Alkaline gel electrophoresis, pulsed field gel electrophoresis, and quantitative PCR analyses (QPCR) of the nuclear (nDNA) and mitochondrial (mtDNA) genomes were used to assess DNA integrity in the spermatozoa of three species exposed to oxidative stress. In human and murine spermatozoa, the mtDNA was significantly more susceptible to H2O2-mediated damage than nDNA. In both eutherian species, exposure to 250 mu M H2O2 induced around 0.6 lesions/10 kb of mtDNA. The mtDNA of human spermatozoa was particularly vulnerable to oxidative stress; 0.25, 1, and 5 mM H2O2 inducing DNA damage equivalent to 0.62, 1.34, and 1.42 lesions/ 10 kb, respectively. Such results emphasize the diagnostic significance of mtDNA as a biomarker of oxidative stress in the male germ line. In contrast, no damage could be detected by QPCR in the nDNA of either eutherian species, on exposure to H2O2 at doses as high as 5 mM. However, electrophoretic analysis indicated that severe oxidative stress could induce detectable nDNA fragmentation in human, but not murine spermatozoa. The mtDNA of tammar wallaby spermatozoa was relatively resistant to oxidative stress, only exhibiting damage (0.6 lesions/10 kb DNA) on exposure to 5 mM H2O2. By contrast, the nDNA of wallaby spermatozoa was significantly more susceptible to this oxidant than the other species. Such vulnerability is consistent with the lack of disulfide cross-linking in marsupial sperm chromatin and suggests that chromatin condensation during epididymal maturation may be important in establishing the resistance of these cells to the genotoxic effects of reactive oxygen species.
Molecular Reproduction and Development Vol. 71, no. 1, p. 77-87