FLUOROMETRIC ANALYSIS OF ENDOCYTOSIS AND LYSOSOMAL PROTEOLYSIS IN THERAT VISCERAL YOLK-SAC DURING WHOLE-EMBRYO CULTURE

Using spectrofluorimetry and fluorescence microscopy, we analyzed the uptake and degradation of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-albumin) by the rat visceral yolk sac (VYS) durin g whole embryo culture. Rat conceptuses exposed continuously to FITC-a lbumin had linear increases of both acid-soluble and acid-insoluble FI TC fluorescence in the VYS. Smaller amounts of FITC fluorescence that were nearly all acid soluble accumulated in the extraembryonic fluid, while the embryo proper did not accumulate a significant amount of flu orescence. During a chase period following a pulse exposure to FITC al bumin, FITC fluorescence in the VYS decreased linearly, while that in the extraembryonic fluid and culture medium increased. Addition of pro teinase inhibitors to the culture medium together with FITC-albumin in creased acid-insoluble FITC-fluorescence in the VYS tissue but decreas ed acid-soluble fluorescent degradation products in the yolk sac, extr aembryonic fluid, and the culture medium. Fluorescence microscopy of y olk sacs exposed to FITC-albumin revealed that the fluorescence was lo calized in apical vacuoles of the yolk sac epithelium and decreased su bstantially during a chase period. In conceptuses exposed to proteinas e inhibitors, the yolk sac epithelium had enlarged vacuoles containing FITC-fluorescence whose clearance in pulse-chase experiments was effe ctively blocked. Overall, these data suggest that FITC-albumin resembl es I-125-albumin in its processing by the VYS and that the fluorescent protein is an attractive alternative tracer molecule for studies of t he effects of embryotoxicants on yolk sac function during whole embryo culture. (C) 1997 Wiley-Liss, Inc.