高寒环境相关调节因子对牦牛卵母细胞成熟及附植前不同来源胚胎发育调控的作用机制

Many factors relate with high altitude were regulated by animals living in high altitude for maintain balance of physiological environment, which combined with the factors secreted during in vitro maturation of oocytes and embryonic development, and the development of themselves was regulated. The yak will be as object of study in this project, and the expression of HIF-1α, HSP70, Ref-1 and some factors relate with development were detected with methods of cDNA microarrays and real time PCR during in vitro maturation of oocytes and development of preimplantation embryo from in vivo, in vitro fertilization and nuclear transfer. The regulatory factors was screened; The expression of HIF-1α, HSP70 and Ref-1were inhibited or induced through add some ralation factors or using the method of RNAi, the role of cytokines and apoptin was analysed during in vitro maturation of oocytes and embryonic development, the quality of oocytes and embryo was detected using methods of immunofluorescence and electron microscope; The environment which similar to plateau will be establishment through changed temperature and concentration of oxygen, the expression of HIF-1α, HSP70 and Ref-1 was analysed, and the quality of oocytes and embryo was evaluated，the role of them to oocytes and embryo in plateau environment will be clarified. This project will be help to enhance the assisted reproductive technology, and improve economic benefits and the efficiency of reproduction of yak. which achieved through regulate and control the expression of HIF-1α, HSP70 and Ref-1.efficiency of reproduction of yak,

高寒物种可调节相关因子使自身生理环境平衡，这些因子可与哺乳动物卵母细胞成熟和胚胎发育分泌的多种细胞因子结合，调控自身的发育。本项目拟以高原牦牛为研究对象，通过基因芯片和实时荧光定量PCR检测牦牛卵母细胞、附植前体内胚胎、体外受精胚胎、核移植胚胎发育各阶段HIF-1α、HSP70及Ref-1的表达差异，筛选相关调控因子；添加相关因子或应用RNAi 技术分别诱导或抑制 HIF-1α、HSP70或Ref-1的表达，分析与发育相关促生长因子、凋亡蛋白的调控作用，评估卵母细胞成熟及胚胎的发育质量；构建卵母细胞成熟和胚胎生长的相似高寒环境，分析高寒调节因子的表达差异及与胚胎发育之间的关系，阐明其对高寒环境适应性及三种因子之间的作用关系。本项目有助于通过调控高寒因子信号通路克服高寒环境的影响，改善牦牛辅助繁殖技术，提高牦牛繁殖效率和经济利益。

牦牛作为青藏高原地区重要物种资源，优化其辅助繁殖技术，提高体外生产牦牛胚胎的质量与繁殖性能对我国青藏高原地区畜牧业的发展至关重要。为了阐明牦牛高寒环境相关因子参与牦牛生殖生理及早期胚胎发育的调控机制，试图通过生长因子改善牦牛卵母细胞和胚胎冷冻技术及体外发育能力。本项目通过分析牦牛高寒环境相关基因特征，检测其在不同繁殖周期生殖系统、早期胚胎中的动态表达。通过相关生长因子的作用，分析早期胚胎发育过程中其对高寒环境相关基因的调控及与胚胎发育能力的关联性，同时分析其对生殖相关体细胞的作用及信号通路，利用生长因子作用提高牦牛胚胎对高寒环境应激的适应性。主要研究成果为：（1）牦牛高寒环境相关调控基因在进化中具有高度保守性，同时也存在种属特异性，与其生长环境特性相关；（2）牦牛高寒环境相关调控基因在繁殖周期主要生殖器官和不同时期胚胎中的表达情况分析，推测出其参与了母牦牛妊娠的调控，同时在牦牛胚胎发育中起着重要作用；（3）生长因子IGF-I在牦牛成熟卵母细胞CT玻璃化冷冻及解冻后对牦牛胚胎发育产生一定影响，通过解冻后调控凋亡相关基因Bax、Bcl-2、CIRP以及AQP3基因和蛋白，以此提高牦牛IVF胚胎发育能力；（4）EGF调控牦牛胚胎Cx43表达水平，提高胚胎相邻细胞间连接与交流，同时调控HSP90基因，以适应体外培养应激环境；（5）生长因子及细胞因子缺乏可引起细胞凋亡及应激反应相关基因的表达增加，添加CSF2在牦牛体外胚胎培养体系中，可部分缓解由这种体外培养缺陷造成的压力；（6）HSP90的表达水平与冷冻后的卵母细胞和胚胎发育能力紧密相关；（7）从分离纯化培养的牦牛子宫内膜上皮细胞上对激素-生长因子-粘附因子调节系统进行研究，并阐明雌二醇及孕酮与EGF和IGF-1与MUC1和PGF之间的表达量诱导关系。研究成果为通过相关生理学调控提高牦牛等高寒环境哺乳动物胚胎体外发育提供了新的科学突破点，改善了牦牛辅助繁殖技术，有助于牦牛种质资源的保护和改良等相关技术的改进。