cAMP-dependent regulation of CYP19 gene in rabbit preovulatory granulosa cells and corpus luteum.

Transcription of the CYP19 gene encoding the aromatase P450 enzyme in ovarian cells is under the control of the two gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), via modulation of intracellular cyclic 3',5'-adenosine monophosphate (cAMP) levels. Primary cultures of rabbit ovarian cells were used to identify the functional regions of the ovarian promoter (PII) that are responsive to the gonadotropic secondary messenger and to estradiol. Transfection experiments in granulosa and luteal cells with deleted constructs of the PII promoter show that the region between -274 and -193bp is critical for cAMP-dependent transcriptional activity. A comparison of PII activities in granulosa and small luteal cells highlights a 50% decrease consecutive to the LH surge. Sequence analysis of the above mentioned region revealed the presence of a cAMP responsive element like sequence (CLS) and of a nuclear receptor element A (NREA). Binding of CREB to CLS has been shown using granulosa and luteal cells nuclear extracts. In addition, we identified the expression of NR5A1 (Steroidogenic Factor 1) and NR5A2 (Liver Receptor Homologue 1) in granulosa and luteal cells. However, the binding to NREA is observed only with granulosa cells nuclear extracts. Data suggest that the NR5A factors are not the main regulators of CYP19 gene, in contrast with the others genes of streroidogenesis enzymes, and additional sites may play an important role during the post-LH surge down-regulation of CYP19 transcription.

The absence of a functional nuclear receptor element A (NREA) in the promoter II of the aromatase P450 gene in rabbit granulosa cells.

Aromatase protein is synthesized in response to gonadotropins that activate expression of their target genes via the cAMP second messenger system. The -882/+103 bp region of the rabbit ovarian promoter (PII) was ligated to a luciferase vector and transfected into granulosa cells to elucidated the mechanism by which cAMP stimulates transcription. Deletions and mutational experiments indicate that (i) a cAMP-response element-like sequence (CLS) present at -208 to -200 bp is the main element required for the activation of the rabbit PII by cAMP and that (ii) both nuclear receptor element sites; NREA (-133/-126 bp) and NREB (-188/-181 bp) do not participate to the cAMP-dependent activity of the PII. The replacement of the specific rabbit NREA site by the human NREA site increases two-fold the cAMP response and indicates that trans-activating factors are present in rabbit granulosa cells. This study shows for the first time an efficient aromatase transcription occurs in granulosa cells in absence of a consensus NREA site. In addition a comparative study has been performed on the sheep aromatase promoter where sites deviate from rabbit. Mutagenesis experiments suggest that some of them are involved in the cAMP-induced response of the rabbit PII.

Differential regulation of two 3' end variants of P450 aromatase transcripts and of a new truncated aromatase protein in rabbit preovulatory granulosa cells.

In rabbit granulosa cells, two cytochrome P450 aromatase (P450 arom) mRNAs issued from promoter II were described: a full-length and a truncated transcript. Western blot analysis showed two P450 arom proteins with apparent molecular masses of 53 and 46 kDa, which are consistent with the predicted theoretical sizes of proteins encoded by these two transcripts. To examine the involvement of the truncated transcript in the regulation of P450 arom gene expression, the level of each transcript was specifically quantified in cultured granulosa cells by competitive quantitative RT-PCR. FSH induced a dose-dependent increase in both estradiol production and P450 arom mRNAs levels with a much more enhancement in the full-length mRNA. The half-life of the transcripts could not explain this differential regulation. Upon dibutyryl cAMP stimulation, the full-length mRNA was less abundant than the truncated one. In contrast, Western blot analysis revealed a stimulation of the 53-kDa protein content, whereas the 46-kDa protein amount was apparently unaffected. TGF beta in FSH-stimulated conditions decreased both estradiol production and P450 arom transcripts levels. TGF beta did not modify estradiol production and aromatase protein amounts induced by dibutyryl cAMP, whereas the two P450 arom mRNAs levels were increased. In conclusion, we report for the first time that a protein encoded by a truncated P450 arom mRNA could be involved in the regulation of estrogen production. Moreover, we show that the two P450 arom mRNAs are regulated in a differential manner, probably through hormonal control of the alternative splicing.