DNA double strand breaks repair pathways in mouse male germ cells

Abstract

DNA double strand breaks (DSBs) are induced by ionizing radiation, and during meiotic recombination. DSBs are repaired via two main pathways, homologous recombination (HR) and non homologous end-joining (NHEJ). There are three main types of male germ cells, spermatogonia, spermatocytes and spermatids. Spermatogonia include stem cells and subsequent types of proliferating spermatogonia and express many DNA repair proteins including gamma-H2AX (a marker for DSBs) and p53 binding protein1 (53BP1) but not MDC1 (mediator of DNA damage checkpoint). MDC1 is an important protein that functions in both HR and NHEJ. In the lack of MDC1, spermatogonia differ from proliferating somatic cells, and this may explain why our studies revealed a slow and incomplete repair of DSBs in spermatogonia. In early spermatocytes (leptotene, zygotene and early pachytene stages of the meiotic prophase) during meiotic recombination DSB are induced. Our findings indicate that, the error prone repair via NHEJ does not take place in early spermatocytes, also not to repair radiation induced DSBs. First, these cells do not express 53BP1, an important protein in NHEJ. Second, this notion could be further established by studying the repair of recombination related DSBs and of DSBs induced by irradiation in scid mice (deficient for NHEJ) and in Rad54/Rad54B deficient mice, potentially deficient for HR. Repair of DSBs in early spermatocytes in scid mice was similar to that in wild type mice, however the repair was significantly lower in Rad54/Rad54B deficient mice. Hence, DSB repair in early spermatocytes is exclusively carried out through HR. In late spermatocytes (mid-pachytene through diplotene stages) all important repair proteins were found and using Rad54/Rad54B deficient mice, and scid mice, we could establish that both NHEJ and HR pathways are active in these cells, NHEJ probably being the most important. The meiotic divisions render round spermatids that elongate and transform into spermatozoa. In spermatids, HR is not possible as thy are haploid and earlier results indicate that in these cells DNA-PKcs, a protein indispensable for classical NHEJ, is not expressed or at very low levels. Nevertheless, after irradiation gamma-H2AX foci disappear in these cells indicating that these cells are capable of DSB repair. Using scid mice and mice in which PARP1 protein function was inhibited, we could establish that in spermatids some residual classical NHEJ takes place and that about half of the repair takes place through the recently discovered alternative pathway for NHEJ, involving the PARP1/XRCC1 proteins. Nevertheless, DSB repair in round spermatids is slow and incomplete. We also found that, in contrast to other somatic cells in the testis, Sertoli cells, the supporting somatic cells in the seminiferous epithelium, express several DNA repair proteins and repair DSBs, as shown by the in vivo disappearance of 53BP1 foci after irradiation and the results of the comet assay. This, together with our finding that adult Sertoli cells resume proliferation in culture indicates that adult Sertoli cells can no longer be regarded as a classical example of terminally differentiated cells that are no longer able to proliferate and do not repair DNA DSBs.