Endocytosis in mouse blastocysts: characterization and quantification of the fluid phase component.

Abstract

Fluid phase endocytosis in mouse blastocysts was characterized using the fluid phase marker, 3H-dextran, which did not bind to the membrane. This nonsaturable uptake occurred via an energy-requiring process, with only 20% accountable by diffusion as indicated by analysis at 4 degrees C. Insulin stimulated uptake of 3H-dextran by 30% (P < 0.05) over the first hr. The rate of uptake then decreased in both control and insulin-treated blastocysts. However, by 2 hr, insulin-treated blastocysts contained 38% more 3H-dextran (38%; P < 0.01) than control blastocysts. Incubation of blastocysts in protein-free medium increased 3H-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 blastocyst volume/min for uptake in the presence of 0.1 g BSA/l and 10 g BSA/l, respectively. Confocal microscopic studies of fluorescently labelled dextran uptake in blastocysts, cultured in the absence of BSA, showed an increase in weak fluorescence labelling in the trophectoderm cells 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 endocytosis 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 maintain the rate of supply of protein nutrient to trophectoderm cells.(ABSTRACT TRUNCATED AT 250 WORDS)