Studies on the expression of 80-kDa human sperm antigen in rat testis and epididymis.

The 80-kDa human sperm antigen (HSA) has demonstrated to be a promising candidate for development of an antifertility vaccine because it is a sperm-specific, conserved, and immunogenic protein. The present study demonstrates the androgen-regulated expression of 80-kDa HSA in testis and epididymis of rat by immunohistochemistry (IHC), using its specific antibodies. Developmental expression of 80-kDa HSA was investigated on days 10, 20, 40, 60, and 90 of age in the testis and epididymis by IHC, and relative staining intensity was estimated by image analysis using BIOVIS software. On days 10 and 20, no significant staining was observed in the testis and epididymis, whereas it gradually increased from day 40 onwards. The highest staining was seen on day 90 in both testis and epididymis. Gradual increase in expression of 80-kDa HSA after day 40 suggests that it is possibly regulated by androgen. To study the androgen-regulated expression of 80-kDa, adult male rats were treated with 75 mg/kg body weight of ethylene dimethane sulfonate (EDS), which selectively destroys Leydig cells and thus induces complete androgen withdrawal. It was observed that the staining intensity decreased following EDS treatment in rat testis as well as epididymis, and it was regained after supplementation with dihydrotestosterone. Increased expression during sexual maturation at the time of testosterone surge and its regulation by antiandrogen/androgen treatment suggest androgen-dependent expression of 80-kDa HSA in rat testis and epididymis.

Antifertility effect of passive administration of antibodies to 80 kDa human sperm antigen and its synthetic peptides in male and female rats.

PROBLEM: A human sperm antigen of molecular size of about 80 kDa (80 kDa HSA) has been reported to be sperm-specific, conserved and responsible for inducing immunological infertility. The partial N-terminal amino acid sequence of 80 kDa HSA (peptide NT) and its peptides obtained by enzymatic digestion with endoproteinase Lys-C (peptides 1-4) and with endoproteinase Glu-C (peptides 5 and 6) did not show sequence homology with any of the proteins of the GenBank. The peptides NT, 1, 2, 3 and 4 were synthesized, conjugated to keyhole limpet hemocyanin and used as an immunogen to raise the antibodies in rabbits. Peptide 3 did not elicit significant antibody titer while peptides NT, 1, 2 and 4 elicited significant antibody titer and immunobiologically mimicked the native protein. METHOD OF STUDY: Effects of passive administration of two injections each of 200 microL of antibodies or 10 and 40 microg purified immunoglobulins to 80 kDa HSA, peptides NT, 1, 2 and 4 on fertility in male and female rats were investigated. RESULTS: Passive administration of antibodies to 80 kDa HSA and its peptides NT, 1, 2 and 4 resulted in agglutination of epididymal spermatozoa with loss of motility but had no effect on sperm count or weights of the reproductive organs. These animals failed to impregnate normal female rats. Passive administration of these antibodies to female rats also resulted in infertility. The presence of antibodies was detected by enzyme-linked immunosorbent assay in uterine secretions of animals treated with antipeptide antibody. The presence of agglutinated spermatozoa was observed in the post-coital vaginal smears of these animals. The immunized females were found to be ovulating normally and the number of corpora lutea were unaltered. Of the four antipeptide antibodies studied, antibodies to peptides NT and 1 were most effective in inhibiting fertility both in male as well as female rats. Hence, the antifertility studies were further confirmed by passive administration of 10 and 40 microg of purified immunoglobulins of antibodies to NT and 1, which resulted in a dose-dependent inhibition of fertility in male and female rats. CONCLUSIONS: The study demonstrated that the synthetic peptides of 80 kDa HSA immunobiologically mimicked the native protein and impaired fertility following passive administration of antipeptide antibodies and hence, suggested the suitability of synthetic peptides of 80 kDa HSA as candidates for development of antifertility vaccine.

Studies with Synthetic Peptides of 80 kDa Human Sperm Antigen (80 kDa HSA).

PROBLEM: The 80 kDa human sperm antigen (HSA) is a sperm-specific and conserved antigen, capable of inducing immunological infertility. Partial N-terminal amino acid sequences of 80 kDa HSA (Peptide NT) and its peptides obtained by digestion with endoproteinase Lys-C (peptides 1-4) and endoproteinase Glu-C (peptides 5-6) did not show any sequence homology with reported known proteins deposited in the Gen-Bank. These sequenced peptides were synthesized and conjugated to key hole limpet haemocyanin (KLH) and evaluated for its antifertility effects. The present communication describes the characterization of these peptides and their antibodies. METHOD OF STUDY: Peptides NT, 1, 2, 3 and 4 were synthesized and conjugated to KLH. Antibodies to KLH conjugated peptides were raised in rabbits by active immunization and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) using sperm extract coated wells. The binding specificity of the synthetic peptides or purified 80 kDa HSA to their antibodies was assessed in the presence of various doses of respective synthetic peptides or 80 kDa HSA. The binding specificity was further confirmed by Western blot analysis. Antipeptide antibodies were also checked for sperm agglutinating activity, in-vitro. RESULTS: Active immunization of rabbits elicited significant antibody titers against the synthetic peptides, except for peptide 3. Antipeptide antibodies specifically recognized the native protein in an ELISA and induced in-vitro agglutination of human, rat and monkey sperm. In addition, Western blot analysis showed that these antipeptide antibodies specifically bind to the 80 kDa HSA band of the sperm extract. CONCLUSION: Synthetic peptides of 80 kDa HSA are immunogenic and antibodies raised against these peptides recognize the native protein detected by ELISA, Western blot analysis. In addition, they possess sperm agglutinating activity. These findings suggest that they are promising candidates in the development of immunocontraceptives.