颗粒细胞中EGFR巴豆酰化修饰调控卵母细胞体外成熟的作用和机制研究

In vitro maturation of human immature oocytes (IVM) is an important assisted reproductive technology and is significantly valuable for a range of clinical applications and social benefits. However, the much lower clinical pregnancy rates than traditional IVF limited its further development and extensive use, mainly resulting from the limited knowledge on the mechanisms of oocyte in vitro maturation. Based on the online data analysis and conformation using clinical samples, we found that over the in vitro maturation of human oocytes, there are different lysine crotonylation levels in cumulus cells, which surround the oocyte and play a crucial role in maintaining oocyte meiotic arrest. The increased lysine crotonylation can significantly activate the EGFR pathway in cultured cumulus cells, as well as the total and phosphorylated EGFR. Moreover, the lysine crotonylation level was related with the level of EGFR and EGFR pathway in cumulus cells from clinical samples. The crotonylation site on EGFR has been proved as an important ATP binding site for EGFR auto-phosphorylation, suggesting that crotonylation on EGFR may affect the EGFR pathway. Taken together, we suggest the hypothesis that lysine crotonylation of EGFR in cumulus cells might play an important role in in vitro maturation of human oocytes through EGFR pathway. Our study was aimed to explore the functions and mechanisms of EGFR crotonylation in human oocytes maturation and to improve the in vitro culture of human oocytes.

较低的卵母细胞成熟率和临床妊娠率是限制卵母细胞体外成熟技术（IVM）应用的关键难题，而其主要原因是卵母细胞体外成熟机制尚未阐明。卵丘颗粒细胞是卵母细胞周围的重要支持细胞，并且颗粒细胞中EGFR通路的激活在卵母细胞成熟中发挥关键作用。通过网上数据分析和临床样本验证，我们发现成熟卵母细胞对应的颗粒细胞中总体巴豆酰化修饰显著增强。进一步研究显示：巴豆酰化修饰可以提高颗粒细胞中EGFR总体水平和磷酸化水平，进而增强EGFR通路的活化，并且巴豆酰化修饰与EGFR通路分子在临床样本中也存在正相关。鉴于EGFR上存在巴豆酰化修饰位点，我们提出：EGFR的巴豆酰化修饰是否通过激活EGFR通路促进了卵母细胞的体外成熟？本项目拟采用免疫共沉淀、siRNA和小鼠IVM模型等方法，阐明EGFR巴豆酰化修饰在卵母细胞体外成熟中的作用及机制，为完善卵母细胞体外培养体系提供科学依据。

人卵母细胞体外成熟（IVM）技术作为重要的辅助生殖方式，具有显著的优势和应用价值，然而不同于卵母细胞体内成熟环境，IVM中存在显著的卵母细胞成熟障碍，而卵母细胞减数分裂阻滞的机制并不清楚。我们研究发现人卵母细胞体外成熟过程中GV阻滞的卵母细胞的辅助性卵丘颗粒细胞巴豆酰化修饰水平较低，而EP300的低表达与巴豆酰化水平低相关。EP300的表达与IVM中颗粒细胞的拓展密切相关。利用人原代卵丘颗粒细胞进行细胞实验，我们进一步发现外源性巴豆酰化底物可以促进颗粒细胞的增殖，抑制颗粒细胞的凋亡。EP300的干扰和激活也相应的调控了颗粒细胞的增殖和凋亡过程。为了探索颗粒细胞中巴豆酰化的关键靶蛋白，我们发现利用人颗粒细胞进行了巴豆酰化蛋白测序，发现其中ANXA2是关键性的巴豆酰化蛋白。巴豆酰化增强了ANXA2与EGFR的结合，促进了EGFR通路的激活。我们的研究确定了颗粒细胞中把巴豆酰化修饰是人卵母细胞体外成熟障碍的重要因素，并且确定了巴豆酰化通过ANXA2-EGFR调控颗粒细胞功能的机制，从而提出外源性巴豆酰化底物和EP300激活剂作为卵母细胞体外成熟体系优化的潜在应用。