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© 1998 Oxford University Press

other

Altered Nuclear Activation Parameters of Rat Sperm Treated in Vitro with Chromatin-Damaging Agents

Dennis E. Sawyer*, Gilbert R. Hillman{dagger}, Tatsuo Uchida{ddagger} and David B. Brown*,1

*Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch Galveston, Texas 77555-0645 {dagger}Department of Pharmacology and Toxicology, University of Texas Medical Branch Galveston, Texas 77555-0645 {ddagger}Office of Biostatistics, University of Texas Medical Branch Galveston, Texas 77555-0645

Received August 14, 1997; accepted March 30, 1998

Standard semen measures do not assess the genetic integrity of sperm. A human sperm activation assay (HSAA) has proven very useful for assessing sperm quality and predicting pregnancy outcome. The HSAA involves incubating permeabilized sperm in cytoplasmic extracts of Xenopus laevis frog eggs. The extracts activate sperm nuclei, which undergo chromatin decondensation, DNA synthesis, and chromatin recondensation, mimicking events that occur after fertilization in vivo. However, no animal model sperm activation assay has been reported. We hypothesize that sperm activation assays will be useful for studying molecular mechanisms of sperm DNA repair by egg cytoplasm and for screening sperm for damaged DNA. Thus, the objectives of this study were to develop an in vitro rat sperm activation assay (RSAA) using cytoplasmic extracts of X. laevis frog eggs and to determine how chemically damaging the sperm chromatin would affect two sperm activation parameters, chromatin decondensation and DNA synthesis. We incubated demembranated rat sperm in a cytoplasmic extract of X. laevis frog eggs supplemented with tritiated thymidine triphosphate ([3H]TTP). The activated sperm nuclei underwent chromatin decondensation and DNA synthesis. Decondensation kinetics 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 [3H]TTP. To investigate how chemical damage affects nuclear activation, we treated rat sperm in vitro with ethylene glycolbis(sulfosuccinimidylsuccinate; SEGS), a reversible crosslinking agent, or hydroxylamine (HA), a DNA base modifier. Treatment with SEGS blocked decondensation in a dose-dependent manner. In contrast, treatment with HA enhanced decondensation, induced gross chromatin abnormalities, and increased [3H]TTP incorporation into activated sperm nuclei, responses consistent with an attempt by the egg cytoplasm to repair DNA damage. These results suggest that the RSAA may be useful for detecting damaged sperm chromatin as a result of toxicant exposure.

Key Words: sperm nuclear activation; Xenopus egg extract; sperm genotoxicity; DNA repair.


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