Tet1对鸡胚原始生殖细胞减数分裂启动的作用及调控机制的研究

Before the initiation of meiosis, primordial germ cells (PGC) undergo a series of epigenetic reprogramming steps during embryogenesis, including initial genome-wide DNA demethylation and restitution that establishes the germline epigenetic ground state. It was reported that the 5-methylcytosine (5mC) dioxygenases Tet1 play crucial role in the DNA demethylation and stem cells reprogramming, however the mechanism of demethylation in PGC regulated by Tet1 thus affecting meiosis process remains unclear. In the present study, to investigate the role of Tet1 in regulating meiotic initiation in female chicken PGC, the in vitro culture model of PGC and ovary tissue were established. Moreover, siRNAs targeting to Tet1 were used to knockdown the expression of Tet1. The target of the present project is to clarify the role and molecular mechanism of Tet1 in regulating oocyte meiotic initiation in the three levels of cell, tissue and animal experiments. In order to study whether Tet1 deficiency affects the developmental process of germ cells and the expression of a subset of meiotic genes, we focus on the regulating mechanism of Tet1 in genome-wide demethylation of PGC. Then reveal the regulating signaling network of female germ cells from mitosis to meiosis transition, and clarify the mechanism regulating by Tet1 in the process of germ cell development. These researches have important implications for understanding the whole regulatory networks of germ cells in early development. Furthermore, it will provide a technology platform for studying functional genes effecting germ cell development.

原始生殖细胞（PGC）在减数分裂前将经历一系列的表观重编程过程，包括DNA甲基化擦除和重新建立。研究发现Tet1在DNA脱甲基过程和干细胞重新编程方面起关键作用，但Tet1如何调节DNA去甲基化从而影响减数分裂的机制尚不清楚？本项目以阐明Tet1对雌性PGC减数分裂启动的作用及调控机理为目标，通过建立鸡胚PGC及卵巢组织体外培养模型和LV-shTet1基因敲减鸡胚模型，在细胞、组织、动物个体三个层面上系统研究减数分裂启动前后甲基化修饰模式的变化；重点研究Tet1在此过程中如何调控DNA去甲基化，进而激活减数分裂相关基因，以及沉默Tet1基因后对生殖细胞发育的影响；揭示Tet1调控雌性生殖细胞从有丝分裂向减数分裂转变的信号转导机制，阐明Tet1调控生殖细胞发育进程的机理。这些研究对从整体上解析生殖细胞早期发育的调控网络具有重要意义，亦为建立研究生殖细胞发育相关功能基因模型提供一个技术平台。

生殖细胞作为遗传信息传递的载体，在发育生物学及畜牧业生产等领域具有广阔的应用前景。原始生殖细胞（PGC）发育过程中在遗传学和表观遗传学方面受到严格的调控。表观重编程过程，包括DNA甲基化擦除和重新建立。本项目从在体和离体两个层面，研究鸡胚PGC不同阶段发育过程中的DNA甲基化修饰变化及Tet1对该过程的调控作用。1）鸡胚PGC生理发育过程中的甲基化情况。随着PGC迁移，全基因组DNA中5mC在E3.5后迅速下降，E4.5逐渐趋于平稳，维持在较低水平，而5hmC水平则逐渐升高，在E5.5达到顶峰，随后急剧下降，这些结果表明PGC在E3.5-E6.5发生了广泛的去甲基化作用；生殖细胞发育相关基因Dazl启动子区域的甲基化水平逐渐降低，而Dazl mRNA的表达则上调；2）Tet1对PGC甲基化的分子调控机理。敲减Tet1基因，PGC克隆团变小，结构松散，而PGC DNA甲基化程度提高。Dazl启动子区甲基化程度显著上升，而Dazl mRNA的表达量显著下降；多能性相关基因Nanog、Sox2和PouV，生殖细胞相关基因Cav和Piwi表达水平显著下降，Dnmt1、Dnmt3a和Dnmt3b表达均无显著变化；3）激活素A可通过激活TGF-β/SMAD信号通路显著提高PGC的体外存活和增殖能力。添加activin A，可显著降低PGC中Dazl基因甲基化水平，提高PGC的体外存活能力。4）Tet1敲减的ESC细胞向PGCLC诱导分化的研究。结果表明Tet1敲减后，PGCLC的形成效率降低，去甲基化比例下降。5）双酚A通过低甲基化Dazl和Stra8基因及ERβ信号通过加速卵母细胞减数分裂进程，进而促进卵巢及原始卵泡的形成及发育。研究结果可为进一步优化生殖细胞体外诱导分化和成熟及理解表观遗传修饰对生殖细胞命运选择提供重要信息，对从整体上解析生殖细胞早期发育的调控网络具有重要意义。