5羟甲基胞嘧啶（5hmC）在猪早期胚胎发育和体细胞重编程中的研究

Development from separate parental germ cells through fertilization and proceeding to a fully functioning adult animal occurs through an intricate program of transcriptional and chromatin changes. Epigenetic alterations such as DNA methylation are an important part of this process.De novo DNA methylation and maintainance is carried out by a group of enzymes called DNA methyltransferases.(Dnmts).And research suggested the existence of mammalian enzymatic activities capable of erasing or modifying pre-existing DNA methylation patterns.However the identity of such enzymes has been enigmatic.Human ten-eleven translocation 1 (TET1) and mouse Tet proteins have recently been identified to have the capacity to convert 5mC to 5hmC (5-hydroxymethylcytosine), raising the possibility that 5mC distribution can be dynamically regulated by the Tet family of DNA hydroxylases. Emerging evidence suggests that Tet protein-mediated 5mC oxidation may also contribute to dynamic changes in global or locus-specific 5mC levels by facilitating both passive and active DNA demethylation.Untill now the role of Tet protein mediated 5mc to 5hmc in the procine oocyte maturation and early stage embyo development was not determined. In this study we aimed to invesitegate the dynamic change of 5hmc and 5hmc regulted gene network during the oocyte maturation and perimplantation stage embryos (IVF, SCNT and PA). The function and expression pattern of Tet family will be studied. We hope this study will enhance our understanding of epigenetic regulation and molecular events in the pig embryo development wich will faiclitate the efficiency of pig ART and genetic modification ability.

DNA甲基化的动态变化在哺乳动物胚胎的正常发育中有非常关键的作用。DNA甲基化建立和维持的机制已经非常清楚，但控制DNA去甲基化的酶却一直是个谜。最新发现的Tet家族蛋白（Tet1， Tet2和Tet3）具有将5mC转化为5hmC的能力使人们意识到Tet家族蛋白在去甲基化中的作用。已经证明第六碱基-5hmC在早期胚胎发育、干细胞的分化等过程中起到非常重要的作用。但有关猪卵母细胞成熟和早期胚胎发育过程中5hmC的动态变化和调控以及Tet家族蛋白表达与功能尚未见报道。本项目将研究猪卵母细胞成熟和早期胚胎发育过程中Tet家族蛋白介导的5mC向5hmC的转化，检测5hmC和Tet家族蛋白在不同来源的胚胎（IVF、SCNT和PA）不同发育阶段的动态变化和全基因组水平的定位，深入理解猪胚胎发育过程和体细胞重编程过程中的表观遗传调控和分子事件，从而提高猪的辅助生殖效率和遗传修饰能力。

DNA甲基化的建立，维持和去甲基化等动态变化在哺乳动物胚胎的正常发育中有非常关键的作用。TET家族蛋白（TET1，TET2和TET3）能够催化5mC发生氧化转化为5hmC。但有关猪卵母细胞成熟和早期胚胎发育过程中5hmC的动态变化和调控以及TET家族蛋白表达与功能尚未见报道。.本项目主要研究内容及结果如下：（1）绘制5mC，5hmC和TET家族蛋白在猪的卵母细胞成熟和早期胚胎发育过程中的表达图谱，探究猪卵母细胞成熟和早期胚胎发育中TET家族蛋白介导的5mC向5hmC转化的模式。结果显示：原核阶段存在同小鼠中相似的不对称去甲基化，从原核阶段到囊胚阶段5hmC的丰度是逐渐增加的，敲低TET3基因的表达会降低猪早期胚胎发育到囊胚的能力(9.4±2.6 vs. 6.85±0.85)。（2）研究了小分子化合物BIX-01294对猪早期胚胎发育过程中5mC向5hmC转化的影响，并对其调控机理进行了初步探讨。研究结果发现，G9a抑制剂-BIX-01294，可以通过降低组蛋白修饰H3K9me2的水平来促进雌雄原核的去甲基化，表明组蛋白修饰H3K9me2参与到猪早期胚胎发育过程中DNA的去甲基化，其作用机制主要是通过提高TET2和TET3的表达和降低DNMT3b的表达实现对5mC的去甲基化进程的促进。（3）利用Solexa测序对猪不同发育阶段的卵母细胞（GV和MII）、以及发育不同时期的胚胎进行了miRNA表达谱分析，比较了卵母细胞成熟过程、早期胚胎发育过程中的miRNA表达差异。研究结果显示，miR-27b-3p在猪的GV期和MII期卵母细胞中的表达水平存在极显著的差异，另外miR-27b-3p可能通过靶向PPARγ调节猪卵母细胞中脂肪酸代谢。利用PPARγ激动剂（罗格列酮）处理GV期卵母细胞，可极显著提高卵母细胞的成熟率（56.46% Vs. 50.99%, P＜0.01）和早期胚胎发育率（22.34% Vs. 16.86%, P＜0.01），从而揭示了miR-27b-3p可通过负调控PPARγ基因来调节猪的卵母细胞成熟和早期胚胎发育。.通过本研究明确了DNA甲基化修饰5mC和5hmC在猪卵母细胞成熟和早期胚胎发育过程中的作用，深入了解猪早期胚胎发育过程中表观遗传修饰的调控机制，并进一步探究了是否可以通过调控5hmC的表达水平来提高胚胎的体外发育能力，推动猪基因组遗传修饰的目的。