Regulation of aromatase gene expression in Leydig cells and germ cells.

The ability of the testis to convert irreversibly androgens into estrogens is related to the presence of a microsomal enzymatic complex named aromatase. Although somatic cells and germ cells (GC) have the capacity to produce estrogens the regulation of the CYP19 gene expression in adult rat testicular cells and specially in freshly purified Leydig cells, pachytene spermatocytes (PS) and round spermatids (RS) is not fully understood. In the present study we have analyzed the putative effects of steroid hormones, transforming growth factor beta (TGFbeta), cytokine (tumor necrosis factor alpha, TNFalpha) and dexamethasone (Dex) on CYP19 expression in these purified testicular cells from adult rat. In parallel the biological role of seminiferous tubules and Sertoli cells conditioned media on the expression of aromatase was studied. Using a highly specific quantitative competitive RT-PCR we established that testosterone (T) enhances CYP19 gene expression in Leydig cells and germ cells, and augments the estradiol outputs. The non-aromatizable androgen 5alpha-DHT induces the same effect as T on P450 aromatase (P450arom) gene expression but was inefficient on the estradiol output. In PS and RS an inhibitory effect on CYP19 gene transcription was observed with TGFbeta (1 ng/ml) alone or in combination with T. Conversely, the addition of TNFalpha (20 ng/ml) increases the P450arom transcription in PS although an inhibitory effect is observed in RS. Together with T, TNFalpha decreases the amount of P450arom mRNA in PS and RS. In PS we found that Dex regulates positively CYP19 expression and negatively in RS. Furthermore in PS a synergistic effect of Dex and TNFalpha on P450arom mRNA expression was observed whereas an additive one was recorded for RS. Therefore in germ cells TNFalpha likely enhances expression of aromatase through promoter PI.4 in PS, possibly via an AP1 site upstream the GAS element, while in RS TNFalpha requires glucocorticoids as a co-stimulator to increase CYP19 gene expression. Finally in presence of seminiferous tubules or Sertoli cell conditioned media, the amount of aromatase transcripts is increased in both Leydig cells and germ cells therefore suggesting that other locally produced modulators, yet unknown, but from Sertoli cell origin, are concerned in the regulation of the aromatase gene expression in rat testicular cells. In summary, using an in vitro model of mature rat Leydig cells, pachytene spermatocytes and round spermatids, we have shown that several factors direct the expression of the aromatase gene and it is obvious that not only promoter PII but also promoter PI.4 are concerned.