Proliferative and steroidogenic capabilities of rat granulosa cells of different sizes

Equine chorionic gonadotrophin stimulates both rat granulosa cell mitoses and oestradiol secretion. However, the mitotic potential of oestradiol-secreting granulosa cells is not known. In the first study, granulosa cells of different sizes were isolated and their ability to secrete oestradiol and proliferate in vitro was determined. Granulosa cells were harvested from equine chorionic gonadotrophin-primed immature rats, separated on a 15-45% Percoll gradient, and collected in 12 fractions. An enriched population of small granulosa cells (44 +/- 1 micron2) was collected in fractions 3 and 4 and an enriched population of large granulosa cells (97 +/- 2 microns2) in fractions 6-8. When granulosa cells from each fraction were cultured for 24 h in the presence of testosterone, the large cells secreted 50% more oestradiol than did the small cells (P < 0.05). Aromatase was shown, by immunocytochemistry, to be expressed mainly by granulosa cells larger than 73 microns2, with the relative amount of aromatase expressed per cell increasing with increasing cell size. However, not all large granulosa cells expressed aromatase. To test proliferative capacity, cells from each fraction were cultured with testosterone and the mitogen, insulin. This study showed that only small cells were able to undergo insulin-induced mitosis. In a second study, follicles of different sizes were isolated from immature and equine chorionic gonadotrophin-primed immature rats and the granulosa cell size distribution determined for each follicle size. This study confirmed that equine chorionic gonadotrophin altered the size distribution from principally small mitotically competent cells to large oestradiol-secreting cells. Studies in vitro further demonstrated that FSH in the presence of 8-bromo-cAMP stimulated small granulosa cells to differentiate into large cells. It is proposed that changes in the population of granulosa cells could account for both the slower growth rate of large antral follicles compared with small antral follicles and the inverse relationship between follicular oestradiol secretion and DNA synthesis of granulosa cells.