The broad long-term objective is to discover more about the cellular and molecular mechanisms of nutrient supply to the rat embryo and fetus in mid- and late-gestation with the ultimate aim of identifying nutrient supply routes that are particularly vulnerable to perturbations leading to congenital defects and/or intra-uterine growth retardation. The focus of the Application is the delivery of amino acids and iron, and the Specific Aims are: 1) To determine the cellular mechanisms of amino acid supply to the rat embryo at the stage of development after implantation but before its envelopment by the visceral yolk sac; 2) To determine the relative importance of proteins and free amino acids in supplying amino acids to the mid- to late-gestation rat fetus; 3) To determine the respective contributions of the placenta and visceral yolk sac in supplying amino acids to the mid- to late-gestation rat fetus; 4) To study the endocytosis of transferrin by the rat visceral yolk sac, its role in mediating the delivery of iron to the embryo, and the effect of inhibitors of pinocytosis on iron supply to the embryo. It has previously been shown that the amino acid nutrition of the 9.5- to 11.5- day rat embryo does not derive from the free amino acid content of the maternal plasma, but from the degradation of pinocytosed protein in the cells of the visceral yolk sac. Also that several agents that induce congenital defects when administered to the mother at this stage of development inhibit either pinocytosis or intralysosomal proteolysis, and thus interrupt amino acid supply to the embryo. The experiments proposed in this Application seek to determine (a) whether pinocytosis and lysosomal catabolism of maternal protein is a quantitatively important source of amino acids for the embryo/fetus at earlier and later stages of gestation than that previously investigated, and (b) whether other important nutrients (such as iron) are supplied by this route. The techniques to be used will include embryo culture, for the early stages, and in vivo methods for later stages. A variety of radioisotope techniques will be deployed throughout, and both biochemical and cytochemical methods will be used for the studies on yolk-sac handling of transferrin. The studies proposed are of direct relevance to human medicine. At the early stages of gestation the yolk sac is a prominent structure in the human conceptus, and the meager data available on this tissue indicate that its cells are morphologically similar to those of the rat yolk sac and may therefore play a similar role. Although the human yolk sac regresses at 10 to 11 weeks of gestation, the questions we seek to answer concerning the possible contribution of plasma protein to the amino acid nutrition of the mid- to near-term fetus are pertinent to the human. It has long been assumed that maternal free amino acid is the only significant source of amino acids for the fetus. Testing this assumption is overdue.
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