In preliminary studies, a markedly different apparent rate of LH and hCG metabolism within cells of the pseudopregnant rat ovary was observed. Since LH and hCG have markedly different plasma half lives and metabolic clearance rates, in vitro studies using cultured rat granulosa cells need be udertaken. The studies in this proposal are designed to better define the subcellular metabolism of hCG, LH and hybrid molecules of complementary subunits of LH and hCG. The differential effects of LH and hCG on receptor turnover and internalization will also be examined. Internalized hormone will be examined by a variety of techniques including sucrose gradient ultracentrifugation, immunoreactivity, bioactivity, gel filtration, receptor binding and binding to concanavalin A. To better understand if the level of glycosalation of the two gonadotropins is responsible for potential observed differences, hybrid LH/hCG molecules will be studied in the in vitro granulosa culture system. Subcellular sites to which gonadotropin associates following internalization will be determined by classical subcellular fractionation techniques. Membrane fractions will be identified by specific enzymes associated with subcellular organelles. In separate but related studies ovarian LH/hCG receptor turnover rates will be examined using a density shift technique. Ovarian cells will be exposed to amino acids enriched in the dense isotopes 2H, 13C, 15H. Newly synthesized receptor will be """"""""heavier"""""""" than pre-existing receptor binding sites. Scatchard analysis of normal and dense receptor will be performed to assure that the dense amino acids do not alter receptor affinity for gonadotropin. Using the density shift technique, the differential rates of receptor biogenesis and down regulation will be examined in response to LH and hCG since those two hormones appear to have markedly different capacities for inducing down regulation. Moreover, this technique will permit assessment of differential rates of receptor synthesis and catabolism in response to hormonal interaction.
