosylated ZP polypeptides and provide encouragement for consideration of the deglycosylated and/or modified forms of porcine zona pellucida antigens as potential targets for human fertility-regulating vaccine development.