跨膜蛋白Gm364通过非经典Notch途径（Notch/AKT）调控小鼠卵母细胞减数分裂进程的作用及机制研究

Defect of oocyte maturation is one of reasons for female infertility. However, the genetic causes remain largely unknown. We found Gm364, one of transmembrane proteins, might work on mouse oocyte maturation. We have confirmed that in mouse ovaries, Gm364 is much more predominant in oocytes than in granulosa cells, and mainly concentrates on the cell membrane. In oocytes cultured in-vitro, knockdown of Gm364 causes significant meiosis delay mainly characterized by defect of germinal vesicle breakdown. Preliminary results showed that Gm364 may interact with Delta, which is the ligand of Notch signaling. Since Notch have been validated to regulate meiosis maturation through activation of AKT, we suppose that Gm364 regulates oocyte maturation through Notch/AKT signaling by interacting with Delta. We will validate our hypothesis by following work. Firstly, verify the role of Gm364 in female fertility through conditional knock-out mice model; Secondly, study if the regulation of Gm364 in meiosis progression dependent on Notch/AKT signaling; Lastly, find the interaction of Gm364 and Delta through live cell imaging and study the molecular mechanisms. We aim to further study the role(s) of Gm364 in female fertility as well as the underlying mechanism(s), and may find new target for diagnosis and treatment of female infertility related to defect of oocyte maturation.

卵子成熟障碍是女性不孕的重要原因，涉及的遗传学因素仍远未阐明。跨膜蛋白Gm364是调节雌性生育力的候选因子之一，但具体作用尚不清楚。我们前期研究证实Gm364在小鼠卵巢中表达于卵母细胞的细胞膜；敲减Gm364导致减数分裂进程受阻。蛋白质谱分析提示Gm364与Notch途径的配体Delta密切相关，而非经典Notch途径已被证实通过活化AKT调控减数分裂，由此提出假说：Gm364通过介导Delta的转运激活Notch/AKT途径，进而促进减数分裂进程。项目拟通过基因敲除鼠模型明确Gm364在卵子成熟中的作用；利用动物模型和体外细胞实验阐明上述作用是否依赖于Notch/AKT途径；通过体外活细胞实验研究Gm364调节Delta运输的作用及机制并明确Gm364激活Notch途径的作用方式。本课题旨在研究Gm364在小鼠卵母细胞成熟中的作用及机制，可能为卵子成熟障碍相关不孕症的诊治提供新靶标。

目前膜蛋白在雌性减数分裂中的研究较少，我们的研究首次揭示跨膜蛋白Gm364的作用和机制。敲除Gm364后原始卵泡和生长卵泡数目均减少、卵母细胞质量受损且成熟率降低。机制上，Gm364在细胞膜上直接结合泛素连接酶MIB2、通过MIB2泛素化激活Notch2的配体DLL3、促进Notch2的胞内段NICD2剪切释放，最终NICD2直接激活AKT调控卵母细胞减数分裂成熟。结合我们既往几个关于膜蛋白在卵母细胞减数分裂过程中的研究，认为膜信号可不依赖于颗粒细胞而调控卵母细胞成熟。经典的Notch信号途径中，NICD剪切释放到细胞质后进入细胞核作为转录因子发挥作用，而在卵母细胞成熟过程中转录活动停止，暗示Notch途径调控翻译或翻译后修饰，即非经典Notch途径。我们通过体内实验发现Gm364敲除后AKT磷酸化水平降低、体外实验发现NICD2可磷酸化激活AKT，提示NICD2可作为激酶发挥作用。基于既往结构相似的蛋白研究和我们的体内外研究数据，Gm364作为膜蛋白激活NICD2，而NICD2具有激酶活性，AKT为其靶标之一。AKT作为mTOR上游重要的激酶作用与卵泡募集与发育，因而对维持雌性生育力不可或缺。总之，我们证实Gm364维持MIB2正确定位和正常的DLL3配体泛素化水平而调节Notch2途径，随后以非经典Notch途径调控减数分裂，本研究为膜蛋白以及Notch途径在雌性生殖中的作用作出重要补充。