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Homologous recombination (HR) is established as a significant contributor to double-strand break (DSB) repair in mammalian somatic cells; however, its role in mammalian germ cells has not been characterized, although being conservative in nature it is anticipated to be the major pathway in germ cells. The germ cell system has inherent limitations by which intact cell approaches are not feasible. The present study, therefore, investigates HR-mediated DSB repair in mouse germ cell extracts by using an in vitro plasmid recombination assay based on functional rescue of a neomycin (neo) gene. A significantly high-fold increase in neo+ (Kan(R)) colonies following incubation of two plasmid substrates (neo delta1 and neo delta2) with testicular extracts demonstrated the extracts' ability to catalyze intermolecular recombination. A significant enhancement in recombinants upon linearization of one of the plasmids suggested the existence of an HR-mediated DSB repair activity. Comparison of the activity at sequential developmental stages, spermatogonia, spermatocytes and spermatids revealed its presence at all the stages; spermatocyte being the most proficient stage. Further, restriction analysis of recombinant plasmids indicated the predominance of gene conversion in enriched spermatocytes (mostly pachytenes), in contrast to gonial and spermatid extracts that showed higher reciprocal exchange. In conclusion, this study demonstrates HR repair activity at all stages of male germ cells, suggesting an important role of HR-mediated DSB repair during mammalian spermatogenesis. Further, the observed preference of gene conversion over reciprocal exchange at spermatocyte stage correlates with the close association of gene conversion with the meiotic recombination program.

Accurate quantification of DNA double strand breaks (DSB) in testicular germ cells is difficult because of cellular heterogeneity and the presence of endogenous γH2AX. Here, we used confocal microscopy to quantify DNA damage and repair kinetics following γ-irradiation (0.5-4 Gy) in three major mouse male germ cell stages, early and late pachytene spermatocytes and round spermatids (RSs), following a defined post irradiation time course. Dose-response curves showing linear best fit validated γH2AX focus as a rapid biodosimetric tool in these substages in response to whole body in vivo exposure. Stage specific foci yield/dose and repair kinetics demonstrated differential radiosensitivity and repair efficiency: early pachytenes (EP) repaired most rapidly and completely followed by late pachytene (LP) and RSs. Repair kinetics for all three stages followed 'exponential decay' in response to each radiation dose. In pachytenes immediate colocalisation of γH2AX and 53BP1, which participates in non-homologous end-joining repair pathway, was followed by dissociation from the major focal area of γH2AX by 4 h demonstrating ongoing DSB repair. These results confirm the differential radiosensitivity and repair kinetics of DSBs in male germ cells at different stages. Taken together, our results provide a simple and accurate method for assessing DNA damage and repair kinetics during spermatogenesis.

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