BOD1、LIMA1基因在卵母细胞成熟与早期胚胎发育中的作用与机制

Maternal genes play an important role in meiosis, spindle assembly associated maternal genes are particularly critical for the euploid maintenance and embryonic development. Applicant's previous studies systematically characterized the transcriptome of human primordial germ cells (PGCs) and pre-implantation embryos at single-cell and single-base resolutions. Two genes (BOD1, LIMA1) were found in a similar expression pattern with maternal genes, which are highly expressed in eggs and early embryos, but low in early PGC (pre-meiotic) and after 8-cell (post-ZGA) stages, suggesting that they may play a crucial role in egg maturation and early embryo development process. It has been reported that these two genes are crucial for spindle assembly and chromosome arrangement, but their role in meiosis and embryonic development remains unknown. This project intends to knock down the expression of BOD1 and LIMA1 in mouse eggs and observe the maturation and euploidy of the eggs, embryo development potential. With donated human immature eggs, the functions of these two genes are to be verified. Furthermore，the cause of repeated eggs maturation arrest and early embryo developmental arrest is planned to evaluated in IVF clinic. The study will help understand the mechanisms of egg and early embryo development and provide new ideas for clinical diagnosis and treatment.

母源基因在减数分裂中发挥重要作用，而纺锤体组装相关的母源基因对卵和胚胎的染色体倍性维持及胚胎发育潜能尤为关键。申请者前期对人类着床前早期胚胎，以及原始生殖细胞的转录组进行系统分析，研究发现BOD1、LIMA1在卵和早期胚胎高表达，而在早期雌性原始生殖细胞（减数分裂前）和八细胞后（合子基因激活）的阶段表达量均较低，提示其在卵成熟及早期胚胎发育过程发挥重要作用。有文献报道这两个基因对有丝分裂过程纺锤体组装和染色体排列具有重要的作用，但在卵母细胞减数分裂及胚胎发育过程中的作用未见相关报道。本项目拟利用小鼠卵母细胞体外敲低BOD1、LIMA1表达，观察卵成熟、胚胎发育及整倍体性，用捐赠人未成熟卵进行功能验证，并探讨其在反复卵成熟障碍、早期胚胎发育阻滞中作用。该研究将有助于理解卵及早期胚胎发育机制，为生殖医学临床疑难病例诊疗提供新思路和手段。

卵母细胞减数分裂和早期胚胎发育是复杂的调控过程，是保证正常卵子发生和维持女性生殖健康的关键环节。通过深入了解减数分裂及卵母细胞成熟机制、早期胚胎调控机制，将为卵母细胞成熟障碍、反复胚胎发育阻滞等相关疾病的诊疗提供理论依据。本课题围绕关键基因在卵母细胞成熟及早期胚胎发育中的调控机制开展研究。首先对LIMA1、NUP37、YPEL5及MTHFR在卵母细胞成熟及早期胚胎发育中的作用进行研究，同时发现PLCζ导致卵母细胞激活失败的新突变位点：1）LIMA1是一个细胞骨架相关蛋白，参与细胞分裂，敲降后导致小鼠卵母细胞异常极体排出率增加；2）NUP37是核孔复合体的构成成分之一，参与控制RNA和蛋白质的核质转运。Nup37基因敲降导致卵母细胞第一极体的异常排出率增加，但不影响染色体分离，同时在早期胚胎中通过调控YAP-TEAD信号通路影响早期胚胎发育潜能；3）YPEL5是一个蛋白编码基因，能够与RanBPM及RanBP10结合，它们作为重要的支架蛋白，能够与多种蛋白质相互作用以调节其功能。Ypel5基因在卵母细胞成熟过程中与纺锤体共定位，且其敲降导致卵母细胞第一极体排除率降低；4）MTHFR催化5,10-亚甲基四氢叶酸还原为5-甲基四氢叶酸，是叶酸代谢的关键酶。人类母源MTHFR的C677T/A1298C联合多态性导致MTHFR酶活性降低，且与卵母细胞质量、胚胎发育潜能等相关； 5）钙振荡是卵母细胞激活过程中的重要事件，精子携带的PLCζ是诱导卵母细胞激活的重要物质。PLCζ R553P突变导致ICSI后卵母细胞激活失败，发生反复受精障碍，提示其C2结构域对于该蛋白功能的正常发挥至关重要。.此外，进一步针对植入前胚胎遗传学检测的难题，课题开展过程中不断开发新方法及策略，如scHaplotyper，能够为临床上胚胎单基因疾病、平衡易位的诊断提供帮助。