纺锤体组装相关的母源基因在早期胚胎发育中的作用机制

Maternal genes play critical roles in the initial phases of embryonic development. Spindle assembly is crucial for embryonic euploidy and pre-implantation embryo development. However, the role of spindle organization associated maternal gene hasn’t been clearly known. Applicant has applied single-cell RNA sequencing (RNA-seq) analysis to individual cells from human preimplantation embryos. More than 20,000 maternal expressed genes were detected, involved three spindle organization associated maternal genes, TUBB8, NUF2 and BUB1B. All of them are highly expressed in mature oocyte, zygote and 2-cell embryo, but their expression significantly decreased in 4-cell embryo. After 8-cell stage, the expression of these genes could hardly been detected. The changes of gene expression indicate that TUBB8, NUF2 and BUB1B are essential during egg maturation and early embryo development. Based on the previous study, applicant will deregulate the expression of these genes by intracytoplasmic siRNA injection to mouse oocytes( include inmature and mature oocytes), then observe the progress of egg maturation , embryonic development, as well as spindle assembly, and detect the genome of oocytes. The conclusion will be verified with human inmature oocyte, to find the pathogenic mechanism of repeatedly egg maturation arrest or pre-implantation embryo development arrest and to provide a scientific basis for clinical diagnosis and treatment.

母源基因对早期胚胎发育起重要的作用，纺锤体的组装对卵母细胞成熟、染色体正常倍性的维持及胚胎早期发育至关重要，但纺锤体组装相关的母源基因在人类早期胚胎发育中的作用机制还缺乏深入研究。申请者前期利用单细胞转录组测序技术对人类早期胚胎进行研究，检测出2万多个母源基因，发现其中有三个纺锤体组装相关基因TUBB8、NUF2、BUB1B，在成熟卵、原核期及二细胞期胚胎均有高表达，但在四细胞期明显下降，八细胞期及之后表达量基本为零，提示其在卵成熟及胚胎早期发育过程中发挥重要作用。本项目拟在此基础上，降低或增加小鼠卵母细胞相应基因表达，观察卵母细胞成熟及胚胎发育情况以及纺锤体组装与卵母细胞遗传信息，并进一步利用人未成熟卵进行验证，从而初步探讨临床上反复卵不成熟、早期胚胎发育停滞的原因。该研究将有助于进一步加深对人类早期胚胎发育的认识，为生殖医学临床上疑难病例诊疗提供新的思路和手段。

纺锤体组装相关母源基因对早期胚胎发育至关重要，参与卵母细胞成熟及早期胚胎发育过程中染色体正常倍性的维持。为深入研究纺锤体组装相关母源基因在早期胚胎发育过程中的作用，本课题围绕关键基因母源基因对卵母细胞成熟及早期胚胎发育机制研究展开。.首先，将Nuf2、Pcid2作为目的基因，探讨其在卵母细胞成熟及早期胚胎发育过程中的作用机制，并对临床上反复卵母细胞成熟障碍及胚胎发育阻滞家系进行已知纺锤体组装相关基因的验证：1）Nuf2基因是纺锤体组装相关母源基因，其编码蛋白参与着丝粒与纺锤体的连接，并介导染色体的正确分离。本课题对Nuf2基因在小鼠植入前早期胚胎发育过程中的调控作用进行了研究，研究发现Nuf2敲降后影响小鼠早期胚胎纺锤体组装和囊胚形成；2）Pcid2是酵母蛋白Thp1的同源物，参与到mRNA由核到胞质的输出过程。本课题研究发现在卵母细胞发育过程中Pcid2的mRNA和蛋白均有较高水平表达，且Pcid2蛋白在卵母细胞成熟过程中与纺锤体共定位。Pcid2敲降后卵母细胞第一极体排出率明显降低；3）纳入反复卵母细胞成熟障碍及胚胎发育阻滞家系20余例，通过对家系成员外周血进行全外显子测序，筛查到2个TUBB8突变家系，均为新发突变位点。该研究为辅助生殖过程中反复失败家系的遗传诊断提供了新的依据。此外，项目还探讨了人类卵母细胞成熟及胚胎发育过程母源基因的建立与表达模式及其潜在功能。