Induction of Luteinizing Hormone Receptors by Adenosine 3',5'-Monophosphate in Cultured Granulosa Cells

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Abstract
The induction of LH receptors by exogenous and endogenous cAMP was examined in granulosa cells from ovaries of hypophysectomized diethylstilbestrol-implanted rats. During the 96-h culture period, FSH stimulated cAMP production, protein synthesis, progesterone formation, and the expression of LH receptors. cAMP accumulation and LH receptor formation were stimulated by 2.5–10 ng FSH, and were maximal at 72 h of culture with 175 ng FSH. Progesterone production peaked after 48 h of culture, but required 25 ng FSH to initiate steroidogenesis and 375 ng for the maximal response. In cells cultured with 5 mM 8-bromo-cAMP (8-Br-cAMP), progesterone production at 48 h was similar to that stimulated by FSH, and LH receptor formation was one third as much as with FSH. Removal of 8-BrcAMP after 48 h of culture caused a fall in LH receptors and progesterone production at 96 h, indicating that continual presence of the cyclic nucleotide was required for the maintenance of granulosa cell function. The LH receptors induced by FSH and 8-Br-cAMP were functionally active, since LH raised progesterone production 15-fold in FSH-treated cells and 3-fold after 8-Br-cAMP treatment. The phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine elevated FSH-stimulated cAMP and progesterone accumulation, but inhibited LH receptor formation and the incorporation of [3H]leucine into granulosa cell protein in a concentration-dependent manner. Simultaneous addition of 8-Br-cAMP or choleragen with increasing concentrations of FSH indicated that enhancement of cAMP accumulation or cAMPdependent pathways synergistically increased LH receptor formation and progesterone production. Incubation of granulosa cells with both FSH and choleragen also resulted in supraadditive accumulation of cAMP. These results indicate that cAMP is required for both the induction and maintenance of LH receptors and steroidogenic enzymes during differentiation of the ovarian granulosa cell.

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