Foxo3b but not Foxo3a activates cyp19a1a in Epinephelus coioides

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   Figure 1

   Phylogenetic analysis of the orange-spotted grouper Foxo3a and Foxo3b with Foxo homologs in other vertebrates. The phylogenetic tree was constructed by MEGA 6.0 using the neighbor-joining method. Data were resampled by 1000 bootstrap replicates. The orange-spotted grouper Foxo3a and Foxo3b were shown in bold. The protein sequences were downloaded from Entrez (NCBI): Cattle, Bos taurus; Human, Homo sapiens; Mouse, Mus musculus; Pig, Sus scrofa; Frog, Xenopus laevis; Medaka, Oryzias latipes; Tilapia, Oreochromis niloticus; Platyfish, Xiphophorus maculatus; Zebrafish, Danio rerio.
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   Figure 2

   Tissue distributions of foxo3a and foxo3b mRNAs in the female orange-spotted grouper as analyzed by RT-PCR. M: 100 bp DNA ladder marker (Takara); a, olfactory bulb; b, telencephalon; c, mesencephalon; d, cerebellum; e, pituitary; f, hypothalamus; g, medulla oblongata; h, ovary; I, retina; j, thymus gland; k, head kidney; l, spleen; m, muscle; n, skin; o, liver; p, stomach; q, foregut; r, midgut; s, hindgut; t, fat; u, kidney; v, heart; w, blood; x, swim bladder; y, gill filament; z, gill arch; RT-, RT minus (no addition of reverse transcriptase); NC, negative control (water used as template).
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   Figure 3

   Western blotting analysis of immunoreactive Foxo3a and Foxo3b in tissues of the female orange-spotted grouper. The tissue homogenates (500μg) from the brain, ovary, spleen, and liver were separated on 12% SDS–PAGE gels, transferred to polyvinylidenefluoride membranes, and then immunoreacted with: (A) the mouse anti-grouper Foxo3a antiserum (1:1000); (B) anti-Foxo3a antiserum pre-absorbed by excessive recombinant full-length Foxo3a; (C) the mouse anti-grouper Foxo3b antiserum (1:1000); (D) anti-Foxo3b antiserum pre-absorbed by excessive recombinant full-length Foxo3b; (E) mouse anti-ACTB monoclonal antibody (1:500; catalog number: 66009-1-Ig; ProteinTech Group, Inc.). The secondary antibodies were 1:1000 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) (catalog number: 115-035-003; Jackson ImmunoResearch Laboratories). The blots were visualized using the BeyoECL Plus Kit (Beyotime). The molecular standards identified with relative molecular mass (M_r×10⁻³) were shown to the left of images.
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   Figure 4

   Localization of immunoreactive Foxo3a and Foxo3b signals in the ovary of the orange-spotted grouper during the ovarian development. Foxo3a immunostaining (A–E), Foxo3b immunostaining (F–J), and HE staining (K–O) were performed on sections from the same ovarian tissue at each stage. The mouse anti-Foxo3a antiserum and mouse anti-Foxo3b antiserum at a dilution of 1:200 were used as the primary antiserum, respectively. The secondary antibody was 1:500 diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L) (catalog number: 115-035-003; Jackson ImmunoResearch Laboratories, Inc). The IHC sections were visualized by DAB chromogen. The insets in C, D, E, H, I, and J are higher magnification of the boxed areas within each image, respectively. OO, oogonia; PGO, primary growth oocytes; FC, follicular cells. Scale bar is 25μm.
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   Figure 5

   Immunoreactive levels of Foxo3a and Foxo3b in the follicular cells during the ovarian development of the orange-spotted grouper. CO, cortical-alveolus stage follicle; EV, early vitellogenic stage follicle; MV, mid-vitellogenic stage follicle; MO, mature stage follicle. Each bar represents mean ± s.e.m. (n=3). *P < 0.05 vs CO (except Foxo3b between EV and CO) and MO. ^#P < 0.05 vs CO, EV, and MO.
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   Figure 6

