Steroidogenic factor-1 as a positive regulator of rat granulosa cell differentiation and a negative regulator of mitosis.
Ovarian follicles contain small nonaromatase-expressing and large aromatase-expressing granulosa cells (GCs). The present studies were designed to determine whether small GCs can differentiate into large GCs and/or express aromatase. Additional studies were conducted to assess the role of steroidogenic factor-1 (SF-1), an orphan nuclear receptor, in regulating GC differentiation and proliferation. For these studies, small GCs were isolated from immature rats by Percoll gradient centrifugation and cultured for up to 48 h with FSH and/or 8-bromo-cAMP (8-br-cAMP). FSH/8-br-cAMP induced a 2-fold increase in SF-1 messenger RNA levels within 4 h. This increase was maintained throughout the culture period. By 24 h 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 that 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 nonsense oligonucleotide (SF-1 NS). The SF-1 AS, but not the SF-1 NS, prevented FSH/8-br-cAMP from increasing 1) SF-1 messenger RNA levels, 2) transcription of a SF-1(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 cultured 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 together, these data demonstrate that SF-1 is expressed in both small and large GCs, and enhanced SF-1 expression is part of the molecular mechanism associated with GC differentiation. Interestingly, SF-1 not only regulates differentiation, but also inhibits TPA-induced GC mitosis.
Shapiro
D B
DB
Department of Obstetrics, University of Connecticut Health Center, Farmington, 06030, USA.
Pappalardo
A
A
White
B A
BA
Peluso
J J
JJ
D000818:Animals; D001141:Aromatase / Q000378:metabolism; D001483:Base Sequence; D002454:Cell Differentiation / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000235:genetics / Q000494:pharmacology*; D005260:Female; D051717:Fushi Tarazu Transcription Factors; D006107:Granulosa Cells / Q000166:cytology*; D018398:Homeodomain Proteins; D008938:Mitosis / Q000187:drug effects*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000235:genetics / Q000494:pharmacology*
D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D015345:Oligonucleotide Probes; D016376:Oligonucleotides, Antisense; D018160:Receptors, Cytoplasmic and Nuclear; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; C515060:steroidogenic factor 1, rat; D001141:Aromatase