利用卵母细胞特异性基因敲除小鼠研究母源性UHRF1蛋白的功能

UHRF1 is an important epigenetic regulation factor. Our previous studies indicated that this protein was highly expressed in the mouse oocyte. Growth and development of oocyte, oocyte meiotic maturation and subsequent early embryonic development, are all the processes precisely controlled by epigenetic mechanisms. Thus, we speculate UHRF1 protein plays critical roles during these development processes. However, due to embryonic lethality of Uhrf1 knockout mice, the functional awareness of maternal UHRF1 protein is still blank. In this study, we intend to take advantage of the oocyte-specific knockout mouse model, to explore the fuction of oocyte expressed UHRF1 protein. In previous study, we have introduced Uhrf1 knockout mice from EUCOMM and used them as a basis to construct oocyte-specific Uhrf1 knockout mice (Uhrf1 CKO mice). So far, Uhrf1 CKO mice was made successfully, the fertility test results showed that the CKO females were completely infertile, however, in the CKO females, we detected follicles at all stages of develpoment and we found similar numbers of ovulated oocytes compared to those from control animals, suggesting that the loss of Uhrf1 has no gross effects on follicular development. Further examination showed that in the fully-grown oocytes collected from Uhrf1 CKO mice, 5mC staining was significantly reduced and the SINE-1 and IAP retrotransposons was derepressed, indicating that the oocyte quality of Uhrf1 CKO mice was poor. In this study, we will explore the influence of Uhrf1 deletion on the processes of oocyte maturation and early embryo development in-depth. This study will not only fill a vacancy in our knowledge about the funciton of maternal UHRF1 protein, but also enhance the understanding of the epigenetic regulation of oocyte and early embryo development.

UHRF1蛋白是一个重要的表观遗传调控因子。我们的前期研究表明，该蛋白在卵母细胞中表达丰度很高。而卵细胞的发育、成熟及后续的早期胚胎发育过程，正是受到表观遗传精确调控的，因而我们推测该蛋白将在其中发挥重要作用。但由于Uhrf1全敲小鼠胚胎致死，到目前为止，对卵细胞中表达的UHRF1蛋白的功能认识尚属空白。本研究中，我们拟利用卵母细胞特异性敲除小鼠，探究母源性UHRF1蛋白的功能。前期工作中，已成功构建了Uhrf1 CKO小鼠，CKO雌鼠完全不育，对其卵巢形态的观察及超排卵实验结果表明其卵泡发育过程未受影响。进一步研究结果显示CKO小鼠卵细胞中5mC水平显著降低，而反转座基因表达异常增加，提示其卵细胞质量异常。本研究将在此基础上,深入探讨Uhrf1敲除对卵细胞成熟及后续早期胚胎发育过程的影响，弥补对母源性UHRF1蛋白功能认识的空白，并增进对卵细胞及早期胚胎发育表观遗传调控的理解。

UHRF1蛋白是从本实验室构建的小鼠卵母细胞蛋白表达谱中筛选得到的, 对其在卵母细胞及植入前早期胚胎中的表达研究提示,该蛋白作为一个母源性因子在卵母细胞和早期胚胎发育过程中发挥作用。在本项目的支持下，我们对该蛋白在早期胚胎中的作用及其作用机制展开了深入探讨。我们利用卵母细胞特异性敲除小鼠，探究母源性 UHRF1 蛋白的功能并揭示了母源性UHRF1在小鼠卵母细胞质量决定中的关键作用。前期工作中，已成功构建了Uhrf1Zp3cKO小鼠并发现cKO雌鼠完全不育。进一步研究发现Uhrf1Zp3cKO小鼠卵母细胞质量明显下降，并从形态学、遗传学、后续发育潜能、表观遗传修饰以及转录水平多个角度来探究UHRF1对小鼠卵母细胞质量的调控。研究发现，缺乏UHRF1的MII期卵母细胞卵周隙增加、纺锤体异常率以及染色体非整倍体率显著增加，Uhrf1Zp3cKO的GV期卵母细胞以及母源缺失UHRF1的2-cell胚胎DNA损伤信号增多。同时、我们也发现与已有文献报道一致，UHRF1缺失的卵细胞及早期胚胎中存在DNA甲基化缺陷，除此之外，Uhrf1Zp3cKO的GV、MII期卵母细胞的H3K9me2水平下降，而H4K12ac水平上升。MII期卵母细胞的RNA-seq结果提示，缺乏UHRF1的卵母细胞发生了大规模的转录水平的改变，差异基因的GO分析显示在染色体分离、DNA损伤等生物学事件富集。综上所述，我们的研究结果表明UHRF1在卵母细胞的质量决定中起着重要作用。进一步探讨母源性UHRF1的生物学功能和潜在机制将增强我们对母源控制卵母细胞和早期胚胎发育的认识。