Analysis of In Vitro Developmental Block of Rat Embryos: Assessment from the View Point of Oxygen Toxicity.

We have already reported that the block to development in cultured 1-cell rat embryos was overcome using HECM-1, a protein-free chemically defined medium without glucose nor phosphate. In addition, we showed the beneficial effect of low oxygen tension in rat embryos. In this study, in order to clarify the reason why in vitro developmental block of rat embryos is over-come in HECM-1, we examined the effects of phosphate, glucose and L-cysteine on the in Vitro development of rat embryos. Furthermore, we examined their effects on the production of H2O2 in embryos by the fluorimetric method. Addition of KH2PO4 even at only 1 μM completely inhibited the development of rat (Wistar) embryos either from the 1-cell stage or the 2-cell stage. Addition of glucose at the concentration of 1000 μg/ml significantly decreased the blastulation rate. The pro-duction of H2O2 in embryos cultured under 5% O2 was significantly (P<0.01) lower than that of embryos cultured under 20% O2 in HECM-1. H2O2 production in embryos cultured in mKRB was significantly (P<0.01) higher than that in embryos cultured in HECM-1, irrespective of the oxygen tension. Addition of neither phosphate nor glucose to HECM-1 affected the H2O2 production at any concentration examined. L-cysteine significantly decreased the H2O2 production in embryos in mKRB, but developmental block could not be overcome by addition of L-cysteine to mKRB. These results revealed that glucose and phosphate independently inhibited the development of rat em-bryos in vitro, and suggest that the mechanism of in vitro developmental block of rat embryos could not be explained simply in terms of oxygen toxicity.