CHANGES IN CONTENT AND CAMP-DEPENDENT PHOSPHORYLATION OF SPECIFIC PROTEINS IN GRANULOSA-CELLS OF PRE-ANTRAL AND PREOVULATORY OVARIAN FOLLICLES AND IN CORPORA-LUTEA

  • 1 January 1983
    • journal article
    • research article
    • Vol. 258  (8) , 5227-5232
Abstract
The responsiveness of granulosa cells to the gonadotropins and cAMP increases as ovarian follicles mature. To determine if this change in response might be related to either the content or cAMP-dependent phosphorylation of specific proteins, proteins were lableled in 30,000 .times. g supernatant fractions (cytosol) with [.gamma.-32P]ATP in the presence or absence of cAMP. Using 2-dimensional gel electrophoresis, granulosa cells of preantral follicles exhibited low amounts of cAMP-dependent phosphorylation of 2 proteins with apparent MW of 54,000-56,000 and 43,000. Using [32P]8-N3cAMP and photoaffinity labeling procedures, the MW = 54,000-56,000 protein was identified as RII, the regulatory subunit of type II protein kinase. Polychromatic silver staining, as well as the photoaffinity labeling, revealed that RII exists in 3 forms, each of which was also labeled by [.gamma.-32P]ATP. Based on the relative isoelectric points and specific silver staining of highly purified actin and phosphorylated actin, the MW = 43,000 protein was provisionally identified as actin. Five proteins (MW = 37,500, 27,500, 22,500, 19,000 and 15,000), in addition to RII and actin, were phosphorylated in cytosol of granulosa cells from preovulatory follicles. By adding increasing concentrations of exogenous catalytic subunit to the cytosols, the content, as well as the phosphorylation of these proteins, was increased selectively in granulosa cells of antral follicles. By using hypophysectomized rats, these 5 proteins are induced by FSH. Because they were not present in cytosols of thecal cells or corpora lutea, they appear to be specific markers for granulosa cells. The content and phosphorylation of RII was also dramatically increased in cytosols of granulosa cells from antral follicles, while that of actin remained unchanged. Granulosa cell differentiation involves regulation by FSH of specific proteins which are substrates for cAMP-dependent protein kinase. FSH and cAMP appear to regulate the intracellular content and phosphorylation to a cAMP response system in granulosa cells. The extent to which RII and the 5 specific phosphoproteins themselves regulate granulosa cell responsiveness remains to be determined.

This publication has 26 references indexed in Scilit: