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

Non-irradiated and X-irradiated (80 Gy) human spermatozoa were processed fo r in situ DNA breakage detection-FISH (DBD-FISH) of the whole genome, follo wing two alternative variations of the basic technique. In the first, cells were initially incubated in the alkaline unwinding solution for transforma tion of DNA breaks into single-stranded DNA (ssDNA) to be hybridized, follo wed by the lysing solutions for protein removal. In the second, incubation in the lysing solutions was carried out before the denaturation step. The f irst approach yielded two subpopulations. While most sperm nuclei were fain tly labeled and had chromocenters, a small subpopulation was strongly and h omogeneously labeled, due to extensive DNA. breakage. X-ray exposure increa sed the surface and mean fluorescence intensity. Otherwise, when the denatu ration step was performed after protein extraction, all sperm nuclei yielde d strong and dispersed FISH signals. Protein removal allows access of the u nwinding solution to the DNA, which has abundant alkali-labile sites, and t hus gives rise to large areas of ssDNA that are labeled by FISH. X-ray expo sure increased the dispersion of FISH signals but decreased their mean fluo rescence intensity. A linear dose-response was generated using the second e xperimental variant, being 30 Gy the lowest dose for detecting induction of damage by X-rays in mature sperm chromatin. These results indicate that DB D-FISH is not only useful for in situ detection of DNA breakage but also fo r revealing structural features of chromatin. (C) 2000 Elsevier Science B.V . All rights reserved.