Nipbl调控小鼠卵母细胞减数分裂成熟中的作用及机制研究

Oocyte development is the basis of life and essential for the human and animal reproduction. Low quality of oocytes which would lead to the failure fertilization and early embryo development is one of the most important causes for human reproductive disorders. The fidelity of chromosome segregation is the core event during oocyte meiotic maturation and is highly related to the oocyte quality. Aneuploidy caused by the mis-segregated chromosomes is often accompanied by the early embryo death, abortion and birth defects. Cohesin complex and its regulatory molecules play the key roles in the establishment and maintenance of chromosome cohesion which is the prerequisite for the chromosome segregation. Our previous study indicates that cohesin subunit Smc1ꞵ participates in the oocyte meiotic maturation via activation of spindle assembly checkpoint. However, whether other molecules are involved and what are the underlying mechanisms are not fully known. Therefore, taking advantage of in vitro oocyte maturation system and microinjection-mediated knockdown and overexpression technology, combined with the methods in molecular biology, cell biology and biochemistry, we set out to investigate the role of a key regulatory molecule in chromosome cohesion Nipbl in the oocyte meiotic maturation, as well as its regulatory mechanism and molecular network. Our findings will provide the theoretical basis for the evaluation of oocyte quality and the novel candidates for prevention of human reproductive diseases.

卵子发育是生命繁衍的基础，对于人类生殖和动物繁殖至关重要。卵子质量低下会导致受精失败和早期胚胎发育停滞，是造成人类生殖障碍的重要原因之一。其中染色体精确分离是卵子减数分裂成熟过程的核心事件，与卵子质量高度相关。染色体分离错误引起的非整倍体往往伴随早期胚胎致死、流产及出生缺陷等生殖疾病。染色体黏合素Cohesin及其调控因子在染色体黏合建立及维持过程中发挥重要作用，是保证染色体精确分离的前提条件。我们前期研究显示Cohesin亚基Smc1ꞵ通过调节纺锤体检验点活性参与卵子减数分裂成熟过程，但其它因子是否参与其调控机制仍不明确。本项目拟通过卵子体外成熟体系和显微操作介导的基因沉默及过表达技术，结合分子生物学、细胞生物学等手段研究Cohesin通路的关键分子Nipbl在调控卵子成熟过程中作用机制和分子网络，为完善染色体黏合蛋白作为卵子质量评价指标提供理论依据，为预防人类生殖疾病提供新的候选靶标。

卵子发育是生命繁衍的基础，对于人类生殖和动物繁殖至关重要。卵子质量低下会导致受精失败和早期胚胎发育停滞，是造成人类生殖障碍的重要原因之一。其中染色体精确分离是卵子减数分裂成熟过程的核心事件，与卵子质量高度相关。染色体分离错误引起的非整倍体往往伴随早期胚胎致死、流产及出生缺陷等生殖疾病。染色体黏合素Cohesin及其调控因子在染色体黏合建立及维持过程中发挥重要作用，是保证染色体精确分离的前提条件。本项目通过卵子体外成熟体系和显微操作介导的基因沉默及过表达技术，结合分子生物学、细胞生物学等手段研究Cohesin通路的关键分子Nipbl在调控卵子成熟过程中作用机制和分子网络。.在本项目中，我们首先验证了Nipbl在GV期定位在核上，从GVBD到MII, Nipbl分布在卵母细胞的细胞质内。通过RNAi体外敲低实验发现Nipbl缺失会导致卵子成熟障碍，并且伴随纺锤体组装异常、染色体排列紊乱和微管动粒连接错误。这些结果都提示Nipbl参与了卵子的减数分裂进程。运用免疫共沉淀结合质谱分析手段鉴定其潜在互作蛋白。我们筛选到HDAC1，其通过自身的去乙酰化作用影响Eg5的乙酰化水平，进而影响纺锤体的组装，这一结论通过CO-IP实验及挽救实验得到了验证。另外，研究结果显示HDAC8在猪卵母细胞中与纺锤体共定位。HDAC8缺失会破坏纺锤体/染色体结构，进而影响猪卵母细胞的减数分裂进程。最后的结果证明HDAC8缺失导致的表型变化是由于猪卵母细胞中γ-微管蛋白向纺锤体极的募集受损。.本研究结果对了解Nipbl及HDAC8在卵母细胞减数分裂过程中的分子机制提供了理论依据，更好的完善了卵子发育成熟的机制研究，为预防人类生殖疾病提供新的候选靶标。