HIF-1α在氧分压调节牦牛体外受精胚胎发育中的作用及其MAPK信号调控机制

Yak's survival environment is characterized by high altitude and hypoxia. Under the influence of severe environment, yaks have low reproduction efficiency. There is a prominent problem of low blastocyst rate and embryo quality about the techniques of embryo production in vitro such as IVF, SCNT. Considering the feature of yak's living environment and referring to the successful experiences of improving the embryo development potential by low oxygen environment in some animals and human, we designed the present project. In this project, the developmental competence of yak's in vitro fertilized embryos cultured under different oxygen tension would be detect to provide reference for selecting the best oxygen tension for yak's embryo development in vitro and optimizing the in vitro culturing system of yak's embryo. On the other hand, in the present project, the expression of HIF-1αwould be detected in yak's embryos came from low oxygen cultural condition, the developmental competence of embryos in which the expression of HIF-1αbe up-regulated or down-regulated would be inspected, the relationship between the expression of HIF-1αand the factors about the MAPK pathway such as ERK, JNK, P38 would be analyzed. These researches would help us to verified the roles and cellular signal transduction pathway of HIF-1αduring the process of yak's preimplantation embryos response to hypoxia. It also laid a theoretical foundation for us to reveal the molecular mechanism of yak's embryo tolerates to hypoxia.

牦牛的生存环境以高海拔低氧为显著特征。受恶劣环境的影响，牦牛自然繁殖效率低下。体外受精、体细胞核移植等胚胎体外生产技术在牦牛上也存在着囊胚形成率较低、胚胎质量不高的突出问题。考虑到牦牛生产环境的特点，参照其他动物和人上低氧环境提高体外培养胚胎发育潜能的经验，本项目在体外培养的基础上，检测不同氧分压条件下牦牛体外受精胚胎的发育潜能，为筛选适宜于牦牛体外生产胚胎培养的最佳氧分压条件，优化设计牦牛胚胎体外培养体系提供参考依据；采用免疫荧光和qRT-PCR技术检测低氧环境下培养的牦牛胚胎中HIF-1α的表达，观测上调和下调HIF-1α对体外培养牦牛胚胎发育能力的影响，分析HIF-1α与ERK、JNK和P38等MAPK信号相关因子之间的关系，有望探明牦牛胚胎发育早期HIF-1α在低氧适应中的作用及其调控的信号转导通路。为揭示牦牛胚胎发育早期低氧适应的分子机制奠定理论基础。

牦牛是生活在青藏高原高寒牧区的重要家畜资源。受恶劣环境的影响，牦牛自然繁殖效率低下，体外受精、细胞核移植等胚胎体外生产技术很不成熟。本项目采用细胞培养、免疫荧光、qRT-PCR和WB技术，阐释了氧分压调节牦牛体外受精胚胎发育的生理机制。取得了如下研究结果：（1） 成功克隆了牦牛MAPK家族的p38MAPK、ERK1、ERK2、 JNK1和JNK2等主要成员基因完整CDS区的mRNA序列，并在GenBank 中进行了登记。（2）研究发现牦牛MAPK家族主要成员基因在生物进化过程中体现出较高的保守性，其基因和蛋白质在牦牛主要生殖器官中呈现出明显的时空差异性表达特征，其表达量与主要生殖器官在发情周期中的功能活动密切相关。（3）在牦牛卵母细胞体外成熟过程中，5 %的氧气浓度可以促进牦牛卵母细胞的成熟，并可显著提高后续孤雌激活胚胎的发育能力；同时，低氧浓度可能会改变与卵母细胞代谢和发育能力相关基因及其蛋白的表达。（4）牦牛卵母细胞体外成熟过程中，5 %氧气浓度可能通过HIF-1α介导VEGF的表达抑制了COCs的细胞凋亡，促进了卵母细胞的发育。（5）在牦牛体外受精胚胎发育过程中，5 %的氧气浓度降低了胚胎的卵裂率，但显著提高了胚胎的囊胚率，增强了附植前牦牛胚胎的发育能力并抑制了其细胞凋亡。（6）5%氧气浓度下HIF-1α蛋白在囊胚中转入细胞核表达，推测5 %氧气浓度可能通过p38 MAPK基因调控HIF-1α基因的表达，提高了囊胚率，进一步改善了附植前胚胎的发育。. 研究结果为筛选适宜于牦牛体外生产胚胎培养的最佳氧分压条件，优化设计牦牛胚胎体外培养体系提供参考依据；也为进一步完全揭示牦牛胚胎发育早期低氧适应的分子机制奠定理论基础。