STIMULATION OF ENDOCYTOSIS IN MOUSE BLASTOCYSTS BY INSULIN - A QUANTITATIVE MORPHOLOGICAL ANALYSIS

The effects of insulin on the endotytic activity of mouse blastocysts in vitro were investigated using confocal laser scanning microscopy, q uantitative image analysis and electron microscopy. Confocal studies s howed that fluorescein isothiocyanate-labelled markers, dextran (fluid phase) and albumin (combined membrane and fluid phase), were endocyto sed by blastocysts and localized within vesicles (about 2.5 mu m in di ameter) in the outer trophectoderm cells. No labelling was detected in the inner cell mass cells or the blastocoel cavity. Treatment with 17 0 nmol insulin l(-1) stimulated the endocytosis of fluorescently label led dextran in freshly collected blastocysts, increasing mean vesicle diameter per embryo by 15% (P < 0.05) after incubation with insulin fo r 2.5 h and mean vesicle number per embryo by 56% (P < 0.01) after 6 h . Both effects were also evident in blastocysts that had been cultured from the late eight-cell stage. Blastocysts incubated for 6 h with in sulin displayed increased convolutions in the trophectoderm apical mem brane compared with controls, indicating increased membrane activity a nd suggesting macropinosome formation. Collectively, these results sug gest that insulin enhances endocytosis in the trophectoderm by stimula ting uptake at the apical membrane into larger and more numerous endoc ytic vesicles and with some evidence of vesicle fusion. This mechanism may provide a metabolic basis for the stimulation by insulin of biosy nthesis, proliferation and morphological development in early embryos.