The purpose of this project has been to examine the dynamics of hormone storage in pituitary gonadotropes. The renewal proposal will examine key factors that may influence storage patterns and GnRH receptivity in the gonadotrope population. Previous studies have shown that the stimulated gonadotrope population is mostly multihormonal and that monohormonal gonadotropes are found among the smallest cells. Our studies suggest that the gonadotrope population is fluid; perhaps the monohormonal gonadotropes are activated as needed during reproductive life. The renewal period will test this hypothesis in three phases. Phase 1 will focus on cycling female rats. A potent biotinylated analog of GnRH will be localized cytochemically on gonadotropes during key stages of the estrous cycle when changes in the number of GnRH receptors have been reported. Double, protein A gold stains for gonadotropins will then be used to confirm the identity of the GnRH target cells in order to learn if monohormonal gonadotropes are activated during early proestrus. Morphometric studies will show if changes in the number of GnRH receptors reflect changes in the number of sites bound per cell, or the number of cells bound, or both. Phase 2 will focus on the differential storage of LH and FSH in multihormonal and monohormonal gonadotropes. GnRH stimulation of multihormonal gonadotropes results in a shift in gonadotropin stores to the cell periphery coupled with a dissociation of LH and FSH storage pools. The proposed renewal will examine the effect of pulsatile exposure to GnRH with and without steroid pre-treatment. Monohormonal gonadotropes will be studied to learn if they can be stimulated by GnRH to produce the other hormone. Dual stains for gonadotropins and tubulin or calmodulin will be used to determine if changes in cytoskeletal elements accompany the mobilization of gonadotropin stores. Phase 3 of this renewal period will continue the studies of gonadectomy cells. Dual stains for biotinylated GnRH and gonadotropins will be used to examine the effect of steroids on GnRH binding, kinetics of internalization, and differential storage of gonadotropins. Techniques employed during this renewal period will include affinity cytochemistry, immunocytochemistry, radioimmunoassay (gonadotropins), electron microscopy, tissue culture, cell separation, and stereometry. The proposed renewal is designed to explore the fluidity of the gonadotrope population in order to learn more about the role of monohormonal cells and how the multihormonal cells mobilize their different storage pools to effect non-parallel release of LH and FSH.
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