Steroid production, cell proliferation, and apoptosis in cultured bovine antral and mural granulosa cells: development of an in vitro model to study estradiol production.

Abstract

This study was undertaken to characterize the relationship between changes in steroid production, cell cycle activity (ie, cell proliferation) and apoptosis in antral and mural bovine granulosa cells cultured in vitro. This was done to select conditions promoting optimal estradiol production by bovine granulosa cells cultured in completely defined conditions. In the first experiment, antral granulosa cells were cultured over the entire 4 days of the culture period in the presence of either 0, 2, or 10 ng/ml of FSH (chronic conditions) or were maintained under minimal FSH support (0.5 ng/ml FSH) for the first 3 days of culture and then were challenged over the fourth day of culture with either 0, 2, or 10 ng/ml FSH (challenged conditions). Compared with cells exposed to constant FSH levels (chronic conditions), the FSH-induced production of estradiol was higher (P < 0.006) and that of progesterone was lower (P < 0.02) over the last 24 h of culture, when antral granulosa cells were maintained under minimal FSH support during the first 3 days of culture (challenged conditions). In the second experiment, dynamics of estradiol and progesterone productions, conversion of [14C]androstenedione into subsequent steroid metabolites, DNA content, cell cycle activity, and apoptosis (as assessed by flow cytometry) of antral and mural granulosa cells over the first 3 days of culture under minimal FSH support and in response to a challenge with FSH during the last 24 h of culture were evaluated. Estradiol production as well as the conversion of androstenedione into testosterone and estradiol were greater (P < 0.01) in antral than in mural granulosa cells cultured under challenged conditions. A higher proportion of mural than antral granulosa cells were in the proliferative state at the end of culture (P < 0.03). This may be related to the decreased ability of mural cells to produce estradiol. FSH suppressed (P < 0.05) the spontaneous onset of apoptosis in both cell types. These results suggest that functional differences between these two cell compartments need to be considered in studying bovine granulosa cells in vitro. Because of their large (400 to 600%) FSH-induced estradiol production, antral granulosa cells cultured under challenged conditions provide a model that can be used to examine substances for their ability to alter estradiol production and apoptosis in bovine granulosa cells.