不同葡萄糖代谢途径在猪卵母细胞成熟中的作用：用RNA干涉和miRNA基因沉默技术的研究

Studies on the effects of glucose metabolism on oocyte maturation are of great importance for our understanding of oocyte maturation mechanisms and for improving the in vitro maturation system and oocyte developmental potential. Although there are many reports on the effects of glucose metabolism on oocyte nuclear maturation, the pathways of glucose metabolism supporting oocyte nuclear maturation remain to be specified. Reports on the effect of glucose metabolism on ooplasmic maturation are limited. Furthermore, inhibitors or activators were usually used in previous studies to regulate enzyme activities. Because these inhibitors and activators may affect the accuracy of results due to their non-specificity and/or toxicity, new techniques for gene expression regulation of related enzymes must be used to specify more accurately the roles of different pathways of glucose metabolism in supporting oocyte maturation. In addition, studies on oocyte glucose metabolism using RNAi and researches on the role of microRNAs in oocyte glucose metabolism have not been reported. In this project, we will first formulate a maturation medium that can support nuclear maturation but only a limited preimplantation embryo development of pig oocytes without glucose. Then, pig oocytes will be cultured in this medium with glucose supplemented, the activities/expression of the enzymes involved in different pathways of glucose metabolism will be regulated by inhibitors, RNAi or microRNAs, and markers for oocyte nuclear and cytoplasmic maturation will be examined to determine the roles of different pathways of glucose metabolism in supporting maturation of the pig oocytes.

研究葡萄糖代谢在卵母细胞成熟中的作用对于了解卵母细胞成熟机理、改进成熟培养系统和提高卵子质量具有重要意义。尽管关于糖代谢对卵核成熟的作用研究很多，但究竟是哪条糖代谢通路支持卵核成熟还不完全明确。糖代谢对卵胞质成熟作用还鲜有报道。以前的研究都采用抑制剂或激活剂调控相关酶活性，因抑制/激活剂的毒性和非特异性会影响实验结果可靠性，必须用新技术调控糖代谢关键酶的基因表达，才能准确揭示不同代谢途径对卵母细胞成熟的作用。关于运用RNAi技术进行卵母细胞糖代谢研究和miRNA在卵母细胞糖代谢中的作用，还未见报道。本项目将首先建立一个在不含葡萄糖情况下能维持猪卵核成熟但不能支持胞质成熟的培养体系。然后，用此体系在添加葡萄糖的情况下培养猪卵母细胞，并通过抑制剂、RNAi或miRNA下调糖代谢不同途径关键酶的表达/活性，观察卵母细胞核和胞质成熟指标，最终确定不同糖代谢途径对猪卵母细胞成熟的作用。

体外成熟（IVM）卵母细胞的发育能力远不如体内成熟的卵母细胞。尽管有许多研究报道了葡萄糖代谢在卵母细胞核成熟中的作用，但关于葡萄糖代谢在卵母细胞胞质成熟中的作用却鲜有报道。以前的研究未能区分是卵丘细胞（CCs）的葡萄糖代谢，还是去掉卵丘的裸卵（DOs）的葡萄糖代谢维持卵母细胞的成熟。另外，关于卵母细胞糖代谢是否存在物种间差异，还知之甚少。我们的研究目标是通过使用一种在抑制葡萄糖代谢的情况下能维持核成熟但不能支持胞质成熟的特殊培养基以及酶活性抑制剂、RNA干涉基因沉默、microRNA过表达或抑制等技术，回答上述问题。研究结果证明，CCs通过磷酸戊糖通路和糖酵解通路代谢葡萄糖，释放代谢产物，促进猪卵母细胞成熟。丙酮酸和乳酸都是通过单羧酸转运蛋白进入到猪DOs中，而丙酮酸又通过线粒体丙酮酸载体蛋白进一步运送到猪DOs和CCs的线粒体内。在猪DOs和CCs中，丙酮酸通过线粒体电子传递而代谢，而乳酸则通过乳酸脱氢酶催化氧化成丙酮酸而利用。猪和小鼠DOs的葡萄糖、丙酮酸和乳酸代谢对CCs的依赖程度具有显著的种间差异。猪的DOs能够利用由乳酸代谢而来的丙酮酸，但小鼠DOs不能。本研究还建立了磷酸戊糖通路和糖酵解通路差异的CCs模型并筛选了一批参与CCs糖代谢的microRNAs。