Regulation of the aromatase gene expression in mature rat Leydig cells

Besides gonadotrophins and testosterone, numerous intratesticular factors, and among them estrogens play a crucial role in the regulation of spermatogenesis in the mammalian testis. The ability of the male gonad to convert androgens into estrogens is well known; the microsomal enzymatic complex involved in this transformation named aromatase, is composed of a specific cytochrome P450 aromatase (P450arom) and a ubiquitous reductase. Using a highly specific reverse transcription polymerase chain reaction (RT-PCR) method, we have measured the amount of P450arom mRNA in purified rat Leydig cells submitted to different treatments during 24 h. In parallel, the estradiol output was determined by RIA. Whatever the concentrations of testosterone used (as substrate of aromatase activity), we noted an increase of the estradiol production, the maximum being obtained for 200 ng/ml (28%). Related to the P450arom mRNA levels, a decrease was first observed for 50 and 100 ng/ml of testosterone then an increase (20%) for the higher doses (200-500 ng/ml). The addition of oLH (0.1-50 ng/ml) to the Leydig cells culture medium induced a dose-related increase of estradiol output till 10 ng/ml. For 50 ng/ml, a decrease was observed. Dealing with the mRNA levels, we first recorded a diminution for 0.1-1 ng/ml of oLH, which was abolished by the addition of testosterone. Then the mRNA levels were increased and reached a maximum for 5-10 ng/ml of oLH (35 and 50%, respectively, in absence and in presence of testosterone). The addition of oLH (50 ng/ml) induced a large augmentation of the quantity of P450arom mRNA (1.9 and 2.1-fold, respectively, in absence or in presence of testosterone). DbcAMP mimicked the effects of oLH. From these data, we confirm the presence of cAMP response-like elements (CRE) and the existence of androgen responsive elements (ARE) sites on the P450arom gene in rat Leydig cells.