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

We studied the effects of calcium (Ca 2+) 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 × 10 5 small and 5 × 10 3 large cells for 2 and 4 h respectively. Removal of Ca 2+ 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 E 2 (PGE 2), 8-bromo-cyclic 3',5' adenosine monophosphate (8-Br-cAMP) and prostaglandin F 2α (PGF 2α) was impaired ( P < 0.05) by low Ca 2+ concentrations. LH and PGE 2-stimulated cAMP production was not altered by low extracellular Ca 2+ concentrations, and PGF 2α had no effect on cAMP. In contrast, basal as well as LH and forskolin-stimulated P production were attenuated ( P < 0.05) in Ca 2+-deflcient medium in the large cells. However, P production stimulated by 8-Br-cAMP was not altered in Ca 2+-deficient medium. Steroidogenesis in large cells was also dependent on intracellular Ca 2+, since 8- N, N-diethylamineocytyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca 2+ 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 Ca 2+ ions, while hormone-stimulated P production in small cells and both basal and hormone-stimulated P in large cells do require Ca 2+. The inhibitory effect of Ca 2+ 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 Ca 2+-calmodulin/protein kinase C-dependent mechanisms.