G4结构解离酶RHAU在卵子成熟中的调控机制

Human oocyte maturation arrest is a bottleneck for artificial reproductive technology (ART), and is also critical for the patients undergone in vitro fertilization-embryo transfer (IVF-ET). Even though the patients can obtain the oocytes by clinical treatment, however, the immature oocyte can’t be fertilized in vitro in the end. Therefore, the investigation of human oocyte maturation arrest is critical to the clinics. We have generated RHAU conditional knock out mice by cre-loxp system, and specifically knock out RHAU in the oocytes of primordial follicles by crossing with Gdf9 mice, and in the oocytes of primary follicles by crossing with Zp3 mice, respectively. The phenotype showed that the oocytesof knock-out mice are arrested at stage of GV which germinal vesicles can’t break down. In previous study, we have demonstrated RHAU as a G4 DNA resolvase can regulate kit expression in spermatogonia differentiation. Meanwhile, researchers have shown that RHAU is highly and specifically expressed in the testes and ovary of mammals. Based on these important findings, to study the function of RHAU in the oocytes is essential for our project. Consequently, our project will keep to carrying out the following work: analysis of differential genes between the control mice and the experimental ones; analysis the relationship of these genes in vivo and in vitro. Demonstration of these issues will provide new theoretical basis and intervention targets to improve oocytes quality and embryo developmental potential in IVF-ET cycles.

人的卵细胞成熟障碍一直是辅助生殖技术临床的难题，临床上有很多病人难以获得成熟的卵细胞，最终无法正常受精。本课题拟研究一种重要的G4结构解离酶RHAU在卵泡发育中的调控机制。已应用通过Cre/loxp系统在卵母细胞中特异性敲除RHAU的小鼠为模型进行表型分析，发现阳性小鼠的卵细胞都停滞在GV期，生发泡不破裂，不能进行体外受精。之前已对精子发生障碍相关的调控因子的作用和机制有过比较深入的研究。证明了转录因子Kit受RHAU的调控，对精原细胞的分化有至关重要的作用。并有文献报道，RHAU在哺乳动物的的睾丸和卵巢中特异性表达。RHAU在卵母细胞中的作用尚待研究。本课题拟通过现在分子生物学的技术手段找出变化差异基因，体内和体外实验去验证这些基因彼此间的调控关系。该研究有望从遗传学角度阐明部分卵细胞成熟障碍的病因，为临床诊断和治疗提供新的理论基础，具有重要的理论价值和临床意义。

本课题拟通过G4结构解离酶RHAU在卵泡发育中的调控机制，解析RHAU在卵母细胞成熟中的作用机制，由于卵母细胞特异性敲除鼠子代鼠繁殖困难，及本中心丰富的胚胎样本，我们拟研究早期胚胎发育阻滞及碎片化的分子机制。本课题以单细胞测序平台为基础，对160例胚胎，包括四细胞发育阻滞，八细胞发育阻滞，第三天碎片化胚胎，四细胞及八细胞正常胚胎，进行测序并进行生物信息学分析，生物功能富集分析发现其显著差异基因主要聚集在细胞增殖，细胞分化，DNA复制，DNA修复损伤，RNA合成，细胞骨架，线粒体能量代谢等通路。进一步发现部分RNA合成的部分基因ZNF157, MBD3L3, CCNH, KLF10, NOL11, ZNF597, TOPORS, ZIM3可能参与了早期胚胎合子基因组激活，对胚胎正常发育至关重要。本课题揭示了胚胎早期发育的分子机制，为改善辅助生殖技术胚胎质量提供了理论基础。