Cholinergic stimulation, through muscarinic receptors, of oxytocin and progesterone secretion from bovine granulosa cells undergoing spontaneous luteinization in serum-free culture.

Bovine granulosa cells were cultured in a defined serum-free system to examine their responsiveness to acetylcholine (ACh). Continuous exposure to concentrations of ACh between 10(-8)-10(-4) M resulted in dose-dependent increases (up to 6.7-fold) in the secretion of oxytocin and progesterone, with an ED50 of 6.6 microM. Ascorbic acid (0.5 mM), a known stimulator of granulosa secretion, synergized with ACh, resulting in an increase in the amounts of hormone secreted and a 7-fold increase in cellular sensitivity to ACh (ED50 = approximately 0.9 microM). Treatment of cells with ACh for 24 h at various times during a typical 5-day culture resulted in a stimulation that persisted for up to 4 days after removal of ACh. Carbachol (10(-8)-10(-4) M), a receptor antagonist with both antimuscarinic and antinicotinic actions, had no distinct effect on hormone secretion by the cells, but the effects of 10(-5) M ACh could be completely abolished by equimolar or hypomolar concentrations of the specific muscarinic receptor antagonists atropine and scopolamine. Nicotine bitratrate (10(-8)-10(4) M), a dose-dependent nicotinic receptor agonist/antagonist, had no effect on the cells. It is concluded that bovine granulosa cells, exhibiting a luteinized phenotype in culture, are responsive to cholinergic agonists in a specific and saturable manner. The response of the cells is probably mediated through muscarinic receptors and has both medium and long term (persistent) components. These results indicate that cholinergic neurotransmitters may play a direct role in the regulation of ovarian function in the ruminant.