Ambroso J L, Larsen S V, Brabec R K, Harris C
Department of Environmental and Industrial Health, University of Michigan, Ann Arbor 48109, USA.
Teratology. 1997 Sep;56(3):201-9. doi: 10.1002/(SICI)1096-9926(199709)56:3<201::AID-TERA3>3.0.CO;2-3.
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.
我们使用荧光分光光度法和荧光显微镜，分析了大鼠内脏卵黄囊（VYS）在全胚胎培养过程中对异硫氰酸荧光素标记的牛血清白蛋白（FITC-白蛋白）的摄取和降解情况。持续暴露于FITC-白蛋白的大鼠胚胎，其VYS中酸溶性和酸不溶性FITC荧光均呈线性增加。几乎全部为酸溶性的少量FITC荧光积聚在胚外液中，而胚胎本身并未积聚大量荧光。在脉冲暴露于FITC白蛋白后的追踪期内，VYS中的FITC荧光呈线性下降，而胚外液和培养基中的荧光则增加。在培养基中添加蛋白酶抑制剂与FITC-白蛋白一起，可增加VYS组织中酸不溶性FITC荧光，但会减少卵黄囊、胚外液和培养基中酸溶性荧光降解产物。对暴露于FITC-白蛋白的卵黄囊进行荧光显微镜检查发现，荧光定位于卵黄囊上皮的顶端空泡中，并且在追踪期内显著减少。在暴露于蛋白酶抑制剂的胚胎中，卵黄囊上皮有含有FITC荧光的扩大空泡，其在脉冲追踪实验中的清除被有效阻断。总体而言，这些数据表明FITC-白蛋白在VYS的处理过程中类似于125I-白蛋白，并且这种荧光蛋白是研究胚胎毒性物质对全胚胎培养过程中卵黄囊功能影响的一种有吸引力的替代示踪分子。
