An objective sperm cytotoxicity assay for male-specific antisera based on ATP levels of unlysed cells: application to assay of H-Y antigen.

We correlated the decrease in levels of ATP in spermatozoa with the extent of cytotoxicity elicited by antibodies against antigenic components on sperm. In the presence of concentrations of complement which did not cause cytolysis or influence the ATP content of epididymal sperm, addition of heat-in-activated sera from non-immunized mice, rats or rabbits did not result in sperm cytolysis or a fall in ATP content. In contrast, addition of rabbit anti-rat spermatocyte sera, which has previously been shown to react with rat spermatozoa (Tung, P.S. and Fritz, I.B. (1978) Dev. Biol. 64, 297-315), did cause sperm cytolysis and a decrease in ATP content. The titre of this antiserum for 50% cytolysis was between 1 : 128 and 1 : 256, as determined by the fall in ATP content. Using these criteria, we examined the cytotoxicity against sperm of different samples of anti H-Y sera. We examined the influence of monoclonal antibody against H-Y, mouse H-Y antisera and rat H-Y antisera raised in inbred females immunized with spleen cells from males of the same strains. In all cases, anti-H-Y lowered ATP levels and lysed sperm with a cytotoxic titre between 1 : 8 and 1 : 16. Measurements of the decrease in ATP content in sperm have been shown to provide an objective and reliable estimate of the percentage of spermatozoa lysed by H-Y antisera. Cytotoxic activity of H-Y antisera was removed by absorption with spleen cells from male mice but not by absorption with spleen cells from female mice.