Differential effects of calcium on progesterone production in small and large bovine luteal cells

We studied the effects of calcium (Ca²⁺) ions in progesterone (P) production by separated small and large luteal cells. Corpora lutea were collected from 31 heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. P accumulation in cells plus media was determined after incubating 1 × 10⁵ small and 5 × 10³ large cells for 2 and 4 h respectively. Removal of Ca²⁺ from the medium did not influence basal P production in the small cells (P > 0.05). However, stimulation of P by luteinizing hormone (LH), prostaglandin E₂ (PGE₂), 8-bromo-cyclic 3',5' adenosine monophosphate (8-Br-cAMP) and prostaglandin F₂α (PGF₂α) was impaired (P < 0.05) by low Ca²⁺ concentrations. LH and PGE₂-stimulated cAMP production was not altered by low extracellular Ca²⁺ concentrations, and PGF₂α had no effect on cAMP. In contrast, basal as well as LH and forskolin-stimulated P production were attenuated (P < 0.05) in Ca²⁺-deflcient medium in the large cells. However, P production stimulated by 8-Br-cAMP was not altered in Ca²⁺-deficient medium. Steroidogenesis in large cells was also dependent on intracellular Ca²⁺, since 8-N, N-diethylamineocytyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca²⁺ release and/or action, suppressed (P < 0.05) basal, LH and 8-Br-cAMP stimulated P. In contrast, basal P in small cells was not altered by TMB-8; whereas LH-stimulated P was reduced 2-fold (P < 0.05). The calcium ionophore, A23187, inhibited LH-stimulated P in small cells and both basal and agonist-stimulated P in large cells. These studies show that basal P production in small cells does not require Ca²⁺ ions, while hormone-stimulated P production in small cells and both basal and hormone-stimulated P in large cells do require Ca²⁺. The inhibitory effect of Ca²⁺ ion removal was exerted prior to the generation of cAMP in the large cells, but distal to cAMP generation in hormone-stimulated small cells. The calmodulin/protein kinase C antagonist, W-7, also inhibited both basal and hormone-stimulated P production in both small and large luteal cells, indicating that P production in luteal cells also involves Ca²⁺-calmodulin/protein kinase C-dependent mechanisms.