CUL4-DDB1泛素连接酶在小鼠卵母细胞发育和减数分裂中的作用

The mammalian female germ cells differentiate into non-proliferating oocytes at the early stage of ovary development, and then form follicles together with the maternal derived granulosa cells. In the following reproductive life, the oocytes are periodically recruited and undergo meiotic maturation. A large proportion of oocytes die during development due to follicle atresia. However, the molecular mechanisms that regulate follicle development and atresia are not fully understood. Particularly, the signaling network in oocyte that detects/repairs DNA damage and maintains genome stability remains unclear. The applicant aims to investigate the essential genes that are involved in the regulation of oocyte development and meiotic maturation, the maintenance of genome integrity, as well as the crosstalk among genes during these cellular events, using in vivo animal models of conditional knockouts. We discovered recently that deletion of damaged DNA binding protein 1 (DDB1) in the oocyte of primordial follicles leads to meiotic division abnormality, global follicle atresia, and premature ovarian failure. We will further deplete DDB1 in oocytes of primary follicles to study the stage-specific functions of DDB1, by crossing DDB1 floxed mice with the previously reported ZP3-Cre mice. We will also test: 1), if DDB1-depletion causesoocyte death and meiotic maturation defects through its substrate adaptor DCAF1; 2), if the oocyte-specific depletion of DCAF1 phenocopies the defects of DDB1 conditional knockout mice; 3), in addition to maintaining oocyte survival at the primordial follicle stage, if CUL4-DDB1-DCAF1 ubiquitin E3 ligase complex also play key roles in meiotic maturation of fully grown oocytes, particularly their effects on chromosome condensation, chromosome seperation, and spindle assembly; and 4), if DNA topoisomerase II (TOP2) is regulated by DDB1/DCAF1 by ubiquitination, and if this regulation affects the chromosome localization or activity of TOP2. In addition, we aim to investivate if TOP2 is an essential DDB1/DCAF1 target protein for oocyte survival and meiotic maturation

哺乳动物雌性生殖细胞在卵巢发育早期分化为不再增殖的卵母细胞，并与颗粒细胞共同形成卵泡结构。在随后的生殖活动中卵母细胞周期性地发育和成熟，或由于卵泡闭锁而死亡。卵泡发育和闭锁以及卵母细胞减数分裂的分子调节机制还有许多未知环节。特别是在卵母细胞中维持基因组稳定和DNA 损伤修复的分子网络还不清楚。申请人拟通过卵母细胞特异性的基因敲除技术，研究调节卵母细胞发育、维持基因组稳定、参与减数分裂成熟的重要基因，以及这些功能相关基因在上述生理过程中的相互影响。我们已发现，在原始卵泡中敲除DDB1 导致卵母细胞减数分裂异常、卵泡闭锁和卵巢早衰。我们将在卵母细胞发育不同时期敲除DDB1，进一步分析该基因在卵母细胞发育和雌性生殖功能维持中的作用及其分子机理，并研究DDB1 敲除引起的卵母细胞死亡是否通过其下游底物识别分子DCAF，以及细胞周期相关蛋白DNA拓扑异构酶2（TOP2）。

首次研究了CRL4复合体在雌性生殖力维持、卵泡发育、卵母细胞减数分裂和受精卵重编程中的作用，为卵巢早衰和雌性不育的诱因提供新的分子机制。卵巢中正常的卵泡数量和卵母细胞完成减数分裂是雌性生育能力的先决条件。当卵巢中的原始卵泡消耗殆尽以后，更年期到来，失去生育功能。在人类，如果40岁以前发生原始卵泡耗竭，就会导致卵巢早衰，该疾病影响人群中1-2%的女性。长期以来，维持原始卵泡存活的机制了解很少。通过本课题研究发现，CRL4泛素连接酶复合体的多个亚基在卵母细胞中高表达，因此在卵母细胞中特异性地敲除了CRL4泛素连接酶复合体的两个重要成分：DDB1和DCAF1/VPRBP，并分析敲除小鼠表型。发现DDB1和DCAF1敲除的小鼠完全不育，并且敲除的卵母细胞在卵泡存活和发育、卵母细胞减数分裂和卵子受精及胚胎发育等过程中都表现出明显的异常，说明CRL4复合体在雌性生殖中起着至关重要的作用。进一步的机理研究发现，CRL4通过调节TET家族DNA去甲基化酶的作用，调节卵母细胞中基因的表达和受精后雄原核的去甲基化，从而在维持雌性生殖力。如果CRL4的功能缺失，就会导致卵母细胞死亡，发生卵巢早衰。CRL4泛素连接酶除了维持原始卵泡存活以外，也对于受精卵中雄原核的去甲基化和体细胞克隆过程中的基因组重编程起着重要调节作用，是卵胞质中以前未知的一个重编程因子。该研究成果从分子机制上揭示了维持卵母细胞存活和雌性生育能力的新机制，为了解卵巢早衰、妊娠失败等女性不孕不育疾病的病因提供了可能的分子机制。这些结果于2013年发表在《Science》上。