H3K9me2调控雌性生殖细胞有丝分裂向减数分裂转变的作用机制研究

Abnormal gametogenesis is one of the main causes of infertility. Meiosis is a germ cell-specific division that is indispensable for generation of haploid gametes. The precise transition from mitosis to meiosis is the primary condition for meiotic initiation in germ cells. However, the underlying regulatory mechanisms still remain elusive. In this investigation, we preliminarily have found that H3K9me2 plays an important role in the transition from mitosis to meiosis of female germ cells by using an in vitro gonad culture system. To further explore the key factors regulated by H3K9me2 in the transition from mitosis to meiosis of female germ cells, Stella-GFP transgenic mouse model and G9a knock-out mouse model will be used, combined with RNA-seq and ATAC-seq technologies. Furthermore, ChIP-seq will be used to verify whether shutting down the expression of pluripotent genes is directly dependent on H3K9me2 modification in the gene regulatory region, and to clarify the exact function and molecular mechanism of H3K9me2 in regulating the transition from mitosis to meiosis of female germ cells. The findings of this proposed project will provide a new direction for the in-depth study of meiotic initiation theoretically, and a theoretical guidance for the treatment of premature ovarian failure and the improvement of oocyte quality clinically.

配子发生异常是导致不孕不育的主要原因之一。减数分裂是生殖细胞特有的分裂方式，它是单倍体配子形成的关键环节。生殖细胞有丝分裂向减数分裂的精准转变是启动减数分裂的首要条件，但是调控该过程的具体分子机制目前还不清楚。利用小鼠性腺体外培养系统，本项目初步发现H3K9me2在雌性生殖细胞有丝分裂向减数分裂转变过程中发挥重要作用。本项目将利用Stella-GFP转基因小鼠和G9a敲除小鼠模型，结合RNA-seq和ATAC-seq技术深入挖掘雌性生殖细胞有丝分裂向减数分裂转变过程中H3K9me2调控的关键因子。进一步通过ChIP-seq验证多能性相关基因的表达关闭是否直接依赖于基因调控区域的H3K9me2修饰，阐明H3K9me2调控雌性生殖细胞有丝分裂向减数分裂转变的确切功能和分子机制。本项目在理论上将为深入研究减数分裂启动的作用机制提出新的方向，在临床上将为治疗卵巢早衰、提升卵子质量等提供理论指导。

减数分裂是生殖细胞特有的分裂方式，通过减数分裂过程能产生单倍体配子，因此探究该过程的具体分子机制对不育不孕疾病诊治具有重要的意义。减数分裂启动的关键环节是生殖细胞有丝分裂到减数分裂的精准转变。然而，对于该过程的探索仍有待进一步挖掘。在本项目中，我们发现H3K9me2在胚胎期雌性生殖细胞中从E13.5开始高表达，通过体内注射或性腺体外培养系统抑制H3K9me2之后，雌性生殖细胞中多能性相关基因的表达未发生下调，有丝分裂相关基因仍持续高表达，且减数分裂相关基因表达异常，这些结果说明抑制H3K9me2导致生殖细胞有丝分裂向减数分裂的转变发生障碍。接着通过分离生殖细胞进行RNA-seq结合Cut&Run分析，我们发现H3K9me2可能通过调控染色质结构改变进而影响生殖细胞有丝分裂向减数分裂的转变。最后，我们结合两种研究减数分裂启动的经典敲除小鼠模型：Dazl 和Stra8敲除模型，初步探究H3K9me2在这两种模型中的表达动态模式以及作用。通过本项目，将开启生殖细胞减数分裂启动过程研究的新认识，为临床上女性卵巢早衰的治疗提供理论指导。