FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development

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   Figure 1

   3β-HSD, FOXL2 and CYP19 expression in a 45 dpc goat ovary. (A) 3β-HSD mRNA detected by in situ hybridization. (B) CYP19 protein and (C) FOXL2 protein detected by immunohistochemistry. (E and F) Double immunolocalization of FOXL2 (blue nuclear staining) and CYP19 (brown cytosolic staining). Note that all CYP19 positive cells also expressed FOXL2. (D) Negative control, sense probe for 3β-HSD. Higher magnification (×4) of a positive region (inside the rectangle) is presented for A, B, C and F in the upper right corner. A line represents the thinness of the cortical region (co) where the majority of germ cells are located. Scale bar in A (for A–D), 200 μm, scale bars in E and F, 50 μm.
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   Figure 2

   CYP19 promoters used during goat ovarian development. (A) Schematic representation of the three different promoter regions found by 5′ RACE analyses; two distal promoters transcribed exon 1.1 and exon 1.6, and one proximal promoter, with two TATA boxes, transcribed directly exon 2 (exon 2a=distal TATA box, exon 2b=proximal TATA box). Promoter specific primers (5A9, 5A8, 5A7) located in the different 5′ non-coding exons are indicated, with the reverse one (5A4) located in the common coding part of the different CYP19 transcripts. (B) RT-PCR expression studies of aromatase tested for all transcripts, with primers located in exons 9 and 10 (E9/E10) or for transcripts specifically initiated at promoters 1.1 or 2, compared with expression profiles of FOXL2 and SPO11, a meiosis-specific factor. E, exon; Days pp, days post partum.
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   Figure 3

   Transfection assays. (A) Schematic representation of the 1249 bp fragment encompassing CYP19 promoters 2 (2a, 2b), 1.5 and 1.6. Circles represent the putative FOXL2 binding sites with no mismatches (black with a 0 inside), one mismatch (dark gray with a 1 inside) or two mismatches (light gray with a 2 inside) in the first three bases of the consensus. The putative Smad-binding site (gray square) near a FOXL2 degenerated site (empty circle) and the putative HNF3/FOXQ1 (black triangle) are also indicated. The location of the 5′ bases of the five different restricted CYP19 promoters cloned in front of the luciferase reporter gene are indicated by dotted lines (-1104, -507, -180, -110, and -40). (B) Comparative induction test of native goat FOXL2 (gFOXL2) and chimerical Foxl2-VP16 on the reporter construct containing three copies of the GRAS element cloned in front of the prolactin promoter driving the luciferase gene. (C) Multiple analyses of luciferase reporter gene driven by different fragments of CYP19 promoter. The different plasmid constructs (pCYP19-Luc) are indicated in the left margin. In COS7 cells, plasmid constructs were transiently co-transfected with a Foxl2-VP16 expression vector or VP16 vector alone. In sheep granulosa cells (oGC) plasmid constructs were transiently co-transfected with a gFOXL2 expression vector or pSG5 vector alone. In both cases, cells were harvested and assayed for luciferase activity. Results are presented as fold induction obtained with Foxl2-VP16 or gFOXL2 compared with VP16 alone or pSG5 alone, in COS7 or oGC respectively.
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   Figure 4

   Chromatin immunoprecipitation assay. Amplification of the CYP19 promoter was positive only in the chromatin immunoprecipitated with the anti-FOXL2 antibody. No signal was detected with the pre-immune serum. We failed to amplify DNA fragments from IMP3 and G3 PDH genes, not supposed to be targets of FOXL2, which excludes a potential non-specific chromatin adsorption. Human DNA stands for the positive control of PCR.
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   Figure 5

   RT-PCR expression studies of the activin/inhibin system, 3βHSD and both estrogen receptors during goat ovarian development. Note the peak of expression centred on 45 dpc for inhibin-α, activin-βA and 3βHSD, similar to those observed for CYP19. ActRII, activin receptor II.
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   Figure 6

   Activin A (A), c-Kit (B) and ERβ (C) immunolocalization in a 45 dpc goat ovary. Higher magnification (×4) of a positive cortical region (inside the rectangle) is presented in the upper right corner for each experiment. A line represents the thinness of the cortical region (co) where the majority of germ cells (gc) are located. Scale bar 200 μm.
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   Figure 7

   Ovarian development in goat during the 20-day period preceding germ cell meiosis. (A) Upper pictures: transverse sections of goat ovaries at 36, 43, 50 and 56 dpc respectively from left to right (m stands for mesonephros). Lower pictures present a fivefold magnification of the region encompassed in the upper rectangles. Note the crucial increase in the gonad during this period, especially in the cortical area (co) where germ cells (gc) are localized. Scale bars in upper pictures, 100 μm; in lower pictures, 20 μm. (B) Schematic representation of the ‘inverse follicle’ organization of the goat fetal ovary.

• © 2006 Society for Endocrinology