|Budget Amount *help
¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1995 : ¥400,000 (Direct Cost : ¥400,000)
Fiscal Year 1994 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Identification of the epitope recognized by antisperm antibodies is important to clarify for the mechanism of immunological infertility and development of immunocontraceptive vaccine. We are trying the active immunization of experimental animals with synthetic peptides fragments corresponding to the sperm specific antigens. Among them, two peptides, YAL-198 (YAL) and rSMP-230, cause infertility of female rats by active immunization. In this project, we synthesized new sperm antigen peptides originated from the deduced amino acid sequence of the 75kD human sperm antigen reacted antisperm antibody produced in the infertile women. Furthermore, we prepared antibodies to YAL,which is corresponding to the extracellular domain of a human sperm protein interacting monoclonal antibody (YWK-II) which induces agglutination of human and rat sperm, and elucidated the contraceptive mechanism by active immunization with YAL.
1. Synthesis of the extracellular domain of the 75kD human sperm antigen
eptide segments corresponding to the extracellular domain of the 75kD human sperm antigen were synthesized by Fmoc solid phase method. The peptides that composed of 16 residue amino acids were yielded 17mg and 15mg. Amino acid analysis showed that the composition corresponds to the theoretical ratio.
2. Effects of anti-YAL antibodies on fertilization and embryo development.
(1) Rabbit polyclonal and murine monoclonal antibodies (pAb and mAb) were prepared as described previously, and IgG fraction was separated by Protein G column chromatography. The pAb and mAb caused neither agglutination nor immobilization of human spermatozoa. Furthermore, hamster test was not inhibited following exposure of human sperm to anti-YAL antibodies.
(2) We examined the effects of antibodies to YAL on fertilization and development of the fertilized eggs in murine model to elucidate the contraceptive mechanism of the active immunization with YAL.Fertilization rates were not different between in control group and in anti-YAL antibody group. However, the development to 4 cell stage embryo or morula was significantly (p<0.05) inhibited, when exposed to antibodies.
These results suggest that active immunization of YAL causes infertility by impairment of embryo growth, but not by inhibition of fertilization, which is the main etiology of infertile women possessing antisperm antibodies. Less