ALTERED NUCLEAR ACTIVATION PARAMETERS OF RAT SPERM TREATED IN-VITRO WITH CHROMATIN-DAMAGING AGENTS

Standard semen measures do not assess the genetic integrity of sperm. A human sperm activation assay (HSAA) has proven very useful for asses sing sperm quality and predicting pregnancy outcome. The HSAA involves incubating permeabilized sperm in cytoplasmic extracts of Xenopus lae vis frog eggs, The extracts activate sperm nuclei, which undergo chrom atin decondensation, DNA synthesis, and chromatin recondensation, mimi cking events that occur after fertilization in vivo, However, no anima l model sperm activation assay has been reported. We hypothesize that sperm activation assays will be useful for studying molecular mechanis ms of sperm DNA repair by egg cytoplasm and for screening sperm for da maged DNA. Thus, the objectives of this study were to develop an in vi tro rat sperm activation assay (RSAA) using cytoplasmic extracts of X. laevis frog eggs and to determine how chemically damaging the sperm c hromatin would affect two sperm activation parameters, chromatin decon densation and DNA synthesis. We incubated demembranated rat sperm in a cytoplasmic extract of X. laevis frog eggs supplemented with tritiate d thymidine triphosphate ([H-3]TTP). The activated sperm nuclei underw ent chromatin decondensation and DNA synthesis. Decondensation kinetic s were examined using image analysis to measure the size of the sperm nuclei as they decondensed. DNA synthesis kinetics were examined using autoradiography of incorporated [H-3]TTP. To investigate how chemical damage affects nuclear activation, we treated rat sperm in vitro with ethylene glycolbis(sulfosuccinimidyl-succinate; SEGS), a reversible c rosslinking agent, or hydroxylamine (HA), a DNA base modifier. Treatme nt with SEGS blocked decondensation in a dose-dependent manner. In con trast, treatment with HA enhanced decondensation, induced gross chroma tin abnormalities, and increased [H-3]TTP incorporation into activated sperm nuclei, responses consistent with an attempt by the egg cytopla sm to repair DNA damage, These results suggest that the RSAA may be us eful for detecting damaged sperm chromatin as a result of toxicant exp osure. (C) 1998 Society of Toxicology.