Endocrinology, Vol 117, 917-927, Copyright © 1985 by Endocrine Society

ARTICLES

Regulation of the content and phosphorylation of RII by adenosine 3',5'- monophosphate, follicle-stimulating hormone, and estradiol in cultured granulosa cells

SL Ratoosh and JS Richards

The mechanisms by which FSH and cAMP induce receptors for LH (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. To examine the relationship of these events, primary cultures of granulosa cells (10(6) 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 four techniques: 1) immunoblotting using an antibody to bovine heart RII; 2) photoaffinity labeling with [32P]8-azido-cAMP; 3) phosphorylation with [gamma-32P]ATP with or without 2 microM cAMP or with the catalytic subunit of cAMP- dependent protein kinase; and 4) 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 microM), 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 microM) 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 1) RLH [59.3 +/- 7.4 cpm/micrograms DNA (without FSH) to 1171.5 +/- 157 cpm/micrograms DNA (with FSH]) and 2) 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.(ABSTRACT TRUNCATED AT 400 WORDS)

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