PLK1和Aurora-A的SUMO化修饰对年龄相关卵母细胞非整倍性的作用机制研究

In recent years, the contradictions between the increased age of marriage and childbearing and the increasing rate of infertility, abortion and infant deformity associated with the age-related aneuploidy in oocytes has become increasingly prominent. The dynamic changes of age-related microtubules lead to aneuploidy of oocytes, but its molecular mechanism remains unclear. It is found that the overall SUMOylation level of oocyte is related to age, and can regulate the function of PLK1 and Aurora-A，then affect microtubule dynamics. However, the effect of SUMOylation of PLK1 and Aurora-A on microtubule dynamics and age-related aneuploidy in oocytes has not been known. Therefore, in this project, the SUMOylation site mutants were used to analyze the mechanism of SUMOylation on the regulation of PLK1 function, and to confirm the effect of SUMOylated Aurora-A on the dynamics of microtubules in oocytes. Then, comparing the SUMOylation difference of PLK1 and Aurora-A in oocytes at different stages between young mice and old mice, it revealed that the correlation between age and the SUMOylation of these two factors. Finally, it verifies the feasibility of reducing the aneuploidy rate in oocytes from older mice by exogenous compensation method. This project aims to study the molecular mechanism of age-dependent microtubule dynamics and to provide a theoretical basis for preventing or reducing the aneuploidy rate of oocytes from older mice.

近年来，婚育年龄的增长与年龄相关卵母细胞非整倍性相关的不孕、流产和胎儿畸形之间的矛盾日益突出。年龄相关微管动态学改变导致卵母细胞非整倍性，但其分子机理尚不清楚。研究发现，卵母细胞整体的SUMO化水平与年龄相关，且可调节PLK1和Aurora-A功能，影响微管动态学。但尚不知PLK1和Aurora-A的SUMO化修饰对微管动力学和年龄相关卵母细胞非整倍性的作用。为此，本项目首先利用SUMO化位点突变体，解析SUMO化对PLK1功能的调节机制，并确证在卵母细胞中Aurora-A的SUMO化对微管动力学的影响；然后，对比高龄与青年雌鼠不同时期卵母细胞中，PLK1和Aurora-A的SUMO化修饰差异，揭示这2个因子的SUMO化状态与年龄的相关性；最后，验证外源补偿法降低高龄雌鼠卵母细胞非整倍率的可行性。本项目旨在研究年龄影响微管动力学的分子机理，为预防或降低年龄相关卵母细胞非整倍性提供理论基础。

卵母细胞减数分裂中染色体分离错误率远高于精子，且其发生率与女性年龄呈正相关。Cohesin降解、微管动态紊乱等假说可解释卵母细胞染色体分离错误，然二者均无法解释年龄具体经由何种途径、何关键因子作用于上述假说。. 本项目着眼于PLK1和Aurora A的SUMO 化修饰在卵母细胞减数分裂中的作用机制，以期望填补上述空白。实验结果显示，免疫荧光共定位检测和免疫共沉淀实验证实PLK1 和Aurora A均可在MI期卵母细胞中发生由SUMO-1和SUMO-2/3介导的SUMO化修饰。突变PLK1 的SUMO化修饰位点导致MI和AI期卵母细胞中PLK1定位改变或消失；突变Aurora A的SUMO化修饰位点导致MI期卵母细胞纺锤体上SUMO-1的定位改变；二者均可导致纺锤体形态异常率增加，甚至纺锤体形成失败。这表明 PLK1的SUMO化修饰通过直接调控PLK1自身募集，Aurora A的SUMO化修饰通过影响SUMO1在纺锤体上的募集，参与调控纺锤体微管动态和纺锤体形成。对比青年、高龄小鼠PLK1、Aurora A和SUMO化蛋白的免疫荧光强度和蛋白表达水平，发现高龄雌鼠卵母细胞整体的SUMO化水平下调，PLK1和Aurora A的表达和招募水平增强。以上研究结果提示，PLK1和Aurora A的SUMO 化修饰是卵母细胞减数分裂所必须的，年龄可能通过PLK1和Aurora A等关键因子的SUMO 化修饰作用于微管动态紊乱假说。. 本研究运用上述因子的SUMO 化位点突变体开展实验，规避了SUMO 化抑制剂等传统手段只能探讨整个卵母细胞广泛的SUMO 化修饰的局限性，其研究成果具有创新性。该研究符合国家“支持生育体系，应对人口老龄化”的社会需求，为相关研究拓展了关键调节因子SUMO化修饰的新思路，为临床卵母细胞染色体分离错误的预防提供理论研究基础。

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