Male germ cells have been shown to differ in their DNA damage response (DDR) with respect to somatic cells. In addition, DDR pathways are modulated along spermatogenesis, accompanying profound chromatin modifications. Histone H2AX phosphorylation is a fundamental step of DDR. Few data are available on the long-term kinetics of phosphorylated H2AX (γ-H2AX) after in vivo irradiation. We have investigated, by microscopic and flow cytometric immunochemistry, γ-H2AX induction and removal in testicular cells of irradiated mice, in comparison with bone marrow cells. In unirradiated testicular cells, much higher levels of γ-H2AX were measured by flow cytometry with respect to bone marrow cells. Irradiation induced a redistribution of γ-H2AX into discrete foci detectable by microscopy. In irradiated bone marrow, the percentage of labelled cells peaked at 1 h and rapidly declined, in agreement with data on in vitro cell lines. In contrast, spermatocytes and round spermatids showed persistent labelling until 48 h. During this time, in spermatids, topological changes were observed in γ-H2AX foci from a pattern of many uncountable dots to a pattern of few large spots. Observations of testicular sections confirmed this trend in the reduction of foci number in spite of substantially invariable percentages of labelled cells in the analysed timeframe. To assess whether γ-H2AX persistence in testicular cells was due to unrepaired DNA breaks, we performed comet assay and immunofluorescence analysis of Mdc1, a marker of DDR different from γ-H2AX. Comet assay showed that most breaks were repaired within 2 h. Forty-eight hours after irradiation, contrary to γ-H2AX foci that remained detectable in 80% of initially labelled cells, Mdc1 foci were observed in only 20-30% of cells. These data suggest that, at long times after irradiation, mechanisms additional to impairment of DNA break repair may account for the long persistence of γ-H2AX foci in male germ cells.
The hypothesis of transgenerational induction of increased cancer susceptibility after paternal radiation exposure has long been controversial because of inconsistent results and the lack of a mechanistic interpretation. Here, exploiting Ptch1 heterozygous knockout mice, susceptible to spontaneous and radiation-induced medulloblastoma, we show that exposure of paternal germ cells to 1 Gy X-rays, at the spermatogonial stage, increased by a considerable 1.4-fold the offspring susceptibility to medulloblastoma induced by neonatal irradiation. This effect gained further biological significance thanks to a number of supporting data on the immunohistochemical characterization of the target tissue and preneoplastic lesions (PNLs). These results altogether pointed to increased proliferation of cerebellar granule cell precursors and PNLs cells, which favoured the development of frank tumours. The LOH analysis of tumor DNA showed Ptch1 biallelic loss in all tumor samples, suggesting that mechanisms other than interstitial deletions, typical of radiation-induced medulloblastoma, did not account for the observed increased cancer risk. This data was supported by comet analysis showing no differences in DNA damage induction and repair in cerebellar cells as a function of paternal irradiation. Finally, we provide biological plausibility to our results offering evidence of a possible epigenetic mechanism of inheritance based on radiation-induced changes of the microRNA profile of paternal sperm.
Poly(ADP-ribose)polymerase-1 (PARP1) is a nuclear protein implicated in DNA repair, recombination, replication, and chromatin remodeling. The aim of this study was to evaluate possible differences between PARP1−/− and wild-type mice regarding induction and repair of DNA lesions in irradiated male germ cells. Comet assay was applied to detect DNA damage in testicular cells immediately, and two hours after 4 Gy X-ray irradiation. A similar level of spontaneous and radiation-induced DNA damage was observed in PARP1−/− and wild-type mice. Conversely, two hours after irradiation, a significant level of residual damage was observed in PARP1−/− cells only. This finding was particularly evident in round spermatids. To evaluate if PARP1 had also a role in the dynamics of H2AX phosphorylation in round spermatids, in which γ-H2AX foci had been shown to persist after completion of DNA repair, we carried out a parallel analysis of γ-H2AX foci at 0.5, 2, and 48 h after irradiation in wild-type and PARP1−/− mice. No evidence was obtained of an effect of PARP1 depletion on H2AX phosphorylation induction and removal. Our results suggest that, in round spermatids, under the tested experimental conditions, PARP1 has a role in radiation-induced DNA damage repair rather than in long-term chromatin modifications signaled by phosphorylated H2AX.
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