RESUMO
The ovarian promoter of the primate and rodent genes encoding cytochrome P450 aromatase (CYP19A1) are robustly responsive to forskolin in luteinized cell models, whereas the ruminant ovarian promoter is minimally active. We explored this discrepancy by investigating the activity of the bovine ovarian promoter in two bovine granulosa cell models, luteinizing and non-luteinizing cells in vitro. In non-luteinizing cells, both FSH and IGF1 increased abundance of transcripts derived from the ovarian promoter. Comparison of the activity of promoters of several species in response to transcription factors (forskolin, NR5A2, FOXL2) in luteinizing cells demonstrated that a rat ovarian promoter-luciferase reporter was regulated mainly by forskolin (18-fold increase over basal expression) and addition of NR5A2 or FOXL2 had no further effect. Activity of a human promoter was significantly increased by NR5A2 plus forskolin (153-fold) compared with forskolin alone (71-fold over basal); addition of FOXL2 did not significantly increase promoter activity. Forskolin alone provoked minor activation of caprine and bovine promoter reporters (3-fold over basal), and addition of NR5A2 increased activity (7- to 11-fold). When forskolin, NR5A2 and FOXL2 treatments were combined, the activity of the caprine and bovine promoters increased to 20- and 34-fold, respectively. These data suggest that a major reason why CYP19A1 is not expressed in luteinized cells (and the corpus luteum) of ruminants may be the stimulatory effect of FOXL2, which does not appear to be the case in the human and rat.
Assuntos
Aromatase/genética , Células da Granulosa/enzimologia , Modelos Biológicos , Regiões Promotoras Genéticas , Animais , Aromatase/metabolismo , Bovinos , Colforsina/farmacologia , Corpo Lúteo/metabolismo , Estradiol/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Hormônio Foliculoestimulante/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Cabras , Células da Granulosa/efeitos dos fármacos , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Luteinização/efeitos dos fármacos , Luteinização/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores do FSH/metabolismo , Especificidade da Espécie , TransfecçãoRESUMO
In the present study, we determined the potential for post-transcriptional regulation of cytochrome P450 aromatase (Cyp19), cytochrome P450 side-chain cleavage (Cyp11a) and 17beta-hydroxysteroid dehydrogenase I (Hsd17b1) mRNA. Bovine granulosa cells were cultured in non-luteinizing conditions that permit long-term oestradiol secretion. Half-lives of mRNA were measured by Northern and/or reverse transcriptase (RT)-PCR after inhibition of gene transcription. In FSH-stimulated cells, the Cyp11a and Hsd17b1 mRNAs had half-lives greater than 12 h. The half-life of Cyp19 mRNA was significantly shorter at 3 h. The addition of the translation inhibitor cycloheximide to FSH-stimulated cells significantly increased Cyp19 mRNA half-life to approximately 12 h. Stimulation of cells with insulin resulted in Cyp19 mRNA half-life that was double (P<0.05) that in FSH-stimulated cells. We conclude that bovine Cyp19 mRNA is very labile under physiological conditions, and that Cyp19 expression is under hormonal control at a post-transcriptional level.
Assuntos
Aromatase/genética , Regulação Enzimológica da Expressão Gênica , Células da Granulosa/metabolismo , RNA Mensageiro/metabolismo , Animais , Northern Blotting/métodos , Bovinos , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Diclororribofuranosilbenzimidazol/farmacologia , Feminino , Flavonoides/farmacologia , Hormônio Foliculoestimulante/farmacologia , Meia-Vida , Insulina/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estimulação QuímicaRESUMO
The objective of this study was to determine if androgens regulate granulosa cell steroidogenesis at physiological doses found in small bovine follicles. Bovine granulosa cells were cultured under serum-free conditions that permit the induction and maintenance of FSH-dependent estradiol secretion. Increasing androstenedione concentrations from 0.1 to 1 or 10 microM significantly increased estradiol accumulation and cytochrome P450 aromatase (P450arom) mRNA abundance. No increase in progesterone accumulation or abundance of mRNA for P450 side-chain cleavage or 3beta-hydroxysteroid dehydrogenase enzymes was observed. The addition of 0.1, 1, or 10 microM progestins or estrogens had no stimulatory effect on P450arom mRNA levels. An analysis of the 5'-untranslated region of P450arom mRNA transcripts indicated that the majority was derived from Cyp19 ovary-specific promoter 2, with some contribution from promoters 1.1 and 1.5. Transcripts from these three promoters were all significantly increased by androstenedione. Testosterone increased promoter 1.1 and 1.5-derived transcripts, but only promoter 2-derived transcripts at the highest dose tested (100 microM). Dihydrotestosterone (DHT) did not affect Cyp19 expression. Collectively, these data show that androgens may exert specific stimulatory effects on P450arom mRNA concentrations in granulosa cells. Interestingly, different androgens had different effects on Cyp19 promoter usage, suggesting differential regulation of aromatase gene expression in the developing follicle.