RESUMO
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.
Assuntos
Aromatase/biossíntese , Regulação Enzimológica da Expressão Gênica , Células Germinativas/enzimologia , Células Intersticiais do Testículo/enzimologia , Animais , Aromatase/genética , Aromatase/metabolismo , AMP Cíclico/farmacologia , Primers do DNA/genética , Dexametasona/farmacologia , Estradiol/análise , Estradiol/biossíntese , Células Germinativas/fisiologia , Humanos , Células Intersticiais do Testículo/fisiologia , Masculino , Regiões Promotoras Genéticas/genética , Ratos , Ratos Sprague-Dawley , Espermátides/metabolismo , Espermatócitos/metabolismo , Testosterona/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Fator de Crescimento Transformador beta1 , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The aim of the present study was to quantify the promoter II- and I.r-derived transcripts of p450 aromatase gene during follicular stages and during corpus luteum formation in the rabbit. An ovulatory dose of hCG induced, first the disappearance of 90% of aromatase transcripts since 6 h before ovulation, and second a gradual decrease during pseudopregnancy. Individual quantification of both the promoter-derived transcripts showed that promoter II-derived transcript was the main transcript expressed both during follicular phase and pseudopregnancy, but kinetics of disappearance were not similar between both the promoter-derived transcripts. Moreover, hCG up-regulates aromatase expression in vitro in luteal tissue but estradiol, which was without effect on aromatase expression in preovulatory granulosa cells, down-regulates this expression in luteal tissue. In conclusion, the regulation of P450 aromatase in rabbit is mainly under control of promoter II regardless of which cyclic stage is studied. Moreover, we reported an opposite effect of estradiol on aromatase expression in vitro between follicular and luteal cells.
Assuntos
Aromatase/genética , Fase Luteal/metabolismo , RNA Mensageiro/análise , Animais , Gonadotropina Coriônica/farmacologia , Estradiol/farmacologia , Feminino , Fase Folicular , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Células Lúteas/efeitos dos fármacos , Células Lúteas/metabolismo , Regiões Promotoras Genéticas , Pseudogravidez/metabolismo , RNA Mensageiro/efeitos dos fármacos , CoelhosRESUMO
The aim of the present study was to analyse the tissue-specific expression of various promoter-derived transcripts from the gene encoding rabbit aromatase cytochrome P450. A new promoter, named I.r, was identified, and promoters II and I.r were sequenced. Promoter I.r-derived transcripts were found in preovulatory granulosa cells, corpus luteum, placenta and adipose tissue. An alternative splice variant of this transcript was found with tissue-specific preference. Tissue-specific expression of promoter-derived variants was studied in the ovary before and after ovulation. While the level of promoter II-derived transcript decreased dramatically after ovulation, that of promoter I.r-derived transcript remained unchanged, indicating that promoter II and promoter I.r were not controlled by a single regulation system. The existence of this dual system of regulation suggests that the rabbit ovary could be a useful model to study the promoter-specific regulation of aromatase.