Ovine placental aromatase: studies of activity levels, kinetic characteristics and effects of aromatase inhibitors

We have measured microsomal steroid aromatase activity in the fetal component of ovine placental cotyledons collected from pregnant ewes between 124 days and 127 days of gestation. Aromatase activity was determined by quantifying the [3H]water by-product when [1 beta-3H(N)] androstenedione was used as substrate. The mean microsomal aromatase activity (+/- SD) was 5.7 +/- 2.2 pmol.min-1.mg protein-1 (n = 12) and was 9% of the aromatase activity of human placental microsomes [mean (+/- SD) of 66.1 +/- 25.0 pmol.min-1.mg protein-1 (n = 7)]. The apparent Km for ovine placental aromatase for androstenedione, at pH 7.4 and 37 degrees C, was 50 nM while the Vmax was 20.6 pmol.min-1.mg protein-1. The respective concentrations effecting 50% inhibition of ovine placental aromatase activity (the I50) for econazole, 4-hydroxyandrostenedione, imazalil, miconazole, ketoconazole and aminoglutethimide were 0.03, 0.05, 0.15, 0.50, 5.0 and 5.5 microM. The order of relative potencies were similar to those obtained for human placental aromatase. Ketoconazole and aminoglutethimide were approx 10 times more potent inhibitors of the sheep enzyme relative to the human. Aromatase activity was not confined to the microsomal fraction of ovine placental tissue but was distributed throughout all the particulate subcellular fractions. The proportionally high activity of the tissue homogenate (1.75 pmol.min-1.mg protein-1) is suggestive that in the last third of pregnancy, aromatase is not rate limiting with regard to placental estrogen production. It would appear, therefore, that the major factor regulating placental estrogen synthesis in ovine pregnancy is the availability of substrate.