DNA double-strand damage and repair following γ-irradiation in isolated spermatogenic cells

Abstract

Various cell types in spermatogenesis exhibit differential sensitivity to radiation-induce DNA damage. The investigation of DNA radiosensitivity in vitro is complicated by the heterogneous population of male germ cells (MGC) present in isolated single-cell suspensions. In the present investigation, the neutral elution technique was used to assess γ-irradiation-induced DNA double-strand damage (DSD) in spermatogonia and preleptotene spermatocytes (SG/PL), pachytene spermatocytes and spermatid spermatocytes, as well as in MGC. In addition, the capability of these cell types to repair DNA double-strand damage was investigated. Based on the well established timing of the rat spermatogenic cycle, the DNA of specific cell populations was labeled using tritiated thymidine. DNA from labeled cells was determined isotopically, whereas total DNA was quantitated using a fluorometric metod. DSD was induced in a dose-dependent manner in the heterogeneous population as well as in the labeled cell populations. SG/PL were more sensitive to γ-irradiation-induced DSD than either the heterogeneous MGC population, pachytene or spermatid spermatocytes. Each cell type exhibited a similar capability to repair DSD following exposure to 3000 rad; repair was rapdi (maximal within 45 min) and incomplete (< 40%). Only pachytene spermatocytes exhibited significant repair following exposure to 6000 rad. Since a difference in sensitivity to radiation-induced DSD was demonstrated, the capability of each cell type to repair a similar initial frequency of strand damage was investigated. SG/PL, pachytene and spermatid spermatocytes differed in their capability to repair similar levels of strand damage. However, the difference in dose required to achieve equal damage may have contributed to other cellular effects, thus altering repair. In summary, a model is described that permits the evaluation of genotoxic responses in specific populations of spermatogenic cells within a heterogeneous cell suspension. The ability of specific cell types to repair γ-irradiation-induced DNA double-strand damage is demonstrated.