DNA Damage in Human and Mouse Spermatozoa after In Vitro-Irradiation Assessed by the Comet Assay

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Abstract
The comet assay is widely employed as a method to measure DNA damage in a wide variety of cell types following genotoxic insult. We have used this method in order to characterise DNA damage in spermatozoa following in vitro irradiation with 137Cs gamma rays. In contrast to somatic cells, the DNA of mammalian spermatozoa is bound by protamine molecules allowing a sixfold more highly compact structure and thus rendering conventional cell lysis protocols ineffective. Therefore, this new method uses an extensive lysis step to ensure effective removal of DNA-associated proteins allowing DNA damage to be scored reproducibly in both murine and human spermatozoa. Mouse spermatozoa collected from the vas deferens at post-mortem or human spermatozoa provided by donors were irradiated with doses of γ-rays from 0–100 Gy using a 137Cs source and then processed for both alkaline and neutral comet assays. Under neutral electrophoresis conditions, which permits the measurement of double-stranded DNA breaks, a linear increase in the amount of DNA damage measured was observed with increasing radiation dose for both murine and human spermatozoa. Similarly, using alkaline electrophoresis conditions to examine DNA single-strand breaks and alkali-labile sites, a linear relationship was also observed for murine sperm but in contrast no such relationship was apparent for human spermatozoa subjected to the same radiation treatments. Interestingly, unirradiated sperm (both human and mouse) showed extensive DNA migration from the nucleus after alkaline assay. Since it is unlikely that the DNA of normal spermatozoa contains high numbers of single-strand breaks and damage was not detected for unirradiated sperm in the neutral assay, it is more likely that this DNA migration is due to the presence of high numbers of alkali labile sites within sperm DNA and that these may be related to the highly condensed structure of spermatozoal DNA. The large radiation doses used in these experiments to produce measurable amounts of DNA damage reflects the high radioresistance of spermatozoa compared to somatic cells and this may also be related to the differences in DNA packaging and conformation. In conclusion, this work shows that the comet assay represents a new method for examining DNA damage in spermatozoa and should be evaluated for use in reproductive toxicity testing.

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