Rat germinal cells require PARP for repair of DNA damage induced by gamma-irradiation and H2O2 treatment

The ability of rat germinal cells to recover from genotoxic stress has been investigated using isolated populations of primary spermatocytes and round spermatids. Using a comet assay at pH 10.0 to assess single strand breakag e (SSB) in DNA, it was found that a high level of damage was induced by 5 G y gamma -irradiation and acute exposure to 50 muM H2O2. This damage was eff ectively repaired during a subsequent recovery period of 1-3 hours culture in vitro but repair was significantly delayed in the presence of the poly(A DP-ribose)polymerase (PARP) inhibitor 3-aminobenzamide (3-ABA). Immunofluor escence detection of PARP with specific antibodies localised the protein to discrete foci within the nucleus of both spermatocytes and spermatids. Pol y(ADP-ribose) (pADPR) could also be detected in spermatid nuclei following gamma -irradiation or H2O2 treatment. Moreover, PARP activation occurs both in spermatocytes and spermatids left to recover after both genotoxic stres ses. The NO donors, 3-morpholino-sydnonimine (SIN-1) and S-nitrosoglutathio ne (SNOG), caused significant SSBs in both spermatocytes and spermatids. Th e effects of SIN-1 could be prevented by exogenous catalase (CAT), but not superoxide dismutase (SOD), in the cell suspensions. SNOG-induced SSBs were insensitive to both CAT and SOD. It is concluded that DNA in spermatocytes and spermatids is sensitive to damage by gamma -irradiation and H2O2 and t hat efficient repair of SSBs requires PARP activity.