This application addresses the problem of prefertilization immunocontraception from the perspective of a defined mammalian sperm surface component and its role in achieving contraception by the antigen's specific antibody interfering with sperm-zona binding. This study will utilize a defined human sperm antigen (hspa18) whose function is to bind human spermatozoa to the zona pellucida. This proposal is organized into six specific aims to address the molecular biology of the antigen and the immunological aspects of preventing fertilization.
The specific aims are: I. The human sperm antigen hspa18 will be characterized by sequencing the human cDNA and the sequence confirmed by partial sequencing of the native molecule. II. The tissue specificity of hspa18 will be determined by Northern blot analysis of RNA from various human and primate organs using the cDNA as a probe. Additionally, a fusion protein to hspa18 will be produced in E. coli. III. Hspa18 has been characterized by the monoclonal antibody MA-51 which inhibits sperm penetration into zona-free hamster oocytes. Therefore, the MA-51 epitope in the hspa18 sequence will be determined. A synthetic peptide of the MA-51 epitope on hspa18 will be made and coupled to KLH for immunization of female mice. IV. The analogous hspa18 antigen in mice has been identified by polyclonal antisera. It will be sequenced from a mouse testis cDNA library to provide a comparative sequence whose fusion protein can be tested as an immunocontraceptive. The mouse model will allow any immunopathological effects to be evaluated. V. Female mice will be actively immunized with the fusion protein from AIM 2, with the synthetic peptide(s) from AIM 3, and with the fusion protein of the mouse antigen sequenced in AIM 4. After insemination, their fertility will be determined. Control mice will be actively immunized with the fusion protein which does not contain the hspa18 or mouse antigen insert molecule (B-galactosidase only) or KLH only. VI. Antisera produced by the females will be used to determine if mouse fertilization in vitro is inhibited.
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