翻译起始因子eIF4E在调节小鼠早期胚胎发育中的作用及其机制研究

Successful completion of preimplantation embryonic development requires the accurate reprogramming of parental genetic information. This is tightly regulated by transcription, translation and post-translation modifications so that embryonic genome activation (EGA) is generated and followed by precise embryonic gene expression to ensure the fate of embryonic development. It's eager to insight into regulatory mechanism of the protein synthesis during different stages of early embryonic development, particularly in initiation of translation. Eukaryotic initiation factor E (eIF4E) is such a key factor controlling the rate-limiting step in more than 95% of the protein synthesis through mRNA's m7G cap-dependent mRNA translation. It is a critical element of eIF4F complex containing eIF4E (cap-binding protein), eIF4A (ATP-dependent mRNA helicase) and eIF4G (scaffold protein). Its activities are mainly regulated by its own phosphorylation at Ser209 and action of eIF4E-binding protein (4E-BP1). eIF4E activities are well described during spermatogenesis and oocytogenesis, and its transcript is detected in mouse preimplantation embryo with higher expression rate at 8-cell stage. However, its roles and regulatory mechanisms in embryonic development are not defined. We have detected different expression patterns of eIF4E and phosphorylated eiF4E Ser209 (p-eIF4E) proteins in preimplantation embryos. More eIF4E protein is detected in mouse 8-cell stage. Zygote has more p-eIF4E than oocyte, but p-eIF4E is absent from 2-cell stage embryo. Embryo culture in several common media causes a dramatic arrest of eIF4E within nucleoli and this is not affected by O2 tension. eIF4E-/- mouse is lethal but eIF4E-/- blastocyst embryo is detected. eIF4E +/-mouse is viable and fertile and produces smaller size of offspring. 4EGI-1 (eIF4E antagonist) prevents the wild-type hybrid zygote development to blastocyst stage in vitro. The aims of this project are: (1) Investigate the hypothesis that the transit action and haemostasis of eIF4E phosphorylation-dependent and non-phosphorylation -dependent (by mTor/4E-BP1) signal transduction pathways are essential and critical mechanisms to regulate preimplantation development; (2) Analyse the significance of eIF4E-/- blastocyst and establishment of eIF4E-/- embryonic stem cell line to determine the eIF4E-dependen profile of mRNA translation; (3) By investigating the effects of embryo culture on eIF4E cellular distribution and eIF4E-dependent protein synthesis, it will provide a study platform to further optimize embryo culture conditions so that the deprivation of gene expression resulting from embryo culture may be rescued and to be consistent with that in vivo. Assisted reproductive technologies (ART) are the powerful means to treat human infertility, but long-term defects are still concerned. Clear understanding of the mechanisms of translation regulation is significant to help achieve good ART outcomes.

翻译起始因子eIF4E是调节真核细胞中95%mRNA 翻译成蛋白质的关键限速因子。它通过磷酸化和非磷酸化两种形式与其他蛋白组成复合物发挥作用。我们前期研究发现，eIF4E和磷酸化eIF4E（p-eIF4E）在小鼠各期早胚中存在表达差异；体外胚胎培养改变了eIF4E的细胞内定位；敲除eIF4E基因的小鼠无纯合子子代，但有纯合子囊胚产生。本项目将系统研究：（1）从2-细胞到4-8细胞胚胎发育过程中，eIF4E非磷酸化到磷酸化转换调控的机制及意义；（2）胚胎体外培养如何改变eIF4E依赖的翻译调控及可能的抢救手段；（3）探讨纯合子囊胚的生物学意义并建立纯合子胚胎干细胞系。最终从分子生物学角度深入理解eIF4E在早期胚胎发育中的调控作用及其机制。同时，研究体外培养对eIF4E依赖性蛋白质合成的影响，为优化辅助生殖培养体系提供实验依据，以期达到胚胎体外培养其基因表达与体内的一致性。

翻译起始因子eIF4E是影响真核细胞基因翻译成蛋白的关键限速因子,体内95%的mRNA翻译与该基因有关。它通过磷酸化和非磷酸化两种形式与其他蛋白组成复合物发挥作用。有文献报道，各期早胚中eIF4E mRNA表达差异显著，8细胞期开始表达量显著增加。本项目的主要研究内容从早胚发育各阶段，系统地研究翻译活跃的2-4细胞期eIF4E的表达及通过磷酸化和非磷酸化的转换调控蛋白的翻译的调节机制，通过eIF4E KO小鼠模型研究其在早胚发育的生理作用。.我们研究发现，eIF4E和磷酸化eIF4E（p-eIF4E）在各期早胚中有显著表达差异。受精卵中p-eIF4E显著高于卵子。在2细胞期eIF4E的活性是通过mTOR/EI4EBP1信号通路调节而不是传统的eIF4E的磷酸化。eIF4E KO的纯合子在囊胚前期致死。eIF4E KO抑制生殖细胞的发育抑制，受精及胚胎的发育。进一步发现，体外受精和胚胎体外培养可以引起eIF4E细胞内的定位表达的改变，而此特征可能作为辅助生殖技术的质量检测标准，用于人类辅助生殖技术临床。