Heterogeneity in granulosa cells of developing rat follicles

The present study was designed to characterize the granulosa cell subpopulations derived from rats in which ovarian growth was induced by diethylstilbestrol (DES) or in which growth and differentiation was induced by pregnant mare's serum gonadotropin (PMSG). In the DES model, immature rats were given two separate injections of 2 mg DES/rat s.c. on 25 and 26 days of age and were killed 24 hr after the second injection. In the PMSG model, rats on day 28 were given 8 IU of PMSG s.c. and were killed 54 hr later. Granulosa cells were isolated from the ovaries, separated on a continuous Percoll density gradient, and divided into 12 fractions. The cells in each fraction were cultured in the presence of androstenedione with or without 20 ng/ml of follicle-stimulating hormone (FSH) and estradiol and progesterone in the incubation medium were measured. In DES-treated rats, granulosa cells in fractions 4 and 5 and fractions 9 and 10 contained about 30–40% of total cell yield and showed high steroidogenic potential. Granulosa cells from fractions 6, 7, and 8, which represent 55–60% of total cell yield, produced relatively low amounts of steroids on a per-million-cell basis. FSH was required for the stimulation of steroidogenesis. In granulosa cells from the PMSG treated rats, aromatase activity appeared maximally induced and incubation with FSH in vitro did not bring about any further increase. However, in vitro incubation with FSH was required for progesterone production. Furthermore, the granulosa cells from the PMSG treated rats also showed much more active estradiol and progesterone synthesis in fractions 9–12 as compared with lower density fractions. These studies suggest functional heterogeneity of granulosa cell populations in their response to FSH-induced steroidogenesis. In addition, it was observed that in both models, there are two major populations of granulosa cells as evidenced by light and electron microscopy. The functional role of small and large granulosa cells in steroidogenesis is unclear; further studies are in progress.