Liu Jin, Zhang Wenchang, Wu Zhiren, Dai Lei, Koji Takehiko
Department of Occupational and Environmental Health, University of Public Health and Key Laboratory of Environment and Health, Fujian Medical University, 350004, China.
Department of Histology and Cell Biology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan.
Acta Histochem Cytochem. 2018 Apr 27;51(2):93-100. doi: 10.1267/ahc.17039. Epub 2018 Apr 24.
For a better understanding of epigenetic regulation of cell differentiation, it is important to analyze DNA methylation at a specific site. In this study, we examined changes in the methylation level of CCGG and GATCG sites during mouse folliculogenesis in paraffin-embedded sections of mouse ovaries. For the purpose, we used a new method, histo endonuclease-linked detection of methylation sites of DNA (HELMET), designed to detect methylation sites of DNA with a specific sequence in a tissue section. Unlike the global level of DNA methylation, which was no change in immunohistochemical staining of 5-methylcytosine throughout folliculogenesis, we found that there were hypermethylation of CCGG and GATCG sites in most of the granulosa cells of tertiary follicles compared to that of primary and secondary follicles. Interestingly, TUNEL-positive granulosa cells, which were frequent in mammalian folliculogenesis, became markedly II-reactive and 3A I-reactive, indicating that the CCGG and GATCG sites may be preferentially demethylated during apoptosis.
为了更好地理解细胞分化的表观遗传调控，分析特定位点的DNA甲基化非常重要。在本研究中，我们检测了小鼠卵巢石蜡包埋切片在卵泡发生过程中CCGG和GATCG位点甲基化水平的变化。为此，我们使用了一种新方法——组织核酸内切酶连接检测DNA甲基化位点（HELMET），该方法旨在检测组织切片中具有特定序列的DNA甲基化位点。与在整个卵泡发生过程中5-甲基胞嘧啶免疫组化染色中DNA甲基化的整体水平没有变化不同，我们发现与初级和次级卵泡相比，三级卵泡的大多数颗粒细胞中CCGG和GATCG位点存在高甲基化。有趣的是，在哺乳动物卵泡发生过程中常见的TUNEL阳性颗粒细胞变得明显呈II反应性和3A I反应性，这表明CCGG和GATCG位点可能在细胞凋亡过程中优先去甲基化。
