In humans the vast majority of maternal-fetal exchanges of nutrients, toxins or harmful organisms is mediated by the placenta. It is therefore of great importance to our understanding of fetal and neonatal nutritional and health problems to ascertain the mechanisms by which the placental trophoblast selectively absorbs substances from the maternal blood. The experiments encompassed in this proposal will examine various structural and functional aspects of placental trophoblast. An important feature of placental development and function is the differentiation of cytotrophoblast into syncytiotrophoblast, accompanied by the process of cell fusion. At present, little is known about the regulation of this critical, but unusual, transition. In the first specific aim, we will take advantage of our ability to isolate a pure cytotrophoblast population and examine the regulation of trophoblast differentiation in vitro, employing a variety of factors known to affect differentiation in other systems, particularly those few where cell fusion occurs. These agents include EGF, insulin, IGF's, prostaglandins, conditioned media from cultures of placental fibroblasts and macrophages, drugs which perturb the cytoskeleton, and extracellular matrix components. We will determine the extent of syncytium formation by a variety of morphological and biochemical techniques. As the trophoblast undergoes differentiation we will examine changes in the distribution and synthesis of two important proteins expressed on the cell surface, transferrin receptor and EGF receptor, and changes in two important cytoskeletal proteins, cytokeratin and desmoplakin. The latter are of particular interest in a cell system undergoing fusion since rearrangement of the cytoskeleton and disappearance of cell junctions would accompany this transition.
Specific aim two will extend our earlier studies of iron metabolism by trophoblast cells. We will examine the transferrin-mediated uptake of iron and the release of iron at different stages of differentiation. Other studies will examine developmental changes in the regulation of transferrin receptor synthesis and distribution. Related studies will define the role of ferritin in trophoblast iron metabolism by examining its uptake, biosynthesis and release. The third specific aim is to demonstrate the distribution of acidic intracellular compartments in placental villi and in isolated trophoblast cells. Acidic intracellular compartments are important locations of receptor-ligand dissociation and sorting and are also the site of iron dissociation from transferrin. Together these aims should result in significant new information on trophoblast differentiation and function.
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