The investigation of some nutritional requirements of rat embryos undergoing organogenesis in vitro
Repeated culture of rat conceptuses of between 101/2 and 111/2 days' gestation in the same sample of rat serum showed that the serum has a finite ability to support embryonic growth and development. After four consecutive cultures of rat conceptuses the serum became exhausted and analysis of it showed that rat conceptuses utilized glucose and certain proteins present within the serum. It was also concluded that rat conceptuses secreted toxic dialysable and non-dialysable components into the medium during culture and these materials affected embryonic growth during subsequent culture of conceptuses in exhausted serum. The nutritional value of free amino acids as a substitute for serum proteins was studied by the addition of leupeptin - a specific lysosomal enzyme inhibitor - to the culture serum. The effect of this was partially reversed if free amino acids were included in the leupeptin-containing serum. The inclusion of both essential and non-essential amino acids in the culture medium was found to be necessary for embryonic growth. A model system for further studies of histiotrophic nutrition (the giant yolk sac) was devised by simple modification of New's culture technique to beyond the usual 48 hours thus allowing for maximum growth of the extra-embryonic membranes in vitro. After 7 extra days' culture in rat serum the giant yolk sac was 2 cm. in diameter and contained approximately 600 micro1 yolk sac fluid. The morphology, junctional permeability, histochemistry and endocytic activity of the giant yolk sac showed this system was, in general, similar to the in vivo yolk sac of a comparable gestational age. The giant yolk sac transported radio-labelled free amino acids which accumulated in the yolk sac fluid and analysis of this fluid showed it to contain 14 major proteins; some of these contained radio-labelled amino acids and were, therefore, synthesised by the giant yolk sac. Finally, giant yolk sac fluid was able to improve the nutritional status of exhausted serum and, therefore, contained components which are vital to normal embryonic growth and development. The major feature of the giant yolk sac system is its capacity to produce the products of histiotrophic nutrition in large quantities for biochemical analysis.