The avian oocyte is a single cell which becomes the yolk of an egg after accumulating large amounts of nutrients from the blood plasma. The uptake of riboflavin by the oocyte is absolutely dependent upon a specific riboflavin-binding protein. Ojective of the proposed research is to understand the mechanism by which trace nutrients are distributed to different tissues with the ovarian follicle of a laying hen as the model system and riboflavin as the specific example. The working hypothesis is that the transport in many cases is mediated by specific nutrient-binding proteins, synthesized in the liver, transported by the blood stream, and taken up by tissues which recognize specific structural features (e.g. attached oligosaccharide or phosphate of the nutrient-binding protein. Egg riboflavin-binding protein is 14% carbohydrate and contains 8 phosphate residues. Both carbohydrate and phosphate have been implicated in the recognition and transport of this phosphoglycoprotein into developing oocytes. The oligosaccharide moieties will be isolated and their structure determined by a combination of chemical and physical methods. Among the latter will be two-dimensional, J-resolved, proton NMR spectroscopy. The sites of phosphate attachment will also be characterized. An in vitro system to measure protein binding to oocyte plasma membranes will be developed and the isolation and characterization of a specific membrane receptor will be attempted. Specific modification of oligosaccharide structures and phosphates will be correlated with their effect on [125I]-riboflavin-binding protein function in vivo and in vitro. The long range goal of this work is to relate the biochemical processes of vitamin metabolism to empirically-determined nutritional requirements for vitamins. In particular this work may have direct importance to fetal nutrition. The plasma of pregnant mammals contain a riboflavin-binding protein homologous to that in laying hens. In analogy to the avian system, this protein is required for fetal development. Thus an understanding of the process of riboflavin transport into chicken oocytes may have applications for improving fetal nutrition or inhibiting fetal growth.
