PRC1调节小鼠卵子减数分裂纺锤体牵拉染色体运动和胞质不对称分裂的机理

During oocyte meiosis maturation, precise control of chromosome pairing and segregation as well as asymmetric division is the prerequisite for embryo development. Spindle is a temporary dynamic structure during oocyte meiosis. Spindle’s proper organization and migration ensures the accurate chromosome separation and asymmetric cytokinesis. Our preliminary results showed that in oocytes with abnormal spindles, protein regulating cytokinesis 1 (PRC1) was severely deficient. We further found that PRC1 participated in chromosome separation and cytokinesis through controlling spindle organization and migration in oocyte. Inhibition or overexpression of PRC1 leads to defect of chromosome alignment and abnormal of polar body extrusion. However, detailed mechanism of PRC1 regulating chromosome dynamic and asymmetric cytokinesis during oocyte meiosis has not been investigated. This project intends to investigate the potential mechanisms of PRC1 in regulating chromosome dynamic and asymmetric cytokinesis by employing approaches of western blot, living cell image, ultra-high resolution microscopy image, functional impair and overexpression, as well as oocyte specific gene knockout mouse. Co-IP and proteomic approach will be employed to figure out molecule(s) as well as the binding site that coordinating with PRC1. This study will provide fundamental data for clinical aneuploidy oocyte screen.

卵子成熟过程中染色体精确配对分离和胞质不对称分裂是胚胎正常发育的先决条件。纺锤体是卵子减数分裂过程中的动态结构。纺锤体正确组装和迁移保证了染色体正常分离和胞质不对称分裂。我们的前期研究结果发现，胞质分裂调控蛋白1（Protein Regulating Cytokinesis 1, PRC1）在纺锤体形态异常的卵子中严重缺失。进一步结果表明，PRC1通过参与卵子纺锤体组装和迁移过程影响染色体分离和胞质分裂。抑制或过表达PRC1会导致染色体错排、极体排放异常。但是关于PRC1调控卵子染色体运动和胞质不对称分裂的详细分子机理尚不清楚。本项目将利用免疫印迹、活细胞示踪、超高分辨成像、功能干扰与过表达、卵子特异性基因敲除小鼠构建等技术，研究PRC1调控卵子染色体分离和胞质不对称分裂的机理；利用Co-IP、蛋白质谱等技术，揭示PRC1互作分子及位点，为临床筛查异倍性卵子提供依据。

在卵母细胞减数分裂成熟过程中，精确控制染色体配对和分离以及细胞质不对称分裂是胚胎发育的前提。纺锤体的正确组装和迁移保证了染色体的精确分离和胞质不对称分裂。胞质分裂蛋白调节因子1（PRC1）是有丝分裂纺锤体组装和细胞质分裂过程中的重要调节因子，但其在卵母细胞减数分裂和早期胚胎发育中的作用尚未完全阐明。在这项研究中，我们检测了PRC1的表达和定位，并揭示了整个减数分裂和有丝分裂过程中与核、纺锤体中区相关的动态模式。PRC1缺失或过表达导致第一极体（PB1）排放和纺锤体迁移失败，同时也会导致非整倍体、动粒微管附着和纺锤体组装缺陷。为了研究PRC1在早期胚胎中的功能，我们将PRC1-MO注入合子和2-细胞期胚胎。合子中PRC1的缺失导致4-细胞、桑椹胚和囊胚的形成失败。PRC1在2-细胞期胚胎的单卵裂球或双卵裂球中的缺失导致细胞分裂失败，表明PRC1在早期胚胎发育中起着不可或缺的作用。免疫共沉淀结果显示PRC1与polo样激酶1（PLK1）相互作用，功能性敲除和挽救实验表明PRC1将PLK1招募到纺锤体中央区，进而调控减数分裂过程中的细胞质分裂。最后，Kif4基因敲减下调PRC1的表达，导致PRC1定位失败。综上所述，我们的数据表明PRC1通过调节染色体动力学和细胞质分裂在卵母细胞成熟和早期胚胎发育中发挥重要作用。这些结果将为不孕症的临床治疗提供基础数据。