In vitro differentiation of bovine granulosa cells obtained from small antral follicles

The present study was undertaken to investigate whether bovine granulosa cells obtained from small antral follicles could differentiate into luteal-like cells in vitro. Granulosa cells obtained from small antral follicles (3-5 mm in diameter) were cultured up to 9 days in the presence or absence of insulin (2 μg/ml), forskolin (10 μM) and insulin (2 μg/ml) plus forskolin (10 μM). On days 1, 3, 5, 7 and 9 of culture, spent media were harvested for hormone assays. Progesterone production in the granulosa cells incubated with insulin plus forskolin increased continuously during the culture period. On the other hand, estradiol-17β production in the granulosa cells sharply decreased on day 3, and then continued to decrease. The size of granulosa cells cultured with insulin plus forskolin for 9 days was significantly larger than that of granulosa cells at the beginning of culture (P<0.001), and the size was similar to that of large luteal cells. Collectively, these data suggest that bovine granulosa cells obtained from small antral follicles can differentiate into large luteal-like cells during culture in the presence of insulin and forskolin.