Expression of EGFP induced by FLE in fetal and postnatal gonads.

<p>The gonads (ovary and testis) of mFLE-EGFP transgenic mice were prepared in E15.5 (A and B, n = 2), P7 (C and G, n = 4), P14 (D and H, n = 2), P21 (E and I, n = 4), and adult stage at P42 or P56 (F and J, n = 4). Whole views of the gonads are shown. B is a fluorescence view of A. The ovaries are delineated by broken lines. Scale bars = 500 μm.</p

Overlapped expression of EGFP driven by BAC-Ad4BP and mCherry by mFLE.

<p>Ad4BP-BAC-EGFP transgenic mice were crossed with mFLE-mCherry transgenic mice to generate double transgenic mice (n = 3). Fluorescence views of the adult ovary are shown (A-C). The ovaries were subjected to immunofluorescence with the antibodies for EGFP (green in D and G) and mCherry (red in E and H). Merged views of EGFP and mCherry are shown in F and I, which are further stained with DAPI (blue). Arrows in F and I indicate theca cells (t) or the interstitial gland (ig). Insets are enlarged views of the areas enclosed by rectangles. t, theca cells; ig, interstitial gland. Scale bars for A-C = 500 μm and those for D-I = 100 μm.</p

Distribution of EGFP-positive cells in mFLE-EGFP gonads.

<p>mFLE-EGFP transgenic mouse ovaries at P7 (A, n = 4), P14 (B, n = 2), P21 (C, n = 4), and adult stage at P42 or P56 (D, F, G-I, M-O, n = 4), and testes at E18.5 (E) and adult stage at P42 or P56 (J-L, P-R) were sectioned and subjected to immunofluorescence with antibodies for EGFP (green), Ad4BP/SF-1 (green in E, and red in others), and 3β-HSD (red). Merged images for Ad4BP/SF-1 and 3β-HSD (E, F), EGFP and Ad4BP/SF-1 (I, L), and EGFP and 3β-HSD (O, R) are shown. A-F, I, L, O, and R were further stained with DAPI (blue). Arrows in E indicate Sertoli cells (Se) or Leydig cells (Le). Arrows in F, I, L, O, and R indicate theca cells (t) or the interstitial gland (ig). Closed white arrowheads in G-L indicate cells double positive for EGFP and Ad4BP/SF-1, while those in M-R indicate cells double positive for EGFP and 3β-HSD. Open arrowheads in G-L indicate single positive cells for Ad4BP/SF-1, while those in M-R indicate single positive cells for 3β-HSD. Insets are enlarged views of the areas enclosed by rectangles. t, theca cells; ig, interstitial gland; gr, granulosa cells; Le, Leydig cell; Se, Sertoli cell; tc, testicular cord; is, interstitial space. Scale bars = 100 μm.</p

Expression of EGFP induced by Ad4BP-BAC-EGFP.

<p>The ovary (A), testis (B), adrenal gland (C), pituitary (D), and VMH (E), in which endogenous <i>Ad4BP/SF-1</i> is expressed, were prepared from adult Ad4BP-BAC-EGFP transgenic mice (n = 3). EGFP expression was observed under a fluorescent microscope. The testis (F-H), adrenal gland (I-K), pituitary (L-N), VMH (O-Q), and spleen (R-T) were sectioned, followed by immunofluorescence with antibodies for EGFP (green in F, I, L, O, and R) and Ad4BP/SF-1 (red in G, J, M, P, and S). Merged views of EGFP and Ad4BP/SF-1 are shown in H, K, N, Q, and T, which are further stained with DAPI (blue). Insets are enlarged views of the areas enclosed by rectangles. st, seminiferous tubule; adc, adrenal cortex; ess, endothelial cell of splenic sinus. Scale bars in A-E = 500 μm and those in F-T = 100 μm.</p

Distribution of EGFP-positive cells in Ad4BP-BAC-EGFP ovary.

<p>The adult ovaries at P42 or P56 were prepared (n = 3) and subjected to immunofluorescence with the antibodies for EGFP (green in A-C and J-L), Ad4BP/SF-1 (red in D-F), and 3β-HSD (red in M-O). Merged views for EGFP and Ad4BP/SF-1 are shown in G, H and I, while those for EGFP and 3β-HSD are shown in P, Q, and R; these are stained simultaneously with DAPI (blue). Arrows in G, H, P and Q indicate theca cells (t) or the interstitial gland (ig). Insets are enlarged views of the areas enclosed by rectangles. f, follicle; t, theca cells; ig, interstitial gland; cl, corpus luteum. Scale bars = 100 μm.</p

Construction of BAC transgene.

<p>Construction of the BAC transgene is shown. The BAC clone (RP23-354G20) used in this study contained <i>Nr6a1</i>, <i>Gpr144-ps</i>, and <i>Psmb7</i> genes in addition to <i>Ad4BP/SF-1</i> as indicated at the top. The directions of gene transcription are indicated by arrows. <i>Ad4BP/SF-1</i> in the BAC was replaced using the targeting vector by recombination using the 5’ and 3’ arms. Ad4BP-BAC-EGFP was finally obtained by FLP-mediated deletion of the 3’ segment of the targeting vector integrated into the BAC. Detailed procedures are described in the Materials and Methods. <i>Nr6a1</i>, nuclear receptor subfamily 6, group A, member 1 (<i>Gcnf</i>); <i>Gpr144-ps</i>, G protein-coupled receptor 144, pseudogene; <i>Psmb7</i>, proteasome subunit, beta type 7; Neo, neomycin resistant gene; pA, poly A; FRT, flippase recombination target; FLP, flippase; EGFP, enhanced green fluorescence protein.</p

Expression of ARX, 3β-HSD, LHX9, and AD4BP/SF-1 in interstitial cells of fetal testes.

