Fluorometric analysis of endocytosis and lysosomal proteolysis in the rat visceral yolk sac during whole embryo culture

Using spectrofluorimetry and fluo- rescence microscopy, we analyzed the uptake and degradation of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-albumin) by the rat visceral yolk sac (VYS) during whole embryo culture. Rat concep- tuses exposed continuously to FITC-albumin had linear increases of both acid-soluble and acid-insoluble FITC fluorescence in the VYS. Smaller amounts of FITC fluorescence that were nearly all acid soluble accumu- lated in the extraembryonic fluid, while the embryo proper did not accumulate a significant amount of fluorescence. During a chase period following a pulse exposure to FITC albumin, FITC fluorescence in the VYS decreased linearly, while that in the extraembryonic fluid and culture medium increased. Addition of protein- ase inhibitors to the culture medium together with FITC-albumin increased acid-insoluble FITC-fluores- cence in the VYS tissue but decreased acid-soluble fluorescent degradation products in the yolk sac, extraembryonic fluid, and the culture medium. Fluores- cence microscopy of yolk sacs exposed to FITC- albumin revealed that the fluorescence was localized in apical vacuoles of the yolk sac epithelium and de- creased substantially during a chase period. In concep- tuses exposed to proteinase inhibitors, the yolk sac epithelium had enlarged vacuoles containing FITC- fluorescence whose clearance in pulse-chase experi- ments was effectively blocked. Overall, these data suggest that FITC-albumin resembles 125 I-albumin in its processing by the VYS and that the fluorescent protein is an attractive alternative tracer molecule for studies of the effects of embryotoxicants on yolk sac function during whole embryo culture. Teratology 56:201-209, 1997. r 1997 Wiley-Liss, Inc.