Establishment of Highly Steroidogenic Granulosa Cell Lines by Cotransfection with SV40 and Ha-ras Oncogene: Induction of Steroidogenesis by Cyclic Adenosine 3′-5′-Monophosphate and Its Suppression by Phorbol Ester

We have established new cell lines from preovulatory follicles of PMSG-treated immature rats by cotransfection of primary cells with SV40 DNA and Ha-ras oncogene. These cell lines secrete progesterone and 20α-hydroxy-4-pregnen- 3-one in response to stimulation with 8-bromo-cAMP, forskolin, or cholera toxin at levels similar to primary cultures of granulosa cells (500 ng/10⁶ cells/48 h). 12-O-Tetradecanoylphorbol 13-acetate inhibits progesterone production induced by 8-bromo-cAMP (67%, P < 0.001), forskolin (88%, P < 0.001), and cholera toxin (75%, P < 0.001), in spite of enhancing cAMP accumulation (200%, P < 0.001) in response to forskolin or cholera toxin. LH, FSH, prostaglandins E₁ and E₂, and PRL do not stimulate progesterone production. The β-adrenergic agonist, isoproterenol, stimulates significantly both cAMP accumulation (80%, P < 0.05) and progesterone and 20α-hydroxy-4- pregnen-3-one secretion (70%, P < 0.05). In contrast to primary cultured cells in which progesterone secretion increases within 90 min of stimulation, in the cell lines progesterone secretion becomes evident only after 12–24 h of stimulation. The stimulation of these lines by forskolin produced a maximum rise in intracellular cAMP levels at 45 min which declined to 50% (P < 0.001) of this value within 12 h. It was found that cAMP suppressed growth concomitantly with induction of steroidogenesis when cotransfected cells were examined for growth by total cell protein and [³H]thymidine incorporation to DNA. These granulosa cell lines are uniquely suited for further studies of cAMP induction and the role of oncogenes in steroidogenesis. (Endocrinology127: 2489–2500, 1990)