title: Intracellular regulation of estradiol and progesterone production by cultured bovine granulosa cells.
abstract: The objective of the present study was to investigate the implication of protein kinase A (PKA), protein kinase C (PKC), and receptor protein tyrosine kinase (R-PTK) pathways in the regulation of estradiol (E2) and progesterone (P4) production by bovine granulosa cells. Cells were harvested from bovine follicles (8-15 mm diameter) and cultured without serum for an initial 3 days (37 degrees C; 5% CO(2) in air; D1-D3). On the fourth day of culture (D4), E2 and P4 production were stimulated with FSH (1-6 ng/ml) or forskolin (FSK) in the presence or absence of intracellular effectors of PKA, PKC, and R-PTK. Culture medium was collected and replaced each day. Stimulation of granulosa cell adenylate cyclase activity with FSK (0.06-3.75 microM) mimicked FSH, inducing a quadratic increase (P < 0.001) of E2 production and a continuous elevation of P4 (P < 0.01). Inhibition of R-PTK activity with genistein (25-50 microM) increased the sensitivity of cells to FSH as demonstrated by a leftward shift in the dose response curve (P < 0.001). Treatment with transforming growth factor-alpha (TGFalpha; 0. 1 ng/ml) abolished the FSH-induced E2 production (P < 0.001) and this effect was not reversed (P < 0.001) by FSK or by genistein. Furthermore, the inhibitory effect of TGFalpha on FSH-induced E2 production was reproduced by phorbol 12-myristate 13-acetate (PMA; 1. 25-2.5 microM), a PKC activator (P < 0.001). Interestingly, genistein inhibited P4 production (P < 0.05). From these results, we conclude that E2 production by bovine granulosa cells is mediated by intracellular factors and can be stimulated downstream from the FSH receptor. The results also suggest that stimulation of R-PTK and/or PKC activities, as probably occurs with TGFalpha, negatively affects the PKA pathway, thus decreasing E2 production. Furthermore, inhibition of R-PTK leads to an increase production of E2 and may limit luteinization of bovine granulosa cells.
authors.affiliation: Département des Sciences Animales, Centre de Recherche en Biologie de la Reproduction, Université Laval, Ste-Foy, Québec, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Legault
authors.forename: J L
authors.initials: JL
authors.lastname: Bailey
authors.forename: M A
authors.initials: MA
authors.lastname: Fortier
authors.forename: P
authors.initials: P
authors.lastname: Rouillier
authors.forename: L A
authors.initials: LA
authors.lastname: Guilbault
mesh_terms: D000262:Adenylyl Cyclases / Q000187:drug effects / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000096:biosynthesis*; D005260:Female; D019833:Genistein / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D011374:Progesterone / Q000096:biosynthesis*; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000187:drug effects; D011962:Receptors, FSH / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D016211:Transforming Growth Factor alpha / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D011962:Receptors, FSH; D016211:Transforming Growth Factor alpha; D005576:Colforsin; D011374:Progesterone; D004958:Estradiol; D019833:Genistein; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D000262:Adenylyl Cyclases; D013755:Tetradecanoylphorbol Acetate