Fluorometric analysis of endocytosis and lysosomal proteolysis in the rat 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) during whole embryo culture. Rat conceptuses 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 accumulated 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 proteinase inhibitors to the culture medium together with FITC-albumin increased acid-insoluble FITC-fluorescence in the VYS tissue but decreased acid-soluble fluorescent degradation products in the yolk sac, extraembryonic fluid, and the culture medium. Fluorescence microscopy of yolk sacs exposed to FITC-albumin revealed that the fluorescence was localized in apical vacuoles of the yolk sac epithelium and decreased substantially during a chase period. In conceptuses exposed to proteinase inhibitors, the yolk sac epithelium had enlarged vacuoles containing FITC-fluorescence whose clearance in pulse-chase experiments was effectively blocked. Overall, these data suggest that FITC-albumin resembles 125l-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.
Ambroso
J L
JL
Department of Environmental and Industrial Health, University of Michigan, Ann Arbor 48109, USA.
Larsen
S V
SV
Brabec
R K
RK
Harris
C
C
D000818:Animals; D018929:Cell Culture Techniques; D004622:Embryo, Mammalian / Q000187:drug effects / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D010450:Endopeptidases / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D015017:Yolk Sac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007976:Leupeptins; C039217:fluorescein isothiocyanate bovine serum albumin; D012710:Serum Albumin, Bovine; D010450:Endopeptidases; D016650:Fluorescein-5-isothiocyanate; C032854:leupeptin