人类女性卵子成熟关键因子PATL2的致病机理研究

Female infertility has become one the most influencing disease which brings heavy burdens to families and the society. There are two aspects of the etiology of female infertility: abnormal internal/external environment and genetic mutations. For a long time due to technical limitations, the contribution of genetic factors to this disease has been a blank research area. The applicant reported mutations of TUBB8 gene cause oocyte meiotic arrest in 2016, which is the first time to clarify the contribution of genetic factors to female infertility. The second pathogenic gene of oocyte maturation arrest PATL2 was reported by the research group of the applicant’s mentor, expanding the discovery of this area. More mutation points and phenotypes were illustrated later on, and the research of PATL2 function in reproduction was done in mice, showing the deregulation of genes involving in oocyte maturation related pathways. In this project, we aim to investigate the pathogenic mechanisms of PATL2 using MATQ, one of the newly developed state-of-the-art single cell RNA sequencing technology. We will set loss-of-function and mutation knock-in research models with mouse oocytes, to explore the influence of these mutations to key genes, pathways and the processing and degradation of maternal transcripts in oocyte maturation, fertilization and early embryo development. This research will unravel mechanisms of PATL2 mutations to female infertility, providing evidence for precision medicine of this disease and laying foundations for therapeutic methods.

不孕不育是当今影响女性健康的重要疾病之一，病因可分为内外环境异常和遗传突变两类。遗传突变导致的卵子异常长期以来未被充分认识。申请人2016年报道了TUBB8基因突变导致卵母细胞减数分裂停滞，在国际上首次明确了遗传因素对卵子成熟障碍的致病作用。之后申请人所在课题组又发现了第二个人类卵子成熟障碍致病基因PATL2，之后更多的PATL2突变位点和表型被揭示，并有小鼠中的初步基因功能研究被报道。本研究将通过多肽纳米颗粒转染和显微注射将PATL2敲减及引入突变至小鼠卵子中，观察卵子成熟受精发育的全过程表型，并使用国际上最新的单细胞RNA测序技术MATQ，对各阶段卵母细胞、受精卵和早期胚胎的转录组进行全面精确挖掘，探究PATL2基因突变对卵母细胞成熟和受精发育的关键基因、信号通路和母源 RNA 消耗的影响，从而揭示其突变致病机理与分子机制，为临床治疗奠定理论基础。

卵子成熟障碍和早期胚胎发育停滞是女性不孕的重要表型。PATL2作为典型的致病基因，其在卵母细胞和早期胚胎中特异性表达，以纯合或复杂合突变的形式致病，能够导致包括以GV期停滞为代表的多种表型。作为一个RNA结合蛋白，普遍推测PATL2起到蛋白质翻译的抑制作用，可能在卵子成熟和母源因子的储存、起效和降解中发挥作用。以往研究多从突变位点报道和表型展示的方式入手，而其致病的具体机制、相关通路的上下游变化和分子间相互作用等方面尚未进行深入的挖掘。本研究从分子、细胞和模式动物多个层次，对人类卵子成熟障碍致病基因PATL2的基因功能和突变致病机制进行了深入的挖掘和较为完整的探讨。在分子层面，我们发现并验证了PATL2的互作蛋白——X8和XA1A。在细胞层面，我们通过显微注射技术向GV卵母细胞注射Morpholino特异性敲低Patl2会导致卵母细胞成熟率降低，单细胞转录组测序发现X2和X4的mRNA水平下降。在模式动物层面，Patl2完全性基因敲除小鼠体外受精实验发现，合子在二细胞期出现发育阻滞，提示Patl2作为母源因子当被去除时会影响胚胎的进一步发育。综上所述，我们推测PATL2通过X蛋白相关通路，以及调控特定基因mRNA的稳定性来参与卵子成熟和早期胚胎发育的调控。我们的研究为进一步明确PATL2突变致病机制提供了新的科学依据和潜在的治疗靶点。

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