Spermatocyte responses in vitro to induced DNA damage

Abstract

Spermatocytes normally sustain many meiotically induced double-strand DNA breaks (DSBs) early in meiotic prophase; in autosomal chromatin, these are repaired by initiation of meiotic homologous-recombination processes. Little is known about how spermatocytes respond to environmentally induced DNA damage after recombination-related DSBs have been repaired. The experiments described here tested the hypothesis that, even though actively completing meiotic recombination, pachytene spermatocytes cultured in the absence of testicular somatic cells initiate appropriate chromatin remodeling and cell-cycle responses to environmentally induced DNA damage. Two DNA-damaging agents were employed for in vitro treatment of pachytene spermatocytes: gamma-irradiation and etoposide, a topoisomerase II (TOP2) inhibitor that results in persistent unligated DSBs. Chromatin modifications associated with DSBs were monitored after exposure by labeling surface-spread chromatin with antibodies against RAD51 (which recognizes DSBs) and the phosphorylated variant of histone H2AFX (herein designated by its commonly used symbol, H2AX), gammaH2AX (which modifies chromatin associated with DSBs). Both gammaH2AX and RAD51 were rapidly recruited to irradiation- or etoposide-damaged chromatin. These chromatin modifications imply that spermatocytes recruit active DNA damage responses, even after recombination is substantially completed. Furthermore, irradiation-induced DNA damage inhibited okadaic acid-induced progression of spermatocytes from meiotic prophase to metaphase I (MI), implying efficacy of DNA damage checkpoint mechanisms. Apoptotic responses of spermatocytes with DNA damage differed, with an increase in frequency of early apoptotic spermatocytes after etoposide treatment, but not following irradiation. Taken together, these results demonstrate modification of pachytene spermatocyte chromatin and inhibition of meiotic progress after DNA damage by mechanisms that may ensure gametic genetic integrity.

Mouse Pachytene Checkpoint 2 (Trip13) Is Required for Completing Meiotic Recombination but Not Synapsis

“…Interestingly, we found that OA treatment of Trip13 Gt/Gt spermatocytes could propel them into MI, despite a report that the same did not occur when wild-type pachytene spermatocytes were treated with the DSB-inducing agents gamma radiation or etoposide [53]. It is possible that the nascent induction of DSBs in pachynema evokes a checkpoint response that cannot be bypassed by OA, whereas the post-strand invasion lesions in TRIP13-deficient spermatocytes do not.…”

SPO11-Independent DNA Repair Foci and Their Role in Meiotic Silencing

“…A third possible endogenous source of DSBs is impaired topoisomerase II activity. Inhibition of topoisomerase II activity in pachytene spermatocytes has been found to result in DSB formation, indicating that topoisomerase II is indeed functional in meiocytes [68]. Fourth, endonuclease activity of ORF2, encoded by Line1 transposons, generates DSBs during the transposition of mobile elements in the genome [69]–[71].…”

Cohesin removal precedes topoisomerase IIα-dependent decatenation at centromeres in male mammalian meiosis II

“…This is a potent genotoxic Topo IIα inhibitor used in cancer therapy, whose effect lies in blocking the activity of Topo IIα once it has cut the concatenated DNA molecules, but prior to the occurrence of the religation (Nitiss 2009b). It has been reported that ET-mediated inhibition of Topo IIα during mouse meiosis induces alterations on homologous recombination during prophase I, fragmentation of centromeric satellite DNA, and failures in chromosome segregation during meiotic divisions (Kallio and Lähdetie 1996;Marchetti et al 2001;Russell et al 2004, Matulis andHandel 2006). Altogether, these data suggest that Topo IIα has a role in chromosome segregation during mouse meiotic divisions, as in mitosis.…”