小鼠附植前胚胎耐热应激损伤的ERK1/2信号级联调控机制

Heat stress disturbing animal reproduction which has been increasingly more and more prominent is, in nowadays, becoming a hotspot in the international research. Our previous research revealed that heat stress activated ERK1/2 signal cascade transduction in mouse blastocyst. However, neither at which stage of the preimplantation embryonic development activated or whether it regulates thermotolerance of the embryo to heat stress nor the signal transduction mechanism involved in the process is clear. We hypothesize that ERK1/2 signal pathway participates in regulating or control of embryonic thermotolerance, which is influenced by both internal and external factors. In vitro derived mouse embryos in their various preimplantation development stages (2-4 cells to blastocyst) will be heat-stressed and advanced technologies in modern developmental biology, molecular biology and cytobiology will be employed to study：①developmental acquisition ability of the embryo in thermotolerance to heat stress; ②cascade activation (Ras-ERK) of ERK1/2 signal pathway; ③regulating and control of ERK1/2 signal pathway in thermotolerance of the embryo to heat-stress including antioxidation and antiapoptosis or regulating embryo itself to produce cytoprotective factors; and ④influence of maternal embryokines including colony-stimulating factor 2（CSF2）and insulin-like growth factors-1 (IGF-1) and baicalin (antipyretic and tocolysis) on the embryonic thermotolerance and their regulations to ERK1/2 signal pathway of the embryo under heat stress. The results will reveal activation stage of ERK1/2 signal pathway and its cascade regulating mechanism in thermotolerance of preimplantation mouse embryo under heat stress, which will provide a theoretic base for revealing the mechanism of heat stress disrupting animal reproduction and preventing summer infertility of dairy cows.

热应激对动物生殖影响日益突显，已成国际研究热点。申请者前期研究发现，热应激能级联激活小鼠囊胚ERK1/2信号通路，但在附植前哪个时期激活、是否调控胚胎耐热应激损伤及其机制尚不清楚。本研究拟以ERK1/2信号通路参与调控小鼠胚胎耐热损伤及其受内、外因调节为理论假说，从附植前胚胎不同发育时期入手，以胚胎耐热损伤及其受ERK1/2信号级联调控为研究切入点，利用发育生物学、分子生物学和细胞生物学等方法，研究热应激时小鼠附植前胚胎：①各发育时期获得耐热能力；②ERK1/2信号通路级联（Ras-ERK）激活；③ERK1/2信号通路调控耐热损伤（氧化和凋亡）和调节因子（HSPs）合成；④母源性胚胎因子（CSF2和IGF-1）和黄芩苷（解热安胎）对胚胎耐热的影响及ERK1/2信号通路调控。明确ERK1/2信号激活时期及其对胚胎耐热的级联调控机制，为揭示热应激影响动物生殖机理和预防夏季奶牛不孕提供理论依据。

全球气温升高严重影响哺乳动物生殖功能，热应激破坏雄性精子发生，影响雌性从卵子质量、胚胎发育到妊娠的各个环节，是夏季奶牛妊娠率低的主要原因。减少热应激的影响、寻找应对措施，已成为世界范围内的研究热点课题。本项目以体外培养的小鼠胚胎为实验对象，利用分子生物学、细胞生物学以及RNA-Seq技术等研究手段，观察热应激时胚胎遗传基因差异表达、氧化还原状态、凋亡与抗凋亡平衡、调节分子的动态变化、胚胎组织结构、线粒体分裂与生物发生、ERK1/2和PI3K/Akt/mTOR信号变化以及中药单体对胚胎的耐热机制。结果获得了正常及热应激囊胚的转录组数据，受热后胚胎有704个显著差异基因，主要参与MAPK信号通路、内质网蛋白质加工、氧化磷酸化等通路中，功能涉及调控胚胎发育、外源性细胞凋亡信号通路、呼吸链、能量代谢等过程。中等及以上程度热应激能改变胚胎超微结构，使胚胎细胞发生氧化损伤和凋亡，影响胚胎细胞线粒体质量、线粒体分裂、生物发生以及自噬，降低胚胎质量和发育率。温和热刺激激活自噬、增加线粒体分裂和线粒体自噬水平，提高胚胎质量和孵化率。在8-cell期一次温和热刺激使胚胎在后期发育过程中更耐热，并可提高其囊胚发育率及囊胚孵化率。胚胎耐热性能很可能受ERK1/2和PI3K/Akt/mTOR信号通路的双重调节。更加有意义的是，黄芩苷和白藜芦醇中药单体均可增加小鼠胚胎耐热性能，机制是其可增加受热刺激胚胎细胞的抗氧化、抗凋亡能力，并可启动一系列的线粒体功能活动。本项目实验结果揭示了小鼠附植前胚胎耐热损伤的分子机制及其可能的中药预防方法，为未来动物临床实践中避免热应激性不孕提供理论基础。