STEROIDOGENIC FACTOR-I AS A POSITIVE REGULATOR OF RAT GRANULOSA-CELL DIFFERENTIATION AND A NEGATIVE REGULATOR OF MITOSIS

Ovarian follicles contain small nonaromatase-expressing and large arom atase-expressing granulosa cells (GCs). The present studies were desig ned to determine whether small GCs can differentiate into large GCs an d/or express aromatase. Additional studies were conducted to assess th e role of steroidogenic factor-1 (SF-1), an orphan nuclear receptor, i n regulating GC differentiation and proliferation. For these studies, small GCs were isolated from immature rats by Percoll gradient centrif ugation and cultured for up to 48 h with FSH and/or 8-bromo-cAMP (8-br -cAMP). FSH/8-br-cAMP induced a a-fold increase in SF-1 messenger RNA levels within 4 h. This increase was maintained throughout the culture period. By 24 h of culture, FSH/8-br-cAMP increased the percentage of large GCs. It was not until 48 h of culture with FSH and 8-br-cAMP th at aromatase expression increased. This increase was detected by both Western blot and quantitative immunocytochemistry. 8-br-cAMP alone did not promote GC differentiation. Small GCs were then cultured with FSH /8-br-cAMP in the presence or absence of an antisense oligonucleotide complementary to the putative SF-1 ligand-binding site (SF-1 AS). As a control, small GCs were cultured with FSH/8-br-cAMP and an 18-mer non sense oligonucleotide (SF-1 NS). The SF-1 AS, but not the SF-1 NS, pre vented FSH/8-br-cAMP from increasing 1) SF-1 messenger RNA levels, 2) transcription of a SF-l(x2) promoter/luciferase construct, 3) GC size, and 4) aromatase expression. In a third series of experiments, small GCs were cultured for 24 h in 1) control media supplemented with 2) a mitogen, phorbol ester [12-O-tetraphorbol acetate (TPA)], 3) FSH/8-br- cAMP, or 4) both. TPA increased the number of GCs by 51 +/- 9%. FSH/8- br-cAMP completely blocked TPA-induced mitosis. When small GCs were cu ltured with FSH/cAMP, TPA, and SF-1 AS, the number of GCs increased by 50 +/- 7%. This increase was not observed with SF-1 NS. Taken togethe r, these data demonstrate that SF-1 is expressed in both small and lar ge GCs, and enhanced SF-1 expression is part of the molecular mechanis m associated with GC differentiation. Interestingly, SF-1 not only reg ulates differentiation, but also inhibits TPA-induced GC mitosis.