Matulis Shannon, Handel Mary Ann
Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, USA.
Mol Reprod Dev. 2006 Aug;73(8):1061-72. doi: 10.1002/mrd.20508.
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.
精母细胞通常在减数分裂前期早期会维持许多减数分裂诱导的双链DNA断裂（DSBs）；在常染色体染色质中，这些断裂通过启动减数分裂同源重组过程进行修复。对于重组相关的DSBs修复后，精母细胞如何应对环境诱导的DNA损伤，人们了解甚少。本文所述的实验检验了这样一个假设：即使正在积极完成减数分裂重组，在没有睾丸体细胞的情况下培养的粗线期精母细胞也会启动适当的染色质重塑和细胞周期反应，以应对环境诱导的DNA损伤。使用了两种DNA损伤剂对粗线期精母细胞进行体外处理：γ射线照射和依托泊苷，一种拓扑异构酶II（TOP2）抑制剂，它会导致持续未连接的DSBs。通过用抗RAD51（识别DSBs）抗体和组蛋白H2AFX的磷酸化变体（此处用其常用符号H2AX表示，即γH2AX，它修饰与DSBs相关的染色质）标记表面铺展的染色质，监测暴露后与DSBs相关的染色质修饰。γH2AX和RAD51都迅速被募集到受照射或依托泊苷损伤的染色质上。这些染色质修饰表明，即使重组基本完成后，精母细胞仍会募集活跃的DNA损伤反应。此外, 照射诱导的DNA损伤抑制了冈田酸诱导的精母细胞从减数分裂前期向中期I（MI）的进展，这意味着DNA损伤检查点机制是有效的。DNA损伤的精母细胞的凋亡反应有所不同，依托泊苷处理后早期凋亡精母细胞的频率增加，但照射后没有增加。综上所述，这些结果表明，粗线期精母细胞染色质发生了修饰，并且DNA损伤后减数分裂进程受到抑制，这些机制可能确保配子的遗传完整性。