   The sub-cellular localization of immunoreactive Foxo3a (red) and Foxo3b (red) and their co-localization with Cyp19a1a (yellow) in follicular cells surrounding the early vitellogenic (A–D), mid-vitellogenic (E–H), and mature oocytes (I–L). The mouse antiserum against Foxo3a or Foxo3b (1:100) and rabbit antiserum against Cyp19a1a (1:100) were used as primary antisera. The secondary antibody was Cy3-labeled goat-anti-mouse for Foxo3a or Foxo3b, and FITC-labeled goat-anti-rabbit for Cyp19a1a. DAPI was used to stain the nuclei blue. The images were observed and captured under a confocal microscope under the same conditions. The insets within each image are higher magnification of the boxed areas, respectively. The overlapping of the red with the blue or green color generated a purple or yellow color, respectively. Scale bar is 10µm.
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   Figure 7

   Foxo3a and Foxo3b upregulated the orange-spotted grouper cyp19a1a promoter activities via the Foxo consensus site. (A) Schematic diagram of the serial deletion promoter constructs for the orange-spotted grouper cyp19a1a containing either the wild-type (WT) or mutated (MUT) Foxo site. The mutated Foxo site was indicated by an “X”. The promoter reporter vectors (280 ng/well) were transiently co-transfected into COS-7 cells (B) or TM4 cells (C) with the orange-spotted grouper Foxo3a (black bar) or Foxo3b (diagonal bar) expression vectors, and an internal control vector pRL-TK (20 ng/well). The total amount of plasmid was adjusted to 400 ng/transfection using an empty expression vector (pcDNA3.0). The omission of the expression vector for Foxo3a and Foxo3b was set as the control. Luciferase activities were measured 48 h after transfection, and data were expressed as fold induction relative to the control. Bars represent means±s.e.m. of triplicates. *P < 0.05 for the differences between the indicated groups.
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   Figure 8

   Gel-shift analysis of Foxo3a and Foxo3b binding to the putative Foxo site in the orange-spotted grouper cyp19a1a promoter. (A) Schematic representation of oligonucleotide probes containing the wild-type (W; underlined) or site-mutated (M; italicized) Foxo site. The co-ordinates of the ends of the oligonucleotides and of the binding sites are listed below in base pairs relative to the transcription start site for the cyp19a1a mRNA. (B) Foxo3a binding to the oligonucleotide probe. (C) Foxo3b binding to the oligonucleotide probe. The ³²P end-labeled double-stranded oligonucleotide probe containing a wild-type or mutated Foxo-binding site was incubated with nuclear extracts of COS-7 cells transfected with or without Foxo3 expression vector. Unlabeled wild-type (W) or mutant (M) probe was included in the reaction (×50 excess) as a competitor to verify the specific binding. C7, the nuclear extracts of COS-7 cells; P3, the nuclear extracts of COS-7 cells transfected with pcDNA3.0 plasmid; EcFoxo3a, the nuclear extracts of COS-7 cells transfected with Foxo3a expression vector; EcFoxo3b, the nuclear extracts of COS-7 cells transfected with Foxo3b expression vector; Np, without addition of COS-7 nuclear proteins; Nu, without addition of unlabeled probes.
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   Figure 9

   ChIP analysis of Foxo3a and Foxo3b binding to the proximal promoter region (−246/+67 bp) of the orange-spotted grouper cyp19a1a in the ovary at different developmental stages. The DNA fragments were immunoprecipitated using specific antibodies against Foxo3a, Foxo3b, RNA polymerase II, or normal mouse IgG, and analyzed with PCR amplification of 38 cycles. The PCR products were separated on 2.0% agarose gels and visualized by staining with ethidium bromide. M, DL1000 bp DNA marker (Takara); EVO, early vitellogenic stage ovary; MVO, mid-vitellogenic stage ovary; MO, mature ovary; Li, liver. The experiments were repeated at least twice, and similar results were obtained.

• © 2016 Society for Endocrinology