GTF2H2调控卵母细胞发育的分子机制研究

Although it is well-appreciated that oocyte quality contributes significantly to the success of reproduction and the health of the offspring, little is known about what key factors by which means determine the developmental competence of the oocyte. Through a forward genetics screen, we have identified that the p44 subunit of general transcription factor IIF, GTF2H2, plays a pivotal role in the control of mouse oocyte development and thus affecting its quality. Gtf2h2 mutant females are infertile but the male is normal. Although the mutant can ovulate normally, and the ovulated oocytes are mature and can be fertilized in vitro, no cleavage occurs normally, and all the zygotes eventually die later during culture. GTF2H2 is robustly expressed by oocytes and co-localizes with meiotic spindles during maturation. Given that GTF IIH is essential for the control of transcriptional initiation, nucleotide excision repair of damaged DNAs, and cell cycle progression, we here plan to investigate how GTF2H2 control oocyte development and hence affecting its quality by focusing on these three aspects. The goal of this project proposal is to unravel the causes of diminished developmental competence in Gtf2h2-mutant oocytes, and reveal the key genes that are under the direct control by GTF2H2 and play key roles in determining oocyte developmental competence.

卵母细胞质量于生殖和后代健康的重要性已被广泛认识，然而哪些关键因子通过何种方式影响卵母细胞质量还很不清楚。我们的正向遗传学研究发现通用转录因子GTF IIH的p44亚基-GTF2H2特异影响卵母细胞质量。Gtf2h2基因突变雌鼠不育但雄性正常；其雌鼠虽能正常排卵且受精，但其受精卵很少能够正常卵裂并且很快死亡。GTF2H2 在卵母细胞内优势表达，并且与卵成熟中的纺锤体及微管组织中心共定位。鉴于GTF IIH在细胞转录起始、DNA损伤的核苷酸剪切修复、以及细胞周期调控中的重要功能，本项目将围绕该三个方面并以Gtf2h2基因突变和卵母细胞特异表达GTF2H2-3xDDK的转基因小鼠为模型，对GTF2H2调控卵母细胞发育进而影响卵母细胞发育潜能的作用和机制展开研究；以期解析Gtf2h2基因突变导致卵母细胞发育潜能低下的原因，并揭示卵母细胞内受GTF2H2调控并对卵母细胞质量起决定作用的关键基因。

从高度分化的配子到具有全能性的早期胚胎的转换是一个新生命产生历程中的最为关键的环节。其中最为重要的标志性变化为卵母细胞的胞质将高度特化浓缩的精卵遗传物质重新编程为雌雄原核内疏松的染色质，进而为形成具有全能性的早期胚胎基因组做好准备。这一过程是由卵母细胞内的母源重编程因子的作用下完成的。然而，这些具有重编程功能的母源因子的身份和具体作用机制还不清楚。本项目在利用ENU－诱变正向遗传学从全基因组范围内筛选调控卵子发生关键基因的过程中，发现通用转录因子GTFIIH的组成亚基GTF2H2的编码基因发生突变后导致雌鼠不孕而雄鼠育性不受影响。进一步分析发现Gtf2h2突变导致雌性不孕的直接原因为卵子受精后卵裂以及早期胚胎发育阻滞。本项目首先在利用TALEN基因组编辑技术成功制备Gtf2h2敲除杂合（Gtf2h2+/-）小鼠，并通过等位基因补偿验证Gtf2h2突变的确导致雌鼠不孕的基础上，利用Gtf2h2突变小鼠模型系统研究了GTF2H2在卵母细胞发育成熟特别是卵母细胞-早期胚胎转换过程的作用及分子机制。本项目发现GTF2H2并不是调控卵母细胞的转录活性、DNA损伤修复，以及发育成熟所必需的，而是调控卵母细胞受精之后原核特别是雄原核的重塑以及合子基因组激活的重要母源因子，进而为卵母细胞到早期胚胎转换以及早期卵裂所必需。GTF2H2于受精卵原核内同分子伴侣蛋白HIRA共定位，通过与HIRA相互作用参与原核形成过程中母源组蛋白H3.3的招募与核小体的组装，进而调控两性原核的形成以及早期胚胎基因组的激活和发育。该成果为研究哺乳动物卵子-胚胎转换以及早期胚胎发育的分子机制开辟了新的思路，为阐明人类辅助生殖临床相关不孕疾病的发病机制及诊治奠定了基础。