Progesterone production in granulosa cells of the domestic fowl: effects of incubation media, pH, cell density and some other factors

The objective of this study was to determine the optimal conditions for the short term incubation of chicken granulosa cells. Compared to mechanically-dispersed cells, collagenase-treatment yielded granulosa cells of greater viability and responsiveness to ovine LH (oLH) stimulation. The rate of progesterone secretion by enzyme-dispersed chicken granulosa cells was subsequently compared in five different culture media during incubation periods of up to 24 hr. Under room air as the gas phase, Medium 199 (M199) containing Hank's salts and Ham's F-12 medium (F-12) were the most effective, whereas Krebs-Ringer bicarbonate-buffered glucose solution (KRBG) and Dulbecco's medium were the poorest. When pH and the ionic strength of KRBG was maintained by continuous gassing with O₂:CO₂ (95:5), progesterone production was similar to that obtained with Hepes-buffered M199. The pH optimum was found to be 7.4, although within the range of 6.6–8.5 granulosa cells remained responsive to LH stimulation. The optimal cell density was observed to be 1 × 10⁴ to 5 × 10⁵/ml. Although time course studies showed that both basal and stimulated progesterone production peaked by about 12 hr of incubation regardless of the media composition, the amounts released were significantly greater in M199 and F-12 during more prolonged (up to 24 hr) incubations. Glucose, a key medium ingredient for hormone-stimulated steroidogenesis, could be replace by pyruvate. On the other hand, lactate was inhibitory. It is concluded that the mature ovarian follicles of the domestic fowl are an excellent source of pure granulosa cells that can be obtained in high yield after a brief treatment with collagenase. These cells remain viable up to 24 hr and continue to produce large amounts of progesterone in response to LH when incubated in an appropriate medium and at optimal cell density.