Luteinizing granulosa cells synthesize high concentrations of progesterone, prostaglandin (PG) E(2) and PGF(2 alpha). The objective of this study was to explore the relationship between prostaglandin and progesterone output from human granulosa cells as they undergo functional luteinization in culture. Granulosa cells were partially purified from ovarian follicular aspirates and cultured at a density of 10(5) cells/ml in serum-supplemented DMEM:Ham's F(12) medium for 0, 1 or 2 days. Cells were then switched to serum-free medium for 24 h before measuring hormone concentrations in this spent medium by specific radioimmunoassays. Over the first 3 days in culture, PGF(2 alpha) and PGE(2) production declined progressively by up to 82+/-3% coincident with a 55+/-11% increase in progesterone output. In subsequent experiments, cells were treated for 24 h on the second day of culture with either 0.01 to 10 microM meclofenamic acid or with 10 microM and 100 microM aminoglutethimide. Meclofenamic acid inhibited synthesis of PGF(2 alpha) and PGE(2) by up to 70+/-9% and 64+/-7% respectively without affecting progesterone output. Likewise, 100 microM aminoglutethimide inhibited progesterone production by 62+/-6% without affecting concentrations of either PGF(2 alpha) or PGE(2). We have concluded that the progressive decline in prostaglandin production and the rise in progesterone output from luteinizing human granulosa cells occur independently of each other.

In luteinizing granulosa cells, prostaglandin E₂ (PGE₂) can exert luteotrophic actions, apparently via the cAMP signalling pathway. In addition to stimulating progesterone synthesis, PGE₂ can also stimulate oxidation of the physiological glucocorticoid, cortisol, to its inactive metabolite, cortisone, by the type 1 11β-hydroxysteroid dehydrogenase (11βHSD1) enzyme in human granulosa–lutein cells. Having previously shown these human ovarian cells to express functional G-protein coupled, E-series prostaglandin (PTGER)1, PTGER2 and PTGER4 receptors, the aim of this study was to delineate the roles of PTGER1 and PTGER2 receptors in mediating the effects of PGE₂ on steroidogenesis and cortisol metabolism in human granulosa–lutein cells. PGE₂-stimulated concentration-dependent increases in both progesterone production and cAMP accumulation (by 1.9 ± 0.1- and 18.7 ± 6.8-fold respectively at 3000 nM PGE₂). While a selective PTGER1 antagonist, SC19220, could partially inhibit the steroidogenic response to PGE₂ (by 55.9 ± 4.1% at 1000 nM PGE₂), co-treatment with AH6809, a mixed PTGER1/PTGER2 receptor antagonist, completely abolished the stimulation of progesterone synthesis at all tested concentrations of PGE₂ and suppressed the stimulation of cAMP accumulation. Both PGE₂ and butaprost (a preferential PTGER2 receptor agonist) stimulated concentration-dependent increases in cortisol oxidation by 11βHSD1 (by 42.5 ± 3.1 and 40.0 ± 3.0% respectively, at PGE₂ and butaprost concentrations of 1000 nM). Co-treatment with SC19220 enhanced the ability of both PGE₂ and butaprost to stimulate 11βHSD1 activity (by 30.2 ± 0.2 and 30.5 ± 0.6% respectively), whereas co-treatment with AH6809 completely abolished the 11βHSD1 responses to PGE₂ and butaprost. These findings implicate the PTGER2 receptor–cAMP signalling pathway in the stimulation of progesterone production and 11βHSD1 activity by PGE₂ in human granulosa–lutein cells.