OSFs介导的组蛋白修饰对牦牛不同来源合子基因激活及早期胚胎发育的调控机制

During oocytes maturation, oocyte secreted factors (OSFs) are crucial cytokines. OSFs promote the oocyte development and maturation through autocrine way, and enhance its fertilization and embryo quality. Yak is our target model in this program. In order to figure out the key OSFs during oocytes maturation, the OSFs levels are detected in immature and mature oocytes by single-celled sequencing and proteomics; to explore the Smads pathway of OSFs, the phosphorylation of Smads is measured with the intervention of OSFs at high level by adding exogenous OSFs and low level decreased by RNAi technology, which is also applicated for the evaluation of OSFs on development competence of mature oocytes and subsequent embryos. In addition, zygotes are derived from three different methods: in vitro fertilization (IVF), somatic-cell nuclear transfer (SCNT) and parthenogenetic activation. Histone modification is detected for the regulation of zygotes genome activation and embryo development at early stage. This study will contribute to improve the efficiency and quality of in-vitro yak-embryo production, and optimize the assisted reproductive technology (ART) for yak. In general, achievements in this scientific research would be helpful to promote the economic development of characteristic livestock industry in Silk Road Economic Belt of China.

卵母细胞分泌因子(oocyte secreted factor, OSFs)是卵母细胞成熟必需的细胞因子，以自分泌的方式调节卵母细胞的发育和成熟，并与受精、胚胎质量紧密相关。本项目拟以牦牛为研究对象，通过单细胞测序和蛋白质组学方法比较未成熟和成熟卵母细胞OSFs表达差异，筛选关键OSFs；添加外源OSFs或应用RNAi技术分别诱导和抑制OSFs，检测其下游Smads信号磷酸化水平，评估成熟卵母细胞和其后续胚胎的发育能力；分析IVF、SCNT、孤雌激活三种方法生成的合子和胚胎中与基因激活、重组、发育等相关的组蛋白修饰水平，揭示牦牛不同来源合子基因激活及早期胚胎发育过程中OSFs介导的组蛋白修饰分子调控机制。研究结果有助于提高牦牛胚胎体外生产质量和效率，改善牦牛辅助繁殖技术，推动我国 “丝绸之路经济带”特色畜牧产业的快速发展。

卵母细胞分泌因子(oocyte secreted factor, OSFs)是卵母细胞成熟必需的细胞因子，以自分泌的方式调节卵母细胞的发育和成熟，并与受精、胚胎质量紧密相关。本项目利用利用蛋白质组学iTRAQ技术筛选确定了牦牛卵母细胞成熟过程中的差异蛋白353个，其中219个蛋白上调，134个蛋白下调，研究发现的差异蛋白主要生物学功能集中在细胞分化进程和信号转导过程，且中不同的阶段参与调控的蛋白存在一定的差异。以筛选出的关键OSFs调控靶标，发现FGF10可分别提高卵母细胞的成熟率和冻精的活力参数，同时也可通过成熟卵母细胞和活力精子中精卵融合相关蛋白以及DNA甲基化相关蛋白促进精卵融合，从而提升胚胎的卵裂率，且可以提高囊胚质量和附植潜能。并证实了牦牛移植前胚胎发育发生典型的去甲基化和再次甲基化修饰等组蛋白生理调控作用，维生素C可调控胚胎DNMT1、DNMT3a和TET3的表达水平，确保5-甲基胞嘧啶的建立、维持和转化，且可显著下调多能基因NANOG启动子区域甲基化水平，上调多能基因NANOG、Pou5f1和CDX2的mRNA转录丰度上调。探索生殖激素与OSFs之间的相互作用关系发现雌激素可通过调控卵丘扩散相关因子HAS2、PTGS2、PTX3和主要OSFs中的GDF9、FGF10和BMP15从而促进卵母细胞的后续发育能力。同时项目研究发现IGF-1可通过调控RBM3抑制低温诱导牦牛卵丘细胞中Bax和Bcl-2的表达，降低细胞凋亡水平，为揭示IGF-1和RBM3参与动物机体或细胞免受低温损伤的分子机制提供了关键信息。研究结果有助于提高牦牛胚胎体外生产质量和效率，改善牦牛辅助繁殖技术，推动我国“丝绸之路经济带”特色畜牧产业的快速发展。