ARHGAP26调控小鼠卵母细胞减数分裂进程的机制研究

One of the most important subjects for improving assisted reproduction technology is to clarify the mechanism of oocyte/embryo aneuploidy ocurrence, reduce the aneuploidy, and improve the egg quality. We found a patient with recurrent pregnancy loss, higher rate of embryo aneuploidy and reduced ovarian reserve. Chromosomal Microarray Analysis indicated the deletion of ARHGAP26 in her peripheral blood cells. Her mother died of breast cancer and her aunts also had cancer and recurrent pregnancy loss. Thus we speculated that deletion of ARHGAP26 maybe one of the reason of recurrent pregnancy loss due to aneuploidy. In this study, we will explore the localization and expression of ARHGAP26 in mouse oocyte meiosis. Immunofluorescent analysis showed that ARHGAP26 was localized to the spindles from the pre-MI stage to the MII stage. Secondly, we will examine whether ARHGAP26 influence the alignment of chromosomes and spindle assembly by regulating the expression of RhoA. Thirdly, we will study how ARHGAP26 impact on ovary reserve, oocyte meiotic maturation and early embryo development by generating conditional knock out mice. Fourthly, we plan to confirm whether the normal progress of meiosis could be recovered and aneuploidy be reduced by means of injecting ARHGAP26 mRNA into the GV oocytes from conditional knock out mice. The research not only has theoretical significance, but also is expected to provide medical treatment for recurrent pregnancy loss therapy.

研究女性卵子非整倍体的发生机制、改善卵子质量是目前辅助生殖领域最为重要的课题之一。我们通过分析全基因组芯片报告发现一例复发流产患者，高发非整倍体合并卵巢储备功能减退，缺失抑癌基因ARHGAP26。其母亲及母亲姐妹也有复发流产史。由此我们推测ARHGAP26缺失可能是导致高发非整倍体卵子引起反复流产的原因之一。本研究拟通过小鼠模型来研究：1）ARHGAP26在小鼠卵子减数分裂进程中的定位与表达，初步结果显示ARHGAP26定位在小鼠卵母细胞减数分裂的纺锤体上；2）ARHGAP26是否通过调控RhoA的表达影响减数分裂过程中纺锤体组装和染色体的分离；3）通过构建条件基因敲除鼠，研究该基因缺失对于卵巢功能、卵子成熟和早期胚胎发育的影响；4）将体外转录ARHGAP26 mRNA注入条件基因敲除小鼠GV期卵母细胞，观察其是否减少非整倍体发生。该研究将为复发性流产诊治提供理论依据。

研究女性卵子非整倍体的发生机制、改善卵子质量是目前辅助生殖领域最为重要的课题之一。我们通过分析全基因组芯片报告发现一例复发流产患者，高发非整倍体合并卵巢储备功能减退，缺失抑癌基因ARHGAP26。其母亲及母亲姐妹也有复发流产史。由此我们推测ARHGAP26缺失可能是导致高发非整倍体卵子引起反复流产的原因之一。本研究通过小鼠模型来研究发现：1）ARHGAP26在小鼠卵子减数分裂进程中定位于卵母细胞质膜上；2）通过构建ARHGAP26基因敲除鼠，发现ARHGAP26-/-小鼠相对于野生型小鼠卵巢体积显著降低。ARHGAP26缺失破坏了卵母细胞线粒体的功能，ROS显著升高，卵子退化率升高。通过体外培养小鼠卵母细胞发现ARHGAP26缺失影响了卵母细胞减数分裂成熟过程中的纺锤体的组装和染色体的排列，MII卵中非整倍体率升高。3）通过Co-IP实验确定了cofilin是与ARHGAP26在质膜上直接相互作用的蛋白，当ARHGAP26基因被敲除，影响了cofilin的功能，cofilin的的下游蛋白，线粒体蛋白Drp1无法募集到线粒体上，从而使卵母细胞线粒体功能受损，卵母细胞质量下降。该研究将为复发性流产诊治提供理论依据。