Ovarian follicular development is dependent on hormone (estradiol and FSH) induced changes in granulosa cell function which lead to the amplication of cAMP production, cAMP binding sites and cAMP-dependent protein phosphorylation- events presumed to be required for ovulation and luteinization. Despite the importance of estradiol and FSH action in granulosa cells, the precise target sites and molecular processes regulated by these signals remain largely unknown. One protein whose content is increased at least 5-10 fold in vivo and in vitro by estradiol and FSH (and cAMP) in granulosa cells is R-II, the regulatory subunit of type II cAMP dependent protein kinase. Furthermore, recent observations indicate that granulosa cell R-II may be an ovarian specific isozyme of a multigene family. Based on these considerations we propose to use ovarian R-II as a """"""""model"""""""" protein for analyzing the molecular aspects of estradiol and FSH action in granulosa cells. Accordingly we propose to: 1) purify and characterize the hormone-regulated R-II from rat ovaries and prepare antibodies; 2) construct a rat granulosa cell gene expression library to isolate R-II cDNA; 3) determine the mechanisms by which estradiol and FSH (cAMP) regulate the synthesis of R-II; 4) determine some of the molecular events associated with granulosa cell luteinization; 5) determine the hormonal regulation of receptors for estradiol in cultured granulosa cells. These goals will be accomplished by applying to the rat granulosa cells system tools which have been developed in molecular biology. Specifically, we will isolate poly(A)+ RNA from granulosa cells, prepare a gene expression library in Gammagtll bacteriophage, identify cDNA for R-II by antibody or oligonucleotide screening, subclone the cDNA for R-II and use it as a probe to answer questions about how estradiol and cAMP act to regulate the transcription of the ovarian specific R-II gene. Two granulosa cell culture systems have been established to analyze the actions of estradiol and FSH (and cAMP) of granulosa cell function. Short-term cultures (in serum-free medium) provide a system for analyzing the induction of R-II in differentiating cells in response to FSH and low concentrations of cAMP. The long-term cultures (in low concentrations of serum) provide a system for analyzing the mechanisms by which hCG/LH and high cAMP cause luteinization and a loss of R-II. Using these approaches, we hope to understand the molecular events associated with the synergistic regulation of granulosa cell differentiation by estradiol and FSH.
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