EGG MATURATION: HORMONE RECEPTOR SITES AND UPTAKE

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This project aims to investigate the early interactions between a defined hormone (l-methyladenine = 1-MA) and its target cell (starfish oocyte) that result in dramatic changes of the cytoskeleton and stimulate cell division. It is known that l-MA initially binds at the cell-surface of the oocyte and is transduced to they cytoplasma where is stimulates the completion of maturation divisions. This hormone-oocyte interaction, therefore, represents an important model system for studying hormone-cell surface interactions in general, as well as similar hormone-oocyte interactions in vertebrates including human, and the regulation of cell division. This project represents one of the Principal Investigator's long-term objectives: elucidation of the mechanisms of cell division. Experiments will test specific hypotheses concerning the mophological distribution of l-MA receptor sites on the oocyte surface and the cellular mechanism of l-MA uptake. They will employ molecular probes and markers in a set of morphological analyses. Tritiated-1-MA will be used to localize the initial binding sites of l-MA and reveal its intracellular pathway by autoradiography and light and electron microscopy. Fluid-content markers will be used to explore the existence, pathway and kinetics of endocytosis as the mechanism of hormone uptake in oocytes before and after application of l-MA; these agents include a fluorescent market (Lucifer Yellow CH) and a cytochemical marker (horseradish peroxidase) for microscopic analyses, and will be complemented by kinetic analyses of tritiated-insulin uptake using scintillation counting. These studies will reveal th role of receptor-mediated endocytosis in hormone-oocyte interations. Normal processes will be manipulated with two kinds of inhibitory drugs: antagonists of oocyte maturation and inhibitors of cytoskeletal organization and function. These manipulations will test the roles of the actin- and tubulin-based cytoskeletons in hormone transductiion and will explore the subcellular actions of known antagonists of oocyte maturation.