Glucose metabolism during in vitro maturation of mouse oocytes: An study using RNA interference

Abstract

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.

Effects of glucose metabolism pathways on nuclear and cytoplasmic maturation of pig oocytes

“…For example, by culturing COCs with enzyme inhibitors, Xie et al 26 observed that while glycolysis promoted cytoplasmic maturation mainly by supplying energy, PPP facilitated cytoplasmic maturation to a greater extent through both reducing oxidative stress and supplying energy via providing glycolysis with fructose-6-phosphate. Xie et al 25 showed that although silencing either G6PD or GAPDH gene in CCs significantly impaired maturation of cocultured mouse DOs, silencing G6PD decreased oocyte competence to a greater extent. While silencing either G6PD or GAPDH in CCs decreased ATP contents of cocultured DOs to a similar extent, silencing G6PD significantly increased oxidative stress as well as decreasing ATP contents.…”

“…As far as we know, this is the first report on the pathways by which pyruvate and lactate are utilized during maturation of pig oocytes. Our recent studies on mouse oocytes showed that inhibiting MCT with 4-CIN or suppressing ETC with rotenone in COCs, or knocking down MPC1 or NDUFV1 genes with RNAi in DOs significantly impaired pyruvate utilization and oocyte maturation 25,26 . However, how lactate is metabolized in DOs and how pyruvate and lactate are utilized in CCs to support oocyte maturation was not observed in previous studies.…”

“…To prepare DOs, the selected COCs were digested with 0.25% trypsin for 3 min, and then were pipetted several times to remove CCs. Procedures for mouse handling, oocyte recovery and DOs preparation are exactly those reported previously by this laboratory 25 .…”

“…While 25% pig DOs developed to MII stage (Fig. 3A), none of the mouse DOs matured 25 when cultured with glucose without CCM. When cultured with both glucose and CCM, however, the MII percentage increased to a maximum of about 70% and 95% in pig and mouse oocytes, respectively.…”

“…The DOs obtained were examined under a microscope for maturation and those showing a first polar body were considered mature. Procedures and media used for in vitro maturation of mouse oocytes were exactly those reported previously by this laboratory 25 .…”