Investigation of sperm cytotoxicity as an indicator of ability of antisera to detect male-specific antigen on preimplantation mouse embryos

Abstract

H-Y antisera were produced in C57BL/6 female mice by repeated intraperitoneal injections of syngeneic male spleen cells. Epididymal spermatozoa were incubated in the presence of H-Y antisera and guinea-pig serum as a complement source. Levels of ATP remaining after treatment were used to calculate the amount of specific killing. Sera of different cytotoxic titres were used in an indirect immunofluorescent assay with a fluorescein isothiocyanate-conjugated IgG fraction of goat anti-mouse IgG (Fc fragment specific) as second antibody. Embryos were classified as fluorescent or nonfluorescent, transferred to pseudopregnant recipients, and allowed to develop to term. Of 12 sera tested for sperm cytotoxicity, 5 were different from a nonimmunized control serum (P < 0.05). Percentage specific killing in each of these sera was 7.8 ± 4.2, 11.7 ± 3.0, 26.0 ± 2.2, 27.7 ± 3.7 and 39.2 ± 4.8, respectively (mean ± s.e.m. with three replicates). The 5 sera and an additional one (4.9 ± 1.3% specific killing) were used in the embryo sexing experiment. The accuracy with which these sera correctly identified sex of preimplantation embryos was 60, 46, 74, 73, 74 and 48%, respectively. Correlation coefficients were 0.86 (P < 0.05) for specific sperm cytotoxicity and percentage of nonfluorescent embryos that were female and 0.78 (n.s.) for specific sperm cytotoxicity and percentage of fluorescent embryos that were male. Therefore, although the sperm cytotoxicity test is useful for screening antisera for the study of H-Y antigen expression on preimplantation embryos, nonfluorescent embryos are more accurately classified as females than are fluorescent embryos as male.