DNA breakage detection-FISH (DBD-FISH) in human spermatozoa: technical variants evidence different structural...

Non-irradiated and X-irradiated (80 Gy) human spermatozoa were processed for in situ DNA breakage detection-FISH (DBD-FISH) of the whole genome, following two alternative variations of the basic technique. In the first, cells were initially incubated in the alkaline unwinding solution for transformation of DNA breaks into single-stranded DNA (ssDNA) to be hybridized, followed by the lysing solutions for protein removal. In the second, incubation in the lysing solutions was carried out before the denaturation step. The first approach yielded two subpopulations. While most sperm nuclei were faintly labeled and had chromocenters, a small subpopulation was strongly and homogeneously labeled, due to extensive DNA breakage. X-ray exposure increased the surface and mean fluorescence intensity. Otherwise, when the denaturation step was performed after protein extraction, all sperm nuclei yielded strong and dispersed FISH signals. Protein removal allows access of the unwinding solution to the DNA, which has abundant alkali-labile sites, and thus gives rise to large areas of ssDNA that are labeled by FISH. X-ray exposure increased the dispersion of FISH signals but decreased their mean fluorescence intensity. A linear dose-response was generated using the second experimental variant, being 30 Gy the lowest dose for detecting induction of damage by X-rays in mature sperm chromatin. These results indicate that DBD-FISH is not only useful for in situ detection of DNA breakage but also for revealing structural features of chromatin.