Involvement of glycolytic metabolism in developmental inhibition of rat two-cell embryos by phosphate

To elucidate the mechanism by which phosphate induces developmental inhibition of rat 2-cell embryos, we examined the mutual effects of glucose and other glycolytic and non-glycolytic sugars, the non-metabolizable glucose analogue, and glycolytic inhibitors on the inhibitory effect of phosphate. In the absence of glucose, 30-49% of embryos treated with 10-500 microM phosphate were able to develop to morula and blastocysts. On the other hand, in the presence of 5 mM glucose, 10 microM phosphate decreased the developmental rate of 2-cell embryos to the 4-cell stage and completely inhibited the development beyond the 4-cell stage. In contrast, glucose showed no influence on development in phosphate-free medium. Similarly to glucose, the other glycolytic sugars fructose (5 mM) and mannose (5 mM) enhanced the inhibitory effect of 10 microM phosphate but had no influence in the absence of phosphate. In contrast, the non-glycolytic sugar and non-metabolizable glucose analogue N-acetylglucosamine and 3-O-methylglucose (3-O-MGlc), respectively, did not enhance the effects of phosphate. 2-Deoxyglucose (2DGlc), another glucose analogue that is non-metabolizable but is converted by hexokinase to 2DGlc 6-phosphate, at concentrations as low as 0.1 mM completely inhibited cell cycle progression of 2-cell embryos cultured in glucose-free (Glc(-)) medium with 10 microM phosphate. In contrast, in the absence of phosphate, 2DGlc at the same concentration allowed 55% of 2-cell embryos to develop to morula and blastocyst stages. Addition of an inhibitor of enolase in glycolysis, sodium fluoride (NaF), at 1 mM to the Glc(-) medium also enhanced the inhibitory effects of 10 microM phosphate, whereas 1 mM NaF in the absence of phosphate showed no inhibitory effects on the development of 2-cell embryos to morula and blastocyst stages. From these results, disturbance of glycolysis is a critical reason for the developmental inhibition caused by phosphate in early rat embryos in culture.