College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an City, P. R. China.
Department of Reproductive Medicine, Affiliated Hospital of Qingdao University, Qingdao, Shandong Province, P. R. China.
J Cell Physiol. 2018 Sep;233(9):6952-6964. doi: 10.1002/jcp.26484. Epub 2018 Mar 7.
In previous studies on glucose metabolism during in vitro maturation, intact cumulus-oocyte complexes (COCs) were treated with enzyme inhibitors/activators. Because inhibitors/activators may have non-specificity and/or toxicity, and culture of COCs cannot differentiate whether glucose metabolism of cumulus cells (CCs) or that of the oocyte supports oocyte maturation, results from the previous studies must be verified by silencing genes in either CCs or cumulus-denuded oocytes (DOs). In this study, RNAi was adopted to specify the effects of glucose metabolism in CCs or DOs on oocyte maturation. Although silencing either glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or glucose-6-phosphate dehydrogenase (G6PD) genes in CCs significantly decreased competence of the cocultured DOs, silencing G6PD impaired competence to a greater extent. While silencing G6PD or GAPDH of CCs decreased glutathione and ATP contents of cocultured DOs to similar extents, silencing G6PD increased oxidative stress as well. Analysis on metabolite contents and oxidative stress index and culture of DOs in medium conditioned with gene-silenced CCs indicated that CCs supported oocyte maturation by releasing glucose metabolites. Silencing mitochondrial pyruvate carrier 1 or NADH dehydrogenase (ubiquintone) flavoprotein 1 of DOs significantly impaired their maturation. The results have unequivocally confirmed that CCs promote oocyte maturation by releasing glucose metabolites from both pentose phosphate pathway (PPP) and glycolysis. Pyruvate is transferred into DOs by mitochondrial pyruvate carrier (MPC) and utilized through mitochondrial electron transport to support maturation.
在以往关于体外成熟过程中葡萄糖代谢的研究中，完整的卵丘-卵母细胞复合物（COC）用酶抑制剂/激活剂处理。由于抑制剂/激活剂可能具有非特异性和/或毒性，并且 COC 的培养不能区分卵丘细胞（CC）或卵母细胞的葡萄糖代谢是否支持卵母细胞成熟，因此以前的研究结果必须通过沉默 CC 或去卵丘卵母细胞（DO）中的基因来验证。在这项研究中，采用 RNAi 来确定 CC 或 DO 中的葡萄糖代谢对卵母细胞成熟的影响。尽管沉默 CC 中的甘油醛 3-磷酸脱氢酶（GAPDH）或葡萄糖-6-磷酸脱氢酶（G6PD）基因显著降低了共培养的 DO 的能力，但沉默 G6PD 会更大程度地损害其能力。虽然沉默 CC 中的 G6PD 或 GAPDH 会以相似的程度降低共培养的 DO 的谷胱甘肽和 ATP 含量，但沉默 G6PD 会增加氧化应激。对用基因沉默的 CC 处理的培养基中 DO 的代谢物含量和氧化应激指数以及 DO 的培养分析表明，CC 通过释放葡萄糖代谢物来支持卵母细胞成熟。沉默 DO 中的线粒体丙酮酸载体 1 或 NADH 脱氢酶（泛醌）黄素蛋白 1 会显著损害其成熟。这些结果明确证实，CC 通过从戊糖磷酸途径（PPP）和糖酵解释放葡萄糖代谢物来促进卵母细胞成熟。丙酮酸通过线粒体丙酮酸载体（MPC）转移到 DO 中，并通过线粒体电子传递被利用来支持成熟。
