【摘 要】
：
雌性哺乳动物中,原始生殖细胞在胚胎发育早期即已分化为不再增殖的卵母细胞,进入减数分裂并与体细胞来源的颗粒细胞共同构成卵泡.在性成熟后的动情周期中,卵泡周期性地激
【机 构】
：
浙江大学生命科学研究院,杭州,310058浙江大学生命科学研究院,杭州市西湖区余杭塘路866号,邮编310058
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雌性哺乳动物中,原始生殖细胞在胚胎发育早期即已分化为不再增殖的卵母细胞,进入减数分裂并与体细胞来源的颗粒细胞共同构成卵泡.在性成熟后的动情周期中,卵泡周期性地激活和发育直至排出成熟卵子或者由于闭锁而凋亡.因此卵母细胞减数分裂及其存活的精确调控是雌性生殖力的保障,这些生理过程的遗传缺陷会导致雌性不育和卵巢早衰.泛素化连接酶CUL4-DDB1在有丝分裂中泛素化调节其底物的降解过程,控制细胞周期进程.但这一复合体在减数分裂和不分裂细胞中的作用从来没有被研究过.我们的研究发现,DDB1在卵母细胞减数分裂和抑制卵母细胞死亡方面发挥着至关重要的作用.首先,用Gdf9-Cre转基因小鼠与携带Ddb1 flox等位基因的小鼠杂交,获得在所有卵母细胞中特异性敲除Ddb1的雌鼠(DDB1f/f；Gdf9-Cre).在与野生型雄鼠的杂交实验中,该雌鼠完全不育并且在三月龄就表现出明显的卵巢早衰,卵巢切片中不存在卵母细胞和卵泡结构.而用Zp3-Cre在激活后的初级卵泡卵母细胞中敲除Ddb1不会导致卵巢早衰,但同样雌鼠不育.因此,这些结果说明DDB1不仅在保护雌性卵母细胞储存库和维持雌性生殖力中发挥重要功能,在卵母细胞减数分裂过程中同样不可或缺.然后我们分析以上两种小鼠(DDB1f/f；Gdf9-Cre和DDB1f/f；Zp3-Cre),分别取MII卵母细胞和二细胞胚胎,发现这两种小鼠表现相同,即不能排出第一极体,更不能受精和启动之后的胚胎发育过程.接着我们分别在体内(注射PMSG.和hCG)和体外(体外培养和活细胞观察)细致分析发现DDBI缺失的卵母细胞中GVBD延缓,染色体排列紊乱,纺锤体小且不能排出第一极体.染色体铺片还发现敲除的卵母细胞染色体松散.针对这些表型,我们初步发现泛素化连接酶CCL4-DDB1复合体可能调节细胞周期抑制因子p27 (CDKN1B)和与微管蛋白动态相关的Katanin p60的泛素化降解途径.但是具体的机制还有待进一步研究.接下来的研究中,我们一方面利用质谱技术筛选CUL4-DDB1复合体在卵母细胞中的底物,另一方面在卵母细胞中敲除DCAF1和WDR20A等以寻找该复合体在卵母细胞中降解底物的adaptor蛋白,试图阐述清楚CUL4-DDB1复合体在卵母细胞中发挥功能的具体机制.
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