Purine Nucleotide Synthesis in Cultured Rat Embryos Undergoing Organogenesis

The culture of rat embryos undergoing organogenesis (9.5 to 11.5 days gestation) is widely used as a technique for evaluating potentially teratogenic agents and for the study of organ differentiation (1). During culture the embryo develops from a relatively undifferentiated neurula stage to almost the tail bud stage with 24 or 25 somites and the segregation of tissues into the primordia of the neural, cardiac circulatory and hepatic organs. These changes are associated with rapid growth of the surrounding yolk sac. Total cell number increases approximately two orders of magnitude, the embryo containing some ≅ 3 x 106 cells and the yolk sac ≅ 0.7 × 106 cells at 11.5 days gestation (2). Relatively little basic biochemical and physiological data are available however concerning this experimental system. The pre-implantation mouse embryo contain high levels of activity of both purine salvage enzymes, hypoxanthine and adenine phosphoribosyl-transferases and this activity increases in parallel with post-implantation embryonic development (3). In these studies we have shown that (i) de novo synthesis is the sole source of the purine nucleotides required for in vitro rat embryonic growth during organogenesis (ii) the presence of high levels of activity of purine catabolic enzymes in the homologous serum essential for culture prohibits the salvage of purines (iii) the degradation of culture medium serum protein by the yolk sac is the major source of amino acids for embryonic growth and development (iv) while the 3-carbon atom of serine is the major source of one carbon units for purine ring synthesis there is a significant contribution from the 2-ring carbon atom of tryptophan. No one-carbon units are derived from glycine, histidine or choline (v) there is a high level of GTP in both the embryo and its yolk sac which is reflected in the very low ATP/GTP ratios.