Some aspects of biochemical differentiation in the rat visceral yolk in vivo and in culture.
2015-11-19T08:50:59Z (GMT) by
By culturing 9.5 day rat egg cylinders as closed yolk sac vesicles, after surgical removal of the embryonic disc, it was shown that the cultured yolk sac retained the essential morphological characteristics of the in vivo yolk sac over an eight day culture period, and significant improvements were made in the original culture methodology, with regard to survival rate and serum requirement, by culturing in Medium 199-supplemented whole rat serum, and increasing oxygen tension over the last half of the culture period. Yolk sacs cultured in this manner exhibited the same cellular morphology as in vivo yolk sacs removed after 17.5 days of gestation, but did not show the same degree of haemopoietic development or basement membrane synthesis. However, the cultured yolk sac endodermal cells appeared to be proliferative at 17.5 days, whereas 17.5 day in vivo yolk sacs did not have the same nuclear appearance. The culture system was used to study aspects of carbohydrate metabolism and xenobiotic metabolising capability, and was used to study the expression and control of two genes - alphafetoprotein and insulin-like growth factor II, in rat visceral yolk sac. Study of hexokinase, pyruvate kinase and succinate dehydrogenase activity in cultured and in vivo yolk sacs from 9.5 days to 17.5 days of gestation, indicated a rise in the level of aerobic tricarboxylic acid cycle catabolism from 9.5 days to 13.5 days of gestation in vivo and in culture, as measured by an increase in succinate dehydrogenase activity. Concommitant with this rise in aerobic carbohydrate catabolism, there was a slight decrease in glycolytic activity as measured by hexokinase and pyruvate kinase activities in vivo and in culture. Activities of all three enzymes significantly decreased from 15.5 to 17.5 days in vivo (p 0.05), but there was no observed decrease in carbohydrate catabolism in culture. Expression of cytochrome P-450IA1 gene, measured by substrate-turnover enzyme assay and immunocytochemistry, was localised to the endodermal cells, and found to be inducible in in vivo yolk sacs from 15.5 to 18.5 days, but expressed as constitutive activity from 9.5 to 17.5 days in culture, postulated to be due to the absence of negative regulatory factors of either maternal or fetal derivation. Using in situ hybridisation techniques to qualitatively and quantitatively determine AFP and IGF-II mRNA, expression of AFP mRNA was localised to endodermal cells, whilst IGF-II mRNA was expressed in both cell lineages. Down-regulation of IGF-II mRNA was shown in neonatal liver by glucocorticoids, but these compounds did not suppress expression in the cultured yolk sac. A thousand-fold decrease in detected AFP mRNA between 16.5 days and 21.5 days of gestation in vivo was not paralleled in culture, and preliminary data indicated that this was due to primary or secondary effects of a maternally-derived transcription factor.