MECHANISM OF IMMUNOLOGICAL INFERTILITY DUE TO THE 75kD HUMAN SPERM ANTIGEN AND ITS SIGNIFICANCE AS CONTRACEPTIVE VACCINE

鉴定抗精子抗体识别的表位对于阐明免疫性不孕症的发病机制和研制免疫避孕疫苗具有重要意义。我们正在尝试用与精子特异性抗原相对应的合成肽片段对实验动物进行主动免疫。其中，YAL-198(YAL)和rSMP-230两种多肽通过主动免疫可导致雌性大鼠不育。在本项目中，我们从不育妇女产生的75kD人类精子抗原与抗精子抗体反应的氨基酸序列中合成了新的精子抗原肽。此外，我们还制备了与人精子蛋白相互作用的单抗(YWK-II)胞外区的抗体，该抗体可诱导人和大鼠精子的凝集，并通过主动免疫阐明了YAL的避孕机制。75kD人精子抗原Two p…胞外区的合成用Fmoc固相法合成了更多与75kD人精子抗原胞外段相对应的肽段。由16个氨基酸组成的多肽的得率分别为17 mg和15 mg。氨基酸分析表明，其组成符合理论比例。抗YAL抗体对受精和胚胎发育的影响。(1)制备兔多克隆抗体和鼠单抗，用蛋白G柱层析法分离免疫球蛋白。多克隆抗体和单抗对人精子既不凝集也不固定化。此外，抗YAL抗体对金黄地鼠的受精和受精卵发育的影响也不受抑制。(2)通过检测YAL抗体对小鼠受精和受精卵发育的影响，探讨YAL主动免疫的避孕机制。然而，当暴露于抗体时，4细胞期胚胎或桑椹胚的发育受到显著抑制(p&lt；0.05)。这些结果表明，YAL主动免疫通过损害胚胎生长而导致不孕，而不是通过抑制受精而导致不孕，这是导致抗精子抗体阳性的不孕不育的主要原因。较少

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 antigenTwo p … More 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