Response of porcine granulosa cells isolated from primary and secondary follicles to FSH, 8-bromo-cAMP and epidermal growth factor in vitro

A cell culture system was developed to study the function of porcine granulosa cells from primary and secondary follicles. Primary follicles were isolated from 1- to 3-day-old pigs. Secondary follicles were isolated from 50- to 60-day-old pigs. Follicles were isolated after a digestion for 15 min with 0.25% trypsin followed by 15 min with 1000 U DNAase. Follicles were plated at 100 primary follicles or 30 secondary follicles per well in 48-well plates and cultured in media containing 10% fetal bovine serum (FBS). During initial plating, follicles attached to the plate and cells spread from the point of attachment. This resulted in monolayer cultures of granulosa cells from primary or secondary follicles. On day 4 of culture, media were replaced with 0.5 ml media containing one of the following treatments: control (media only); 10% FBS; 100 ng FSH; 2 mmol 8-bromo-cAMP l−1 or 50 ng epidermal growth factor (EGF). Media and cells were harvested on day 6, after 2 days of treatment. FBS and EGF increased DNA in granulosa cell cultures from primary or secondary follicles (P < 0.01). Treatment with 8-bromo-cAMP increased DNA in granulosa cell cultures from primary but not from secondary follicles (P < 0.05). Conversely, treatment with FSH increased DNA in granulosa cell cultures from secondary but not from primary follicles (P < 0.05). Progesterone release was greater in granulosa cell cultures from primary and secondary follicles treated with 8-bromo-cAMP than in controls (P < 0.001); however, progesterone concentrations were not influenced by FBS, EGF or FSH in granulosa cells from primary or secondary follicles. In summary, EGF and 8-bromo-cAMP stimulated proliferation of granulosa cells from primary and secondary follicles, and 8-bromo-cAMP also stimulated progesterone secretion. In contrast, FSH had no effect on progesterone secretion or proliferation of granulosa cells from primary follicles, but stimulated proliferation of granulosa cells from secondary follicles.