Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro.
The objective of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate cytochrome P450 aromatase (Cyp19) mRNA and oestradiol accumulation in oestrogenic bovine granulosa cells in vitro. Bovine granulosa cells from small follicles (2-4 mm diameter) were cultured for 6 days under non-luteinizing conditions in the presence of insulin at 100 ng/ml, or insulin (10 ng/ml) and FSH (1 ng/ml). On day 4 of culture, specific inhibitors of phosphatidylinositol 3-kinase (PI3K; LY-294002), protein kinase C (PKC; GF-109203X), protein kinase A (PKA; H-89) or mitogen-activated protein (MAP) kinase activation (PD-98059) were added. The addition of PI3K and PKC inhibitors, but not of PKA inhibitor, significantly decreased insulin-stimulated Cyp19 mRNA levels and oestradiol accumulation (P < 0.001). The PKA inhibitor significantly decreased FSH-stimulated Cyp19 mRNA abundance and oestradiol secretion, whereas PI3K and PKC inhibitors decreased oestradiol secretion without affecting Cyp19 mRNA accumulation. Inhibition of MAP kinase pathway significantly increased Cyp19 mRNA abundance in insulin- and FSH-stimulated cells. P450scc mRNA levels and progesterone secretion were not affected by any inhibitor in either experiment. Although FSH stimulates Cyp19 expression predominantly through PKA, oestradiol secretion is altered by PI3K and PKC pathways independently of Cyp19 mRNA levels. In addition, we suggest that Cyp19 is under tonic inhibition mediated through a MAP kinase pathway.
Silva
J M
JM
Centre de recherche en reproduction animale, Faculté de médecine vétérinaire, Université de Montréal, St-Hyacinthe, Quebec, Canada J2S 7C6.
Hamel
M
M
Sahmi
M
M
Price
C A
CA
D000818:Animals; D001141:Aromatase / Q000235:genetics*; D015152:Blotting, Northern / Q000379:methods; D002417:Cattle; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004958:Estradiol / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D005419:Flavonoids / Q000494:pharmacology; D005640:Follicle Stimulating Hormone / Q000502:physiology*; D005498:Follicular Phase; D006107:Granulosa Cells / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D007546:Isoquinolines / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008301:Maleimides / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology
D002867:Chromones; D005419:Flavonoids; D007211:Indoles; D007328:Insulin; D007546:Isoquinolines; D008301:Maleimides; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; D013449:Sulfonamides; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D004958:Estradiol; D005640:Follicle Stimulating Hormone; D001141:Aromatase; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; C070515:bisindolylmaleimide I; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one