In this review article, the correlation of anti-sperm antibodies with infertility in women and the possible development of contraceptive vaccines with antigens of the zona pellucida are reviewed focusing on our own studies.
Anti-sperm antibodies detected by Isojima's sperm immobilization test are frequently found in unexplained infertile women. When the sperm immobilizing antibodies are present in serum, they are also found in the cervical mucus, the peritoneal fluid and even in the follicular fluid and patients with a high titer of the antibodies find it hard to conceive a child. Our clinical studies showed that the sperm immobilizing antibodies impaired sperm passage in the female reproductive tracts from the cervix through the fallopian tubes and fertilization at the level of sperm binding to the zona pellucida. Analysis using monoclonal antibodies as well as patients' anti-sperm antibodies has shown that the carbohydrate moieties of sperm and seminal plasma are major epitopes of these antibodies. In immunofluorescent staining tests, one of the human monoclonal antibodies (MAb H6-3C4) derived from a patient with strong sperm immobilizing antibodies reacted with ejaculated human sperm and the epididymis but not with the testes and other somatic organs, suggesting that the corresponding antigen is secreted from the epididymis and bound to epididymal sperm during their passage through the epididymis. In Western blot analysis, MAb H6-3C4 reacted to antigen molecules of Mr 15-25 kDa present in methanol-chloroform extracts of either ejaculated sperm or seminal plasma. Animo acid sequence analysis of the reactive molecule revealed that the animo acid sequence was identical to the core peptide of CD52, a glycosylphosphatidylinositol (GPI)-anchored protein on lymphocytes.
Furthermore, the epitope for MAb H6-3C4 was found to be the specific N-linked carbohydrate in the male reproductive tract. Recently, we found that CD52 isolated from ejaculated sperm showed anti-complement activity in the classical complement activation pathway.
In mouse experiments, CD52 was localized in the epididymis and vas deferens but not in the testis, liver or kidney. Immunization with CD52 purified from vas deferens produced auto-antibodies in male mice and iso-antibodies in female mice. The antibodies raised in mice showed a strong complement-dependent sperm immobilizing activity but their fertility was not disturbed. Although both female and male CD52-knockout mice were fertile when they were mated with wild-type mice, the mating between CD52-knockout female and male mice resulted in significantly reduced numbers of offspring, suggesting the presence of CD52 in female reproductive organs. In fact, CD52 mRNA and protein were detected in cumulus cells after ovulation and in the endometrium after nidation of embryos. Similar findings were made in humans.
The zona pellucida (ZP) is a unique extracellular matrix composed of at least three major glycoproteins (ZP1, ZP2, ZP3) and plays an important role in the fertilization process. Immunization with ZP2 or ZP3 produced auto-reactive anti-ZP antibodies rendering all the immunized animals infertile due to ovarian failure. To avoid the induction of ovarian failure, an attempt was made to isolate B cell epitope of the fertilization blocking anti-ZP monoclonal antibody, because cellular immune responses were found to be involved in ovarian failure. A chemically synthesized ZP peptide including the B cell epitope was conjugated with diphtheria toxoid (DT) as carrier and injected with Freund's adjuvant into experimental animals. All animals immunized showed a good response with high anti-ZP antibody titers, always accompanied by severe impairment of follicular development. To study directly the effect of anti-ZP antibodies on follicular development, an in vitro culture system for preantral follicles was established in mice.
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