The aim of the proposed research is to study the mechanisms by which developing amphibian oocytes modulate their rate of yolk protein (vitellogenin) endocytosis. Dramatic changes in the capacity for vitellogenin internalization occur at specific times during oocyte growth and maturation, and these transitions are under hormonal control. This system is therefore ideally suited to the examination of the physiological control of receptor-mediated endocytosis, a process of central importance in mediating a eukaryotic cell's interaction with its environment. The number and distribution of vitellogenin receptors will be examined by studying the binding of radiolabelled ligands to oocytes at different phases of their developmental sequence (previtellogenic, vitellogenic, and post-vitellogenic oocytes). The distribution of binding activity during subcellular fractionation will yield information regarding the behavior of receptors in actively endocytic vs. quiescent phases. The effect of gonadotropins (which enhance vitellogenin accumulation over 1-3 days in vivo), insulin (stimulating yolk accumulation in vitro), and progesteron (which inhibits the process within 8-9 hours in vitro) will be examined. The intracellular pathway taken by the ligand en route to yolk platelets will be studied by combining subcellular fractionation and ultrastructural localization with EM tracers, and compared to the pathway taken by insulin. Monoclonal antibodies will be prepared against the receptor, using membrane fractions with high specific binding activity (e.g., plasma membranes) as immunogens. The receptor will be purified by adsorbtion to either the monoclonal antibodies or the ligand coupled to Sepharose, and polyclonal antibodies also prepared. Anti-receptor antibodies will then be used for further analysis of receptor behavior during endocytosis, 1) to confirm the receptor distribution inferred from binding studies, 2) to co-localize the ligand and receptor using immunofluorescence and immunoelectron microscopy, and 3) as one means for determining rates of receptor turnover, by metabolic labelling coupled with immunoprecipitation of the receptor. The technique of density-shift after microinjection of heavy amino acids into cultured oocytes will be used to study modulations in the rates of receptor synthesis and degradation in oocytes (plus/minus) hormones.
