ENDOCYTOSIS IN MOUSE BLASTOCYSTS - CHARACTERIZATION AND QUANTIFICATION OF THE FLUID-PHASE COMPONENT

Fluid phase endocytosis in mouse blastocysts was characterized using t he fluid phase marker, H-3-dextran, which did not bind to the membrane . This nonsaturable uptake occurred via an energy-requiring process, w ith only 20% accountable by diffusion as indicated by analysis at 4 de grees C. Insulin stimulated uptake of H-3-dextran by 30% (P < 0.05) ov er the first hr. The rate of uptake then decreased in both control and insulin-treated blastocysts. However, by 2 hr, insulin-treated blasto cysts contained 38% more H-3-dextran (38%; P < 0.01) than control blas tocysts. Incubation of blastocysts in protein-free medium increased H- 3-dextran uptake to a rate equivalent to 12% of the blastocyst volume/ min (1,500 +/- 240 pliter/hr), compared to 4.5% and 1.5% of the blasto cyst volume/min for uptake in the presence of 0.1 g BSA/l and 10 g BSA /l, respectively. Confocal microscopic studies of fluorescently labell ed dextran uptake in blastocysts, cultured in the absence of BSA, show ed an increase in weak fluorescence labelling in the trophectoderm cel ls of blastocysts, compared to blastocysts cultured in the presence of BSA. There was no diffusion of fluorescence label into the blastocoel cavity. This is consistent with fluid being endocytosed, possibly by a large number of small pinocytic vesicles. Thus fluid-phase endocytos is in blastocysts is stimulated by insulin, increasing the delivery of nutrient-containing fluid into blastocysts. In the absence of protein , embryos also increase fluid uptake, possibly in an attempt to mainta in the rate of supply of protein nutrient to trophectoderm cells. An a nalysis of the rate of protein delivery in both adsorbed and dissolved phases is presented, which reveals the potential for significant cont ributions of both phases of endocytosis to blastocyst metabolism in vi vo. (C) 1995 Wiley-Liss, Inc.