The dynamics of DNA methylation during epigenetic reprogramming of primordial germ cells in medaka (<i>Oryzias latipes</i>)

Primordial germ cells (PGCs) are precursors of eggs and sperm. How the PGCs epigenetically reprogram during early embryonic development in fish is currently unknown. Here we generated a series of PGC methylomes using whole genome bisulfite sequencing across key stages from 8 days post fertilization (dpf) to 25 dpf coinciding with germ cell sex determination and gonadal sex differentiation in medaka (Oryzias latipes) to elucidate the dynamics of DNA methylation during epigenetic reprogramming in germ cells. Our high-resolution DNA methylome maps show a global demethylation taking place in medaka PGCs in a two-step strategy. The first step occurs between the blastula and 8-dpf stages, and the second step occurs between the 10-dpf and 12-dpf stages. Both demethylation processes are global, except for CGI promoters which remain hypomethylated throughout the stage of PGC specification. De novo methylation proceeded at 25-dpf stage with the process in male germ cells superseding female germ cells. Gene expression analysis showed that tet2 maintains high levels of expression during the demethylation stage, while dnmt3ba expression increases during the de novo methylation stage during sexual fate determination in germ cells. The present results suggest that medaka PGCs undergo a bi-phasic epigenetic reprogramming process. Global erasure of DNA methylation marks peaks at 15-dpf and de novo methylation in male germ cells takes precedence over female germ cells at 25 dpf. Results also provide important insights into the developmental window of susceptibility to environmental stressors for multi- and trans-generational health outcomes in fish.原始生殖细胞（primordial germ cells，PGCs）是卵子与精子的前体细胞。目前学界对于鱼类早期胚胎发育过程中，原始生殖细胞的表观遗传重编程机制尚不明晰。本研究以青鳉（Oryzias latipes）为研究对象，利用全基因组亚硫酸氢盐测序技术，对受精后8天（days post fertilization，dpf）至25 dpf的关键阶段的原始生殖细胞进行了一系列甲基化组测序，该阶段覆盖了生殖细胞性别决定与性腺性别分化的关键时期，旨在阐明生殖细胞表观遗传重编程过程中DNA甲基化的动态变化规律。本研究获得的高分辨率DNA甲基化组图谱显示，青鳉原始生殖细胞的全基因组去甲基化以两步模式进行：第一步发生于囊胚期至受精后8天阶段，第二步则发生于受精后10天至12天阶段。上述两处去甲基化过程均为全基因组范围的事件，唯有CpG岛（CGI）启动子区域在原始生殖细胞特化的整个阶段始终保持低甲基化状态。受精后25天阶段开始出现从头甲基化过程，且雄性生殖细胞的从头甲基化进程领先于雌性生殖细胞。基因表达分析结果显示，TET2在去甲基化阶段维持高表达水平，而在生殖细胞性别命运决定过程中，DNMT3BA在从头甲基化阶段的表达量显著上调。本研究结果表明，青鳉原始生殖细胞经历了双相表观遗传重编程过程：DNA甲基化标记的全基因组清除在受精后15天达到峰值，而雄性生殖细胞的从头甲基化进程在受精后25天仍领先于雌性生殖细胞。本研究结果同时为解析鱼类多代及跨代健康效应中，环境应激源易感的发育窗口提供了重要参考依据。