Glucose and phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria

Abstract

While perinuclear clustering of active mitochondria, as revealed by Rhodamine 123 staining and confocal microscopy, is part of normal hamster embryo development, it is not known whether this reorganization is necessary for development. To determine if disruption of mitochondrial organization occurs in developmentally compromised embryos, the intensity of Rhodamine 123 staining was quantitated using NIH Image Software in different regions of cultured hamster 2-cell embryos exposed to either blocking (contains glucose and phosphate) or non-blocking culture conditions. Three regions within each blastomere were defined based on the organization of freshly collected embryos: cortical (ring beneath 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 staining in the intermediate region, with corresponding reduced intensity in the perinuclear region, implicating glucose and phosphate in the redistribution of mitochondria. Glucose and phosphate treatment also selectively reduced the FITC Phalloidin staining of actin microfilaments in the interior of the embryo. Neither cytochalasin D nor colchicine, at doses that blocked the second cleavage, caused redistribution of mitochondria like that seen with glucose and phosphate treatment. Additionally, cytochalasin D was unable to disrupt actin microfilaments in the perinuclear region, although it induced a "clumpy" appearance in both mitochondria and microfilaments. This report not only offers a more mechanistic explanation of the embryo 2-cell block (translocation of mitochondria involved in glucose and phosphate inhibition) but suggests that appropriate cellular organization, including the spatial positioning of the mitochondria, may be a prerequisite for normal development and that the physical organization of the embryo is susceptible to damage by exposure to culture conditions.