Bovine granulosa cells were cultured in collagen (Vitrogen 100)-pretreated wells using defined medium to study the secretion of oxytocin and progesterone under serum-free conditions. Secretion of oxytocin began spontaneously after the first day and was maintained at a high level during a 1 week culture period. Addition of serum to the medium reduced oxytocin concentrations by up t o 90%. There were positive exponential relationships between oxytocin and progesterone concentrations and the inoculated cell density (range, 1.67 to 23.4 x lo5 cells/ml/well). The results indicate that neither serum nor gonadotrophins are required for in vitro differentiation of bovine m-anulosa cells and that addition of serum may attenuate subsequent hormone secretion. This cu'iture system should provide a better in secretion than those previously described.
Methods
Cell preparation and culture Granulosa cells were prepared from healthy, preovulatory, nonluteal phase, bovine follicles as previously described (Jungclas and Luck, '86; Luck and Jungclas, '87). Selected follicles had a diameter of 0.8-1.4 cm, a fluid volume of 0.8-1.8 ml, and yielded 11.5-18.5 x lo6 cells per follicle. Cells were washed in a mixed medium (1: 1 v:v; Dulbecco's modified Eagle's medium: Ham's F12 with bicarbonate buffer, glutamine, penicillin, and streptomycin; Flow, Meckenheim, FRG).
Multiwell culture plates (Nunclon, Wiesbaden, FRG) were pretreated with Vitrogen 100 (Colla-