以特异组蛋白H3K4me3修饰作为卵子老化及卵巢退行性变化的生物标记物和评价指标的研究

In women, oocyte developmental potential and ovarian functions decrease following the aging process. Genetic mutations and accelerated aging frequently cause ovarian dysfunction and infertility. The success rate of assisted reproduction in women over 40 years old is only one-third of that in women younger than 30 years old. However, the mechanism of oocyte aging and age-related ovarian functional degeneration in mammals including human is insufficiently investigated. Previous works by the applicants indicate that the DNA binding protein Cxxc-finger protein 1 (CFP1), a key subunit of SETD1 methyltransferase complex, is essential for trimethylation of histone H3 lysine-4 (H3K4me3) during oocyte development. Loss of Cxxc1, the gene encoding CFP1 protein, results in a decrease of oocyte developmental potential, impaired meiotic maturation, and chronic anovulation in mice. These phenotypes are similar to those observed in aging mice and women. In this subject, the applicants aim to investigate if CFP1-SETD1-mediated H3K4 trimethylation is a key epigenetic modification that prevents oocyte aging and ovarian degenerative changes, and if H3K4me3 can be detected as a biomarker of aging in mammalian oocytes. The applicants also aim to reveal the crucial epigenetic network that affects oocyte aging process, by using the oocyte-specific Cxxc1 knockout mouse strain as a model of advance reproductive aging. Using the chromatin immunoprecipitation (ChIP)- and single cell sequencing techniques, as well as optimized trace protein spectrum assays, the applicants aim to identify key mRNAs, proteins, and chromatin configuration changes that are involved in the oocyte aging process. Taken together, these unique and novel approaches are expected to provide reliable database for further studies of reproductive aging and aging-related woman infertility.

人类卵母细胞质量和卵巢功能伴随年龄增长逐渐下降，40岁以上女性辅助生殖的成功率只有30岁及以下年轻女性成功率的1/3。造成卵母细胞老化并导致卵巢功能下降的机制还有待深入研究。申请人前期工作发现，DNA结合蛋白CFP1作为SETD1组蛋白甲基化酶的重要亚基，介导组蛋白H3第四位赖氨酸三甲基化。CFP1缺失引起小鼠卵子质量降低、减数分裂和排卵障碍，与老化卵母细胞表型类似。本课题拟提出并证明，CFP1-SETD1介导的H3K4me3修饰是延缓卵母细胞老化的关键表观修饰，并且可以作为卵母细胞老化及卵巢退行性变化的生物标记物；利用母源CFP1缺失导致卵子提前老化的小鼠模型，通过染色质免疫共沉淀和微量细胞测序技术，建立卵母细胞关键组蛋白修饰之间的重要调控网络。并结合微量蛋白质谱找到影响卵母细胞衰老的关键蛋白质，为解决卵母细胞老化后质量下降、受孕率低等问题提供数据支持平台。

人类卵母细胞质量和卵巢功能伴随年龄增长逐渐下降，40岁以上女性辅助生殖的成功率只有30岁及以下年轻女性成功率的1/3。造成卵母细胞老化并导致卵巢功能下降的机制还有待深入研究。申请人研究发现，DNA结合蛋白CXXC1作为SETD1组蛋白甲基化酶的重要亚基，介导组蛋白H3第四位赖氨酸三甲基化。CXXC1缺失引起小鼠卵子质量降低、减数分裂和排卵障碍，与老化卵母细胞表型类似。CXXC1-SETD1介导的H3K4me3修饰是延缓卵母细胞老化的关键表观修饰，并且可以作为卵母细胞老化的生物标记物；并且随着年龄增加或Cxxc1缺失，卵母细胞虽然可以启动减数分裂成熟，但是mRNA降解能力减弱，这可能是造成临床中很多大龄患者的卵母细胞虽然完成了核成熟但是受精后发育潜能低下的重要原因。利用母源CXXC1缺失导致卵子提前老化的小鼠模型，通过染色质免疫共沉淀和微量细胞测序技术，建立卵母细胞关键组蛋白修饰之间的重要调控网络。遗传学证据表明，USP16是小鼠卵母细胞中最主要的H2AK119ub1去泛素化酶，卵母细胞中条件性敲除Usp16不会影响其存活、生长以及减数分裂成熟，但会造成卵母细胞成熟过程中母源H2AK119ub1去除异常，损害卵母细胞受精后合子基因组激活和早期胚胎发育潜能。结合起来阐明组蛋白修饰对卵母细胞成熟和早期胚胎发育的影响，为解决卵母细胞老化后质量下降、受孕率低等问题提供数据支持平台。