Song Ning, Endo Daisuke, Song Bin, Shibata Yasuaki, Koji Takehiko
Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan; Department of Anatomy, Histology and Embryology, Shanghai Jiaotong University School of Medicine, 280 South Chongqing Rd., Shanghai 200025, PR China.
Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
Toxicology. 2016 Jun 15;361-362:62-72. doi: 10.1016/j.tox.2016.07.005. Epub 2016 Jul 7.
Mammalian spermatogenesis is a progressive process comprising spermatogonial proliferation, spermatocytic meiosis, and later spermiogenesis, which is considered to be under the regulation of epigenetic parameters. To gain insights into the significance of DNA methylation in early spermatogenesis, 5-azadC was used as a molecular biological tool to mimic the level of DNA methylation in vivo. Since the drug is incorporated into DNA during the S-phase, spermatogonia and spermatocytes would be affected primarily in mouse spermatogenesis. Adult male ICR mice were intraperitoneally injected with 5-azadC at a dose of 0.25mg/kg/day for 10 consecutive days, allowing us to examine its maximum effect on the kinetics of spermatogonia and spermatocytes. In this short-term protocol, 5-azadC induced significant histological abnormalities, such as a marked increase in apoptosis of spermatogonia and spermatocytes, followed by severe loss of spermatids, while after termination of 5-azadC treatment, normal histology was restored in the testis within 35days. Quantification of the methylation level of CCGG sites as well as whole DNA showed spermatogonial hypomethylation, which correlated with increased apoptosis of spermatogonia. Interestingly, the hypomethylated cells were simultaneously positive for tri-methylated histone H3 at K4. On the other hand, no changes in methylation level were found in spermatocytes, but PCNA staining clearly showed disordered accumulation of S-phase spermatocytes, which increased their apoptosis in stage XII. In addition, different immunohistochemical staining pattern was found for DNA methyltransferases (DNMTs); DNMT1was expressed in the majority of all germ cells, but DNMT3a and b were only expressed in spermatogonia. Our results indicate that 5-azadC caused DNA hypomethylation in spermatogonia, but induced prolongation of S-phase in spermatocytes, resulting in the induction of apoptosis in both cases. Thus, 5-azadC affects spermatogenesis at more than one differentiation stage with different mechanisms, probably due to the specific usage of DNMTs.
哺乳动物精子发生是一个渐进的过程，包括精原细胞增殖、精母细胞减数分裂以及随后的精子形成，这一过程被认为受表观遗传参数的调控。为深入了解DNA甲基化在早期精子发生中的重要性，5-氮杂胞苷（5-azadC）被用作一种分子生物学工具来模拟体内DNA甲基化水平。由于该药物在S期掺入DNA，精原细胞和精母细胞在小鼠精子发生过程中会受到主要影响。成年雄性ICR小鼠连续10天腹腔注射剂量为0.25mg/kg/天的5-氮杂胞苷，以便我们研究其对精原细胞和精母细胞动力学的最大影响。在这个短期实验方案中，5-氮杂胞苷诱导了显著的组织学异常，如精原细胞和精母细胞凋亡显著增加，随后精子细胞严重丢失，而在停止5-氮杂胞苷治疗后，睾丸在35天内恢复了正常组织学。对CCGG位点以及全基因组DNA甲基化水平的定量分析显示精原细胞低甲基化，这与精原细胞凋亡增加相关。有趣的是，低甲基化细胞同时在K4位点的组蛋白H3三甲基化呈阳性。另一方面，精母细胞中未发现甲基化水平变化，但增殖细胞核抗原（PCNA）染色清楚显示S期精母细胞无序聚集，这增加了它们在第XII阶段的凋亡。此外，发现DNA甲基转移酶（DNMTs）有不同的免疫组化染色模式；DNMT1在所有生殖细胞的大多数中表达，但DNMT3a和DNMT3b仅在精原细胞中表达。我们的结果表明，5-氮杂胞苷导致精原细胞DNA低甲基化，但诱导精母细胞S期延长，在两种情况下均导致凋亡诱导。因此，5-氮杂胞苷以不同机制在多个分化阶段影响精子发生，这可能是由于DNMTs的特定作用方式。