<p>Expression of ARX (red), AD4BP/SF-1 (blue), and 3β-HSD (green) in fetal testis at E13.0 was examined. Merged images of ARX and 3β-HSD (upper panels of A–E), and ARX and AD4BP/SF-1 (lower panels of A–E) are shown. The arrowhead in A indicates an ARX-strongly positive, AD4BP/SF-1-negative, and 3β-HSD-negative cell. The arrowhead in B indicates an ARX-weakly positive, AD4BP/SF-1-weakly positive, and 3β-HSD-negative cell. The arrowhead in C indicates an ARX-weakly positive, AD4BP/SF-1-weakly positive, and 3β-HSD-weakly positive cell. The arrowhead in D indicates an ARX-weakly positive, AD4BP/SF-1-strongly positive, and 3β-HSD-modestly positive cell. The arrowhead in E indicates an ARX-negative, AD4BP/SF-1-strongly positive, and 3β-HSD-strongly positive cell. Cells showing expression of the marker proteins above are illustrated (F). Expression of LHX9 (green) and ARX (red) in E11.5 male gonads was examined (G-I). Dashed lines in G-I indicate the gonad-mesonephros border. Expressions of MAFB (green) and AD4BP/SF-1 (red), and ARX (green) and AD4BP/SF-1 (red) in E11.5 male gonads were examined on consecutive sections (J–O, and J’–O’ for enlarged views). Arrows in J and L indicate autofluorescence of blood cells. Dashed lines in J’–O’ indicate border between MAFB or ARX-positive cells and AD4BP/SF-1-positive cells. Arrowheads in J’ and L’ indicate MAFB-positive cells just beneath AD4BP/SF-1-positive cells. Scale bars = 100 µm. g, gonad; m, mesonephros.</p

Structural abnormalities induced in <i>Arx</i> KO mouse.

<p>Urogenital systems of wild type (Wild type XY) (A) and <i>Arx</i> KO (<i>Arx</i> KO X*Y) (B) male mice at E18.5 are shown. Arrows indicate the testes. tes, testis; b, bladder; kid, kidney. Intratesticular testosterone levels of wild type (Wild type XY) and <i>Arx</i> KO (<i>Arx</i> KO X*Y) testes were measured at E18.5 (C). The data are indicated as the mean ± SD. *, P<0.05. Expression of <i>Insl3</i> and <i>Vegfa</i> in wild type (Wild type XY) and <i>Arx</i> KO (<i>Arx</i> KO X*Y) testes at E12.5 was determined by quantitative RT-PCR (D and E). The data were standardized using <i>β-actin</i> (Actb) and shown as the mean ± SD. *, P<0.05. Locations of the sections of the gonad are schematically shown with horizontal lines with numerals, 1 to 6 (F). The coelomic blood vessel is indicated with red line. Double immunofluorescent staining for LAMININ (green) and PECAM (red) was performed with the serial sections (1 to 6, corresponding to the numerals in (E)) with 100 µm interval of wild type and <i>Arx</i> KO mouse testes at E12.5 (G). Arrowheads indicate the coelomic blood vessel in wild type, while the corresponding structure could not be observed at this stage in the KO testis. Scale bars = 100 µm.</p

Suppressed proliferation in fetal Leydig cells.

<p>Cell proliferation was evaluated by BrdU incorporation studies with three fetal testes. BrdU labeled fetal testes at E12.5, E14.5 and E16.5 were sectioned and the sections were immunostained with antibodies for 3β-HSD (green) and BrdU (red) (left panels in A; Leydig cell), and with SOX9 (green) and BrdU (red) (right panel in A; Sertoli cell). Enclosed areas are enlarged at the top right in each panel. Arrows indicate cells double-positive for SOX9 and BrdU. Scale bars = 100 µm. Numbers of 3β-HSD and BrdU double-positive cells (closed bars) and 3β-HSD single-positive cells (open bars) (B), and numbers of SOX9 and BrdU double-positive cells (closed bars) and SOX9 single-positive cells (open bars) (C) were counted (n = 3). The numbers of these cells per unit area are plotted. The data are indicated as the mean ± SD. *, P<0.05, **, P<0.01.</p

Expression of ARX in interstitial cells of fetal gonads.

<p>Expression of ARX was examined by immunohistochemistry using an anti-ARX antibody. Wild type male (XY) (A–D) and female (XX) (I–L) gonads of mouse fetuses at E11.5, E12.5, E14.5, and E18.5 were tested. Double immunofluorescent staining for ARX (green) and AD4BP/SF-1 (red) was performed with male (E–H) and female (M-P) gonads at the same stages. Dashed lines indicate the gonad-mesonephros border. Scale bars = 100 µm. Whole gonadal extracts (5 µg) prepared from mouse fetuses of both sexes at E11.5, E12.5, E13.5, E14.5, and E18.5 were subjected to western blot analysis using anti-ARX and anti-α-tubulin antibodies (Q). The location of 50 and 75 kDa protein markers are indicated. Arrowheads in L and P indicate middle part of ovary and mesovarium, respectively. Magnified views of E18.5 testis are shown (R-U). Sections are counterstained by DAPI (blue, T and U). Arrowheads in R-U indicate ARX-positive peritubular myoid cells and arrows in T and U indicate ARX-negative and DAPI-positive (blue) endothelial and unknown interstitial cells, respectively. Scale bars = 25 µm. ad, adrenal; tc, testis cord; bv, blood vessel.</p