GLUCOSE AND PHOSPHATE TOXICITY IN HAMSTER PREIMPLANTATION EMBRYOS INVOLVES DISRUPTION OF CELLULAR-ORGANIZATION, INCLUDING DISTRIBUTION OF ACTIVE MITOCHONDRIA

While perinuclear clustering of active mitochondria, as revealed by Rh odamine 123 staining and confocal microscopy, is part of normal hamste r embryo development, it is not known whether this reorganization is n ecessary for development. To determine if disruption of mitochondrial organization occurs in developmentally compromised embryos, the intens ity of Rhodamine 123 staining was quantitated using NIH Image Software in different regions of cultured hamster 2-cell embryos exposed to ei ther blocking (contains glucose and phosphate) or non-blocking culture conditions. Three regions within each blastomere were defined based o n the organization of freshly collected embryos: cortical (ring beneat h plasma membrane), perinuclear, and intermediate regions. While there was no treatment effect on the total staining intensity, glucose and phosphate treated embryos had significantly higher Rhodamine 123 stain ing in the intermediate region, with corresponding reduced intensity i n the perinuclear region, implicating glucose and phosphate in the red istribution of mitochondria. Glucose and phosphate treatment also sele ctively reduced the FITC Phalloidin staining of actin microfilaments i n the interior of the embryo. Neither cytochalasin D nor colchicine, a t doses that blocked the second cleavage, caused redistribution of mit ochondria like that seen with glucose and phosphate treatment. Additio nally, cytochalasin D was unable to disrupt actin microfilaments in th e perinuclear region, although it induced a ''clumpy'' appearance in b oth the mitochondria and microfilaments. This report not only offers a more mechanistic explanation of the embryo 2-cell block (translocatio n of mitochondria involved in glucose and phosphate inhibition) but su ggests that appropriate cellular organization, including the spatial p ositioning of the mitochondria, may be a prerequisite for normal devel opment and that the physical organization of the embryo is susceptible to damage by exposure to culture conditions. (C) 1997 Wiley-Liss, Inc .