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.