DNA double-strand breaks & poptosis in the testis

Abstract

During spermatogenesis, DNA damage is a naturally occurring event. At a certain stage, during the first meiotic prophase, DNA breaks are endogenously induced and even required for meiotic recombination. We studied these DNA breaks but also used ionizing radiation (IR) to induce DNA double-strand breaks (DSBs). Spermatogenesis is a multi step process that begins with mitotically dividing diploid stem cells and ends with highly differentiated haploid elongated spermatids. Every phase of spermatogenesis is tightly regulated, and during the process the different spermatogenic cells completely change in chromatin structure and response to DNA damage. A proper response to DNA damage is essential for germ cell development and loss-of-function mutations of many DNA damage response proteins lead to abrogation of spermatogenesis. We studied the testicular response to IR and DNA double-strand breaks (DSBs) mainly at two levels: at the spermatogonial level, during which the germ cells undergo mitotic divisions, and at the first meiotic prophase during which DSBs required for meiotic recombination are induced. Firstly, we investigated whether spermatogonia, being connected by cytoplasmic intercellular bridges, can individually undergo apoptosis in response IR or whether they, like during germ cell density, degenerate in interconnected clusters. In response to IR the germ cells were found to individually undergo apoptosis. Secondly, we investigated whether the c-Abl-p73 pathway, and possibly p63, can be an alternative apoptotic pathway as described for p53 deficient mice. Therefore we studied the testicular expression patterns and interactions of these proteins before and after irradiation. c-Abl was found to activate p73, but not p63, in the testis in response to IR and the c-Abl-p73 pathway can be an additional apoptotic pathway for damaged male germ cells. Furthermore, we studied the expression pattern of ?-H2AX, which is commonly used as a marker for DSBs, in the testis before and after irradiation. We also investigated whether ?-H2AX signaling is connected with p53 induced spermatogonial apoptosis and whether DNA-PK is required for p53 signaling and ?-H2AX foci formation and signaling during spermatogenesis. In the testis, ?-H2AX appeared not only to be present at DSBs but also just before the onset of meiosis and at the X&Y chromosomes, spermatogonial ?-H2AX appeared to be connected to p53 and totally independent of DNA-PK. We also studied non-homologues end joining (NHEJ) and the protein complex DNA-PK in the testis. We localized the three subunits of DNA-PK; Ku70, Ku86 and DNA-PKcs, in different cell types during spermatogenesis and found that the Ku heterodimer is absent during meiotic recombination, thereby preventing NHEJ to happen instead of meiotic recombination. Additionally, using DNA-PKcs deficient scid mice, we described a function for DNA-PKcs in the spermatogonial DNA damage response and during meiosis. Finally, we studied expression and activation of ATM, which lies at the basis of most responses to DSBs, during spermatogenesis. Using an antibody specific for phosphorylated ATM we were able to compare ATM activation in response to IR with the presence of endogenously active ATM during the meiosis. ATM phosphorylation in the testis was found to be coherent with p53 induction, however, during meiosis ATM seems to be phosphorylated at different unknown sites