title: Formation of 4-oestrene-3,17-dione (19-norandrostenedione) by porcine granulosa cells in vitro is inhibited by the aromatase inhibitor 4-hydroxyandrostenedione and the cytochrome P-450 inhibitors aminoglutethimide phosphate and ketoconazole.
abstract: The origin and biosynthesis of 4-oestrene-3,17-dione (19-norandrostenedione), a major steroid in porcine ovarian follicular fluid, was investigated by culturing granulosa cells from 4-6 mm follicles of prepubertal gilts with radiolabelled androstenedione and 19-hydroxyandrostenedione. Steroid metabolites were purified by solvent extraction and lipophilic column chromatography, and analysed by C18 reverse-phase high-performance liquid chromatography. 19-Hydroxyandrostenedione, 19-norandrostenedione and oestradiol-17 beta were obtained as major metabolites from androstenedione, while 19-norandrostenedione and oestradiol-17 beta were the major products from 19-hydroxyandrostenedione. Serum alone or serum plus FSH significantly enhanced formation of 19-norandrostenedione and oestradiol-17 beta from each substrate, compared with controls. Micromolar concentrations (1 mumol/l) of 4-hydroxyandrostenedione, an aromatase inhibitor, significantly reduced formation of 19-norandrostenedione and oestradiol-17 beta by granulosa cells cultured with serum and FSH. Formation of 19-norandrostenedione and oestradiol-17 beta from androstenedione and 19-hydroxyandrostenedione was also significantly inhibited by aminoglutethimide phosphate, a cytochrome P-450 inhibitor known to block the conversion of androstenedione to oestrogens. Ketoconazole, an inhibitor of the cytochrome P-450 dependent 17,20-lysase, blocked formation of 19-norandrostenedione and oestradiol-17 beta only at millimolar concentrations. These results suggest that 19-norsteroid and oestrogen formation from C19 aromatizable androgens may share a common or overlapping pathway, and imply that 19-norsteroid and oestrogen synthesis is mediated by cytochrome P-450 dependent enzymes.
authors.affiliation: MRC Group in Reproductive Biology, University of Western Ontario, London, Canada.
authors.forename: M W
authors.initials: MW
authors.lastname: Khalil
authors.forename: P
authors.initials: P
authors.lastname: Morley
authors.forename: M A
authors.initials: MA
authors.lastname: Glasier
authors.forename: D T
authors.initials: DT
authors.lastname: Armstrong
authors.forename: T
authors.initials: T
authors.lastname: Lang
mesh_terms: D000616:Aminoglutethimide / Q000031:analogs & derivatives* / Q000494:pharmacology; D000735:Androstenedione / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D005260:Female; D006107:Granulosa Cells / Q000187:drug effects / Q000378:metabolism*; D007654:Ketoconazole / Q000494:pharmacology*; D013552:Swine
chemical_list: D000616:Aminoglutethimide; C026416:aminoglutethimide phosphate; D000735:Androstenedione; C011482:19-hydroxy-4-androstene-3,17-dione; C014594:formestane; D007654:Ketoconazole; C002828:19-norandrostenedione