Regulation of Ovarian Aromatase: Studies by Aromatase Assays in vitro and in vivo

Abstract: An in vitro method was developed for measuring aromatase, based on binding of competitive aromatase inhibitor [11C]vorozole to the active site of the enzyme. [11C]Vorozole displayed high, specific binding in vitro to human placenta and human granulosa cells (GC), both fresh and frozen/thawed cells, provided correct procedures were used. High, specific binding was also observed in pig and rat ovaries, whereas binding in other tissues was unspecific and usually low. Aromatase concentrations measured by [11C]vorozole binding correlated well to aromatase activity measured by [3H]water release from 1?[3H]androstenedione. In human GC in vitro, low concentrations of 5?-dihydrotestosterone (DHT), but not of other androgens, stimulated aromatase activity measured by [3H]water release but had no effects on aromatase concentration measured by [11C]vorozole binding. DHT may interact with aromatase differently than other androgens, perhaps by changing aromatase affinity to precursor. In the rat estrous cycle, aromatase activity in ovarian homogenate, measured by [3H]water release, together with serum androstenedione and estradiol-17?, peaked between 6 and 13 h after onset of the light period of proestrus, the former activity being independent of radioactive substrate concentration. [11C]Vorozole binding characteristics changed more rapidly than de novo synthesis of the enzyme. [11C]Vorozole binding Kd showed close inverse correlation to aromatase activity in ovarian homogenate and to serum estradiol-17?. Rapid changes in substrate affinity rather than changes in substrate concentration or de novo synthesis of the enzyme may thus be important for regulation of ovarian aromatase. The [11C]vorozole in vivo technique yields additional information compared with traditional in vitro techniques.