Abstract
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.
Highlights
The maintenance of DNA integrity in the paternal genome is of utmost importance for reproduction and it is well known that DNA lesions in germ cells can be transmitted to the generation [1]
A delay of radiation-induced DNA damage repair was shown in cultured rat spermatocytes and spermatids [25], and mouse spermatids in vivo [3] when poly(ADP ribose) metabolism was inhibited by chemical treatment
Cytotoxic effects induced by 4 Gy X-rays on WT and PARP1−/− testis cells were assessed by flow cytometric DNA content analysis 48 h after irradiation
Summary
The maintenance of DNA integrity in the paternal genome is of utmost importance for reproduction and it is well known that DNA lesions in germ cells can be transmitted to the generation [1]. PARP family members in DNA repair processes has been extensively studied in somatic cells, both using chemical inhibitors and mouse and cellular models genetically defective for the enzymes [13,16,17,18,19,20,21,22,23,24] These studies show that inhibition or lack of PARP slows down DNA repair and increase the cytotoxicity of ionizing radiation and alkylating agents. A delay of radiation-induced DNA damage repair was shown in cultured rat spermatocytes and spermatids [25], and mouse spermatids in vivo [3] when poly(ADP ribose) metabolism was inhibited by chemical treatment These studies left unanswered the question of the specific role of PARP1 in the germ cell DNA damage response because of the poor specificity of chemical inhibitors towards different PARP family members [25]. The persistence of γ-H2AX foci after DNA repair was evaluated to assess the role of PARP1 in long-lasting chromatin remodeling [37]
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