Nrf2影响雌性生殖细胞减数分裂启动的机制研究

The entrance of primordial germ cells into meiosis is a crucial event for the formation of primordial follicles. It also affects ovarian reserve directly. Meiosis initiation was regulated by retinoid acid signaling pathway. Nuclear factor-E2 related factor 2 (Nrf2) is a vital transcription factor against oxidative stress. It interacts with retinoid acid (RA) receptor. Our previous studies have shown that Nrf2 was expressed in oocytes and early embryos. The mRNA level of Nrf2 was dynamic during the vital period of meiosis initiation. Inhibited expression of Nrf2 was related to decreased mRNA levels of meiosis initiation-specific genes, reduced number of primordial follicles, and inhibited RA signaling pathway. Clinical investigation indicated that the mRNA level of Nrf2 in granulosa cells was positively associated with ovarian reserve. Therefore, Nrf2 might regulate meiosis initiation and ovarian reserve via RA signaling pathway. In the present study, the effects of Nrf2 on meiosis initiation and ovarian reserve were investigated with in vitro, in vivo and clinical models, by testing the effects of Nrf2 on meiosis initiation-specific genes, chromosome synapsis, primordial follicle formation. Furthermore, the involvement of RA signaling pathway was also tested via the addition of RA to find out the related mechanism. In addition, the present study will provide a new idea for regulating meiosis initiation and increasing ovarian reserve in the future.

生殖细胞减数分裂启动与卵泡形成密切相关，直接影响卵巢储备，该过程受视磺酸（RA）信号通路调节。Nrf2是抗氧化应激关键转录因子，可与RA受体相互结合。本课题组前期结果证明Nrf2表达于各级卵母细胞及早期胚胎，且动态表达于小鼠减数分裂启动关键时间点；下调Nrf2能够抑制减数分裂启动及RA通路关键基因的mRNA水平，减少原始卵泡数；人颗粒细胞中Nrf2水平与卵巢储备正相关。据此提出假说，Nrf2可通过RA信号通路影响减数分裂启动及卵巢储备。为验证假说，本项目拟运用生殖嵴体外培养、肾包囊移植及Nrf2敲除小鼠模型，通过检测Nrf2对减数分裂启动关键基因、染色体联会、原始卵泡形成的影响，阐明Nrf2对减数分裂启动的影响；以人颗粒细胞为模型，研究Nrf2与卵巢储备的相关性；并通过检测干预RA通路对Nrf2效果的影响，探讨Nrf2的作用机制，以期为调控减数分裂启动、改善卵巢储备提供新思路。

生殖细胞减数分裂启动与卵泡形成密切相关，直接影响卵巢储备，该过程受视黄酸（RA）信号通路调节。Nrf2是抗氧化应激关键转录因子，可与多种分子（如RA受体、p21等）相结合。本课题以生殖嵴体外培养为平台，通过qRT-PCR、Western blot等分子生物学方法，证明Nrf2的mRNA及蛋白表达在小鼠雌性生殖嵴减数分裂启动关键时期胚胎11.5d（E11.5）至E14.5d呈动态表达。抑制Nrf2表达能够显著抑制减数分裂启动关键基因Stra8、Sycp3及Dazl的mRNA水平，减少单位面积原始卵泡的数量,抑制卵母细胞发育，并且抑制视黄酸（RA）通路基因视黄酸合成酶（Raldh）及其受体（RARa）的表达，而添加RA则能显著挽救Nrf2抑制剂的效果，然而无法达到完全恢复的效果。另外， Nrf2抑制剂还能上调p53-p21通路，而添加p53抑制剂能够挽救Nrf2抑制剂引起的减数分裂启动关键基因水平下降及卵泡的减少。以上结果表明，抑制Nrf2能够通过调节RA及p53-p21信号通路影响雌性生殖细胞减数分裂启动、卵母细胞发育及原始卵泡形成。