Regulation of the Content and Phosphorylation of RIIby Adenosine 3′,5′-Monophosphate, Follicle-Stimulating Hormone, and Estradiol in Cultured Granulosa Cells*

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Abstract
The mechanisms by which FSH and cAMP induce receptors for LH [luteinizing hormone] (RLH) and increase progesterone (P) production in estradiol (E)-primed ovarian granulosa cells remain unclear, but may involve increases in the regulatory subunit of cAMP-dependent protein kinase II (RII) and the phosphorylation of specific cellular proteins. Primary cultures of granulosa cells (106 cells/ml) from E-treated (1.5 mg/day for 3 days) immature female rats were incubated with 10 nM E with or without FSH (25 ng/ml) for 0-120 h. The cytosolic content of RII was analyzed by 4 techniques: immunoblotting using an Ab to bovine heart RII; photoaffinity labeling with [32P]8-azido-cAMP; phosphorylation with [.gamma.-32P]ATP with or without 2 .mu.M cAMP or with the catalytic subunit of cAMP-dependent protein kinase; and phosphorylation of intact cells with [32P]orthophosphate. All approaches revealed a time-dependent 5- to 6-fold increase in RII content in granulosa cells cultured for 48 h with E and FSH compared to that in cells treated with E alone. The content of RI, the regulatory subunit of protein kinase type I, remained low throughout the culture period regardless of hormone treatment. Granulosa cells were also cultured with E (10 nM) and 8-bromo-cAMP (8-Br-cAMP; 0.25-3 mM) or forskolin (0.5-100 .mu.M), agents that increase intracellular cAMP, for 48 or 72 h. The cytosolic content and phosphorylation of RII were increased by culturing granulosa cells in E and 8-Br-cAMP (1 mM) or forskolin (50 .mu.M) for 48 h. The increase in RII was associated with a FSH-mediated increase in the content and phosphorylation of other cAMP-dependent phosphoproteins. The increases in RII and cAMP-dependent phosphoproteins were associated with specific alterations in granulosa cell function: a FSH-mediated rise in RLH [59.3 .+-. 7.4 cpm/.mu.g DNA (without FSH) to 1171.5 .+-. 157 cpm/.mu.g DNA (with FSH)] And P accumulation in the medium [0.05 .+-. 0.03 ng/ml (without FSH) to 25.3 .+-. 4.6 ng/ml (with FSH)] at 48 h. A dose-dependent increase in the RLH and P accumulation in the medium was observed at 48 h of culture with E and 8 Br-cAMP or E and forskolin. In intact cultured granulosa cells, RII is a major cytosolic phosphopotein whose content and phosphorylation state are regulated by cAMP, and increased phosphorylation of RII may be involved in the molecular events associated with the expression of FSH (cAMP)-mediated differentiation of granulosa cells.

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