PROBLEM: The present study was conducted to investigate the oocyte membrane protein(s) that is involved in sperm binding in the mouse, and whether or not it can be used for the development of a contraceptive vaccine.
METHOD OF STUDY: The zona-free oocytes were treated with Triton X-100 and the extract was analyzed for homogeneity in the sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) after staining with silver nitrate. The appropriate band of 50±4 kD, designated as OM antigen, was excised from the gel and used for the immunization. The female rabbits were immunized with the excised band per se and the female mice were immunized with the OM antigen after conjugation to keyhole limpet hemocyanin (KLH). The affinity-purified antibodies were analyzed in the enzyme-linked immunosorbent assay (ELISA), immunoprecipitation procedure, western blot procedure, indirect immunofluorescence technique (IFT), and sperm-oolemma binding assay. Actively-immunized mice were analyzed for in vivo fertility.
RESULTS: The Triton X-100 extract of zona-free oocytes predominantly showed a single protein band of 50±4 kD in the SDS-PAGE. Active immunization of female rabbits and of female mice with OM antigen raised high antibody titers (ELISA titer>1:4096) that specifically recognized the OM antigen in the immunoprecipitation and Western blot procedures, and reacted with the oocyte in the IFT. These antibodies demonstrated a significant (P<0.05) up to a complete block of sperm-oolemma binding in the in vitro binding assay. Binding of both the acrosome-intact and acrosome-reacted sperm was inhibited. Mice actively immunized with OM antigen also showed a significant reduction in vivo fertility as seen by the 9-day implants in uteri. Preliminary data indicate that the antibodies to OM antigen were tissue-specific and did not react with the specific band in any tissue extract in the western blot procedure.
CONCLUSIONS: These results indicate that the OM antigen is involved in sperm-oolemma binding and constitute an attractive molecule that needs further investigation for examining its utility in the contraceptive vaccine development.