Summary. Female cell-mediated immunity to allogeneic spermatozoa after repeated natural insemination, in the absence of pregnancy, was compared with that after systemic challenge using the cell-mediated microcytotoxicity test to measure cytotoxic cell alloreactivity. After multiple (3–6) inseminations the majority of females (11 out of 13) showed a significant degree of lymphocytotoxicity to male-strain histocompatibility alloantigens in the para-aortic lymph nodes, and to a lesser extent in the spleens, while a single insemination was usually not sufficient to evoke a specific cytotoxic cell response. This differed from the low and highly variable degree of female sensitization after multiple systemic challenge with allogeneic spermatozoa via the intraperitoneal route. By contrast, a single systemic challenge via the footpad proved to be the most highly consistent and effective route for eliciting cell-mediated immunity to male-strain histocompatibility alloantigens in all 9 female mice. This alloreactivity appeared to be directed at alloantigens other than the male-specific H—Y antigen. These findings show that the precise route of immunization is a major factor in the development of female cell-mediated immune responsiveness to allogeneic spermatozoa.
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