Luteotropic and luteolytic responsiveness of ovine luteal cells in long-term culture

Ovine luteal cells were collected and plated 36 h (Day 2) after injection of human chorionic gonadotropin (Day 0) to induce ovulation. Cells were maintained (Days 2-12) in Medium 199 containing 5% calf serum, which was replaced daily. Progesterone secretion was not stimulated (p>0.05) by luteinizing hormone (LH, 10 ng/ml or 100 ng/ml) at any time during culture. However, it was enhanced (p<0.05) with a 24-h pulse of dibutyryl adenosine 3',5'-monophosphate (dbcAMP) during early (2.2-fold stimulation over basal; Days 5,6) or mid- (1.7-fold stimulation over basal; Days 8,9) culture if the pulsing medium contained serum, but not if serum had been withdrawn for 24 h. Continuous exposure of cultures to dbcAMP (2 mM, Days 3-12) resulted in continuously stimulated (p<0.05) progesterone secretion (range 1.8- to 4.1-fold stimulation). An increased (p<0.05) percentage of cells staining positive for 3β-hydroxy-Δ⁵-steroid dehydrogenase -Δ⁵, Δ⁴-isomerase (3βHSD) activity were recovered on Day 12 in cultures incubated (Days 3-12) with dbcAMP. Incubation of cultures continuously with prostaglandin F(2α) (PGF(2α)) produced dose-dependent inhibition (p<0.05) of progesterone secretion. Reduced numbers of 3βHSD-positive cells were recovered from these incubations. These experiments demonstrate luteotropic (dbcAMP) as well as luteolytic (PGF(2α)) effects on ovine luteal cells in long-term culture. This study provides evidence that these cultures will be useful for investigating the development of hormonal regulation of luteal function.