Figure 1.
Expression of ARX in interstitial cells of fetal gonads.
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.
Figure 2.
Expression of Sertoli and Leydig cell marker proteins in Arx KO testes.
Expression of Leydig cell markers, 3β-HSD (A and B), AD4BP/SF-1 (strong signals outside of the testis cords) (C and D), and DAX-1 (strong signals outside of the testis cords) (E and F), and Sertoli cell markers, AD4BP/SF-1 (weak signals in testis cords) (C and D), DAX-1 (weak signals in testis cords) (E and F), MIS (G and H), and WT-1 (I and J) in testes of wild type (Wild type XY) and Arx KO (Arx KO X*Y) mice at E14.5 was investigated by immunohistochemistry. HE staining was also performed for the specimens (K and L). Enclosed areas in A–F are enlarged at the top right in each panel. White and black arrowheads indicate Leydig cells and Sertoli cells, respectively. Dashed lines indicate testis cord. Scale bars = 100 µm.
Figure 3.
Reduction in fetal Leydig cells in Arx KO testes.
Expression of Leydig cell marker 3β-HSD was analyzed in wild type (Wild type XY) (A, C and E) and Arx KO (Arx KO X*Y) (B, D and F) gonads at E12.5, E14.5, and E18.5. Double immunofluorescent staining for 3β-HSD (green) and SOX9 (red) was performed with wild type (G) and Arx KO (H) gonads at E14.5. Dashed lines indicate the gonads. The numbers of Leydig and Sertoli cells were counted using the sections prepared from E14.5 testes and are shown per unit area (I and J). The data are shown as the mean ± SD. * P<0.05. Scale bars = 100 µm.
Figure 4.
Suppressed proliferation in fetal Leydig cells.
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.
Figure 5.
Structural abnormalities induced in Arx KO mouse.
Urogenital systems of wild type (Wild type XY) (A) and Arx KO (Arx 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 Arx KO (Arx KO X*Y) testes were measured at E18.5 (C). The data are indicated as the mean ± SD. *, P<0.05. Expression of Insl3 and Vegfa in wild type (Wild type XY) and Arx KO (Arx KO X*Y) testes at E12.5 was determined by quantitative RT-PCR (D and E). The data were standardized using β-actin (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 Arx 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.
Figure 6.
Expression of gonadal marker genes in Arx KO and Wnt4 KO gonads.
Expression of Dhh (A and A’), Ptch1 (B and B’), Pdgfrα (C and C’), Wnt4 (G-G’’) and Fst (H-H’’) in wild type male (Wild type XY), Arx KO male (Arx KO X*Y) and wild type female (Wild type XX) gonads was examined by in situ hybridization and quantitative RT-PCR (D-F). Quantitative RT-PCR (D–F) was performed at E12.5. The data were standardized using β-actin (Actb) and shown as the mean ± SD. Expression of 3β-HSD and ARX in wild type male (Wild type XY), Wnt4 KO female (Wnt4 KO XX) and wild type female (Wild type XX) gonads was also examined by immunohistochemistry (I–I’’ and J–J’’). Lines and dashed lines indicate the gonadal epithelium and gonad-mesonephros border. Scale bars = 100 µm. g, gonad; m, mesonephros; kid, kidney; ad, adrenal.
Figure 7.
Expression of ARX, 3β-HSD, LHX9, and AD4BP/SF-1 in interstitial cells of fetal testes.
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.