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

To elucidate the mechanism by which phosphate induces developmental inhibit ion of rat 2-cell embryos, we examined the mutual effects of glucose and ot her glycolytic and non-glycolytic sugars, the non-metabolizable glucose ana logue, and glycolytic inhibitors on the inhibitory effect of phosphate. In the absence of glucose, 30-49% of embryos treated with 10-500 muM phosphate were able to develop to morula and blastocysts. On the other hand, in the presence of 5 mM glucose, 10 muM phosphate decreased the developmental rate of 2-cell embryos to the 4-cell stage and completely inhibited the develop ment beyond the 4-cell stage. In contrast, glucose showed no influence on d evelopment in phosphate-free medium. Similarly to glucose, the other glycol ytic sugars fructose (5 mM) and mannose (5 mM) enhanced the inhibitory effe ct of 10 muM 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 c ycle progression of 2-cell embryos cultured in glucose-free (Glc(-)) medium with 10 muM phosphate. In contrast, in the absence of phosphate, 2DGlc at the same concentration allowed 55% of 2-cell embryos to develop to morula a nd blastocyst stages. Addition of an inhibitor of enolase in glycolysis, so dium fluoride (NaF), at 1 mM to the Glc(-) medium also enhanced the inhibit ory effects of 10 muM phosphate, whereas 1 mM NaF in the absence of phospha te showed no inhibitory effects on the development of 2-cell embryos to mor ula 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. J. Exp. Zool. 287:503-509, 2000. (C) 2000 Wil ey-Liss, Inc.