La régulation du gène CYP19A1 dans les cellules de granulosa bovine in vitro

Oestradiol plays an important role in reproduction in general, particularly during follicular growth. Production of estradiol requires the expression of CYP19A1 following stimulation of granulosa cells by follicle-stimulating hormone (FSH) and insulin like growth factor-1 (IGF-1). In cows, there are six promoters (1.1, 1.2, 1.3, 1.4 and 1.5 and 2) that direct transcription of CYP19A1, and promoter 2 (P2) is the major promoter used in granulosa cells. However the effect of FSH and IGF-1 on the activation of these promoters of aromatase remains unclear. Further, the CYP19A1 gene has a very short half-life and a long 3' non-translated region (3'UTR) that suggests post-transcriptional as well as transcriptional regulation. The aim of my PhD project is to study the regulation of the CYP19A1 gene in the cow. This summary is divided into two parts, the transcriptional regulation involving the promoter region and the post-transcriptional regulation involving the 3'UTR.  The first part of my project was to study the transcriptional regulation of CYP19A1 gene; we measured the expression of the different promoters in luteinized or nonluteinized bovine granulosa cells following stimulation of cells with FSH or IGF-1. The results of RT-PCR showed that FSH and IGF-1 increases mRNA levels from both promoters 2 and 1.1 in non luteinized granulosa cells. Subsequent experiments showed that FSH stimulates the promoter 2 via the PKA pathway and IGF-1 stimulated promoter 2 via the PKC pathway. FSH and IGF-1 stimulate the expression of 1.1 via the PI3K pathway. In subsequent studies in luteinized cells with luciferase reporter genes driven by the specific CYP19A1 promoters, FSH stimulated promoter 1.1 in a dose dependent manner but that promoter 2 was weakly activated and not responsive to FSH. We then compared the activity of human, rat, goat and bovine promoters in luteinised bovine granulosa cells. The most significant result is that the bovine (and caprine) P2 depends on several transcription factors (NR5A2, FOXL2) whereas the human and rat promoters largely depend on cAMP. In fact, these data demonstrate a reasonably robust expression of the bovine P2 when treated with forskolin, NR5A2 and FOXL2. FOXL2 appears to be a determinant of promoter activity in ruminants.  The second part of my project was to study the post-transcriptional regulation of the CYP19A1 gene. The objective was to identify the elements required for the regulation of the half-life of CYP19A1 mRNA. To do so, we generated and inserted different fragments of the 3'UTR region of CYP19A1 mRNA in the pGL3promoter vector downstream of the luciferase gene, which was then transfected into luteinized granulosa cells to assess the impact of the 3'UTR sequence on the expression of luciferase reporter gene. We identified a sequence of 200 bp between 926 and 1134 bp of the 3'UTR region of CYP19A1 mRNA that  significantly reduced luciferase activity. The same fragments were inserted into the pGEMTeasy vector for in vitro transcription and the generation of mRNA for UV crosslinking with protein extracts to demonstrate the presence of mRNA/protein complexes. We detected protein complexes of 66 and 80KDA that specifically bound to the 200 pb probe. This protein is expressed in granulosa cells but not in other tissues such as the liver and heart. The use of reporter gene attracted the interest of a company producing equine chorionic gonadotropin (eCG), and an interest was expessed in developing this system to measure the FSH-like bioactivity in eCG, and therefore have a more effective commercial product. In this study, we developed a FSH bioassay system based on the transfection of cells with an the FSH receptor and a colorimetric reporter gene to estimate the activity of FSH in the serum of the mare ; these results may be applicable at the farm / industry.

Keyphrases: Aromatase, 3'UTR, eCG, Promoter, Granulosa cell