牛附植前胚胎获得耐热的MAPK信号调控机制

Heat stress disrupting animal reproduction which has been becoming more and more prominent is an increasingly hotspot of international research in nowadays. Our previous research confirmed that deleterious effects of heat shock on bovine preimplantation embryos become attenuated as development proceeds. However, neither at which stage and how the embryos gain their resistance to heat stress nor signal transmission mechanism involved in the process is clear. We hypothesize that bovine preimplantation embryos gain thermotolerance as development to advanced stage which varies genetically. In vitro derived embryos from three breeds of cow in their various preimplantation development stages (zygote to blastocyst) will be used to study the embryonic response (antioxidation, apoptosis and production of cytoprotective molecules) to heat stress and the regulating mechanism of MAPK signal transmission as well as genetic variation in acquired thermotolerance of bovine preimplantation embryos subject to elevating temperature using advanced technologies in modern developmental biology, molecular biology and cytobiology. Dynamic changes in genetic gene differential expression, the key molecules（JNK, p38 and ERK1/2）expression in the MAPK signal pathway, embryonic redox state, apoptosis and antiapoptosis equilibrium, as well as the cytoprotective molecules (HSPs and IGF-1) will be monitored in the study. The correlations between these measured indicators and embryo thermotolerance will be analyzed to reveal the genetic factors, the development stage when MAPK activates and its signal transmission mechanism in embryo developmental gained thermotolerance. The results 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凸显，成为现今国际研究热点。申请者前期研究表明，牛附植前胚胎耐热力随发育而增加，但在胚胎发育的哪个阶段、如何获得耐热及其细胞信号转导机制尚不清楚。本研究以牛附植前胚胎发育获得耐热并有遗传差异为理论假说，从附植前胚胎不同发育阶段（合子-囊胚）入手，以胚胎热激反应（抗氧化、凋亡和合成调节分子）及MAPK信号调控（信号激活及其调控）为研究切入点，以牛（3个品种）体外受精的胚胎为实验对象，利用发育生物学、分子生物学和细胞生物学等研究手段，观察热应激时胚胎遗传基因差异表达、MAPK信号通路中关键因子（JNK、p38和ERK1/2）表达、氧化还原状态、凋亡与抗凋亡平衡、调节分子（HSPs和IGF-1）的动态变化，分析其与胚胎发育获得耐热的相关性，找出胚胎发育获得耐热的遗传因素，揭示MAPK信号激活时期及其转导机制，为阐明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。

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，是夏季家畜妊娠率低的主要原因。减少热应激的影响、寻找应对措施，已成为目前重要的研究热点课题。本项目以体外培养的胚胎为实验对象，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、MAPK信号通路中关键因子表达、氧化还原状态、凋亡与抗凋亡平衡、调节分子的动态变化。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、内质网中蛋白质加工、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。体外培养胚胎发育率降低，并且GJA1，CDH1和Bcl-2基因表达下调，而HSP70，BAX和DNMT1基因表达上调。热应激能增加胚胎细胞凋亡，使Caspase-3，BAX表达量增高，BCL-2和ERK1/2信号通路中相关因子表达量降低，级联抑制ERKI/2信号通路上（Ras、Raf、MEK1和MEK2）下（c-Fos、AP-1）游因子，抑制ERK1/2信号转导通路，从而介导胚胎细胞凋亡。热应激同时也使囊胚ROS含量增多和线粒体功能损伤，降低囊胚线粒体膜电位稳定并激活细胞和线粒体自噬。黄芩苷能显著上调热应激诱导的GJA1，CDH1，BCL-2和DNMT3a基因表达、下调HSP70，BAX和DNMT1基因表达和ROS含量增多，阻止线粒体膜电位降低和功能损伤，促进ERK1/2信号通路下游c-Fos、AP-1等核转录因子蛋白磷酸化，有助于胚胎耐热。实验结果揭示了附植前胚胎耐热损伤的分子机制及其受热损伤可能的预防方法，为临床实践中避免热应激性不孕提供理论基础。