Figure 1.
Comparative analysis of AR, SOX9 and AMH expression in adult human testes representing androgen-insensitivity syndrome (AIS), Sertoli-cell-only syndrome (SCOS), and normal spermatogenesis.
The localization and expression of AR, SOX9, and AMH proteins were analyzed by immunohistochemical analysis. (A, D, G) AR in normal, SCOS and AIS testes. (B, E, H) SOX9 in normal, SCOS and AIS testes. (C, F, I) AMH in normal, SCOS and AIS testes. No AR expression was detected in AIS testes. Note that all Sertoli (S) and peritubular myoid (M) cell nuclei showed AR expression in SCOS and normal samples. Leydig (L) cells with AR-positive nuclei were not consistently detected in SCOS. SOX9 expression was predominantly observed in the Sertoli cells. AMH was strongly expressed in the Sertoli cell cytoplasm of AIS and SCOS testes. The corresponding histograms show the mean relative intensities of DAB in the human testes biopsies. Automated acquisition and tissue cytometric cell measurement analysis were performed using the TissueFAXS system (TissueGnostics, Vienna, Austria) and HistoQuest (TissueGnostics) Analysis Software. There was an inverse correlation between the immunostaining density of AR and those of SOX9 and AMH in human testes. Magnification ×400; bar =20 µm. (J) The AR, SOX9, AMH and (K) several sertoli cell markers gene transcripts in testicular tissues from men between control and SCOS group were analyzed by real-time RT-PCR. The mRNA expression of SOX9 and AMH gene had an increasing trend, but AR gene transcript had a decreasing trend in the SCOS group compared to the normal spermatogenesis group. All data are representative of at least three independent experiments and each bar represents mean ± SD. Concomitant detection of 18S/ GAPDH mRNA served as a reference for relative quantification. *P <0.05.
Figure 2.
Immunohistochemical analysis of mouse testes.
(A) AR, SOX9 and AMH immunostaining of testes obtained from wild-type and AR knockout (bottom panel) mice. Note that all Sertoli (S) cell nuclei, peritubular myoid (M) cell nuclei and Leydig (L) cells were AR-positive in wild-type mouse testes. Sertoli (S) cell nuclei showing SOX9 expression and Sertoli (S) cell cytoplasm with weaker AMH immunostaining for a wild-type mouse are shown in the upper panel. Sertoli (S) cell nuclei showing SOX9 distribution in an AR knockout mouse. Specific and robust AMH immunostaining was detected exclusively in the cytoplasm of Sertoli (S) cells in AR-knockout mouse testes with deficient spermatogenesis. Magnification ×400; bar = 20 µm. (B) Quantitative evaluation of AR ,SOX9 AMH in testicular tissues from wild-type and AR-knockout mice by real time RT-PCR. Each bar represents the mean ± SD; results were normalized with respect to 18S/GAPDH mRNA expression. SOX9 and AMH mRNA expression was significantly higher in testes from AR-knockout mice compared to controls. (C) SOX9 and AMH protein expression was significantly higher in testes from AR-knockout mice compared to controls by Western blot. (D) Co-immunostaining for AR and SOX9 Sertoli (S) cell nuclei of wild-type mouse testis. Magnification ×400. bar = 20 µm.
Figure 3.
The expressions of SOX9 and AMH are repressed by androgen/AR.
(A and B) TM4 cells transfected with AR RNAi and treated with or without DHT for 24~48 hours were analyzed by Western blot and quantitative real-time RT-PCR. (C and D) C3H10T1/2 cells transfected with pcDNA3-flag-AR plasmid, followed by incubation with or without 1x10-8 M DHT for 24~48 hours. The expression of AR, SOX9 and AMH were analyzed by Western blot and quantitative real-time RT-PCR. The optical densities obtained for protein and mRNA expression from vector with vehicle treatment were normalized using GAPDH,β-tubulin or β-actin expression levels and set as 1. All data are representative of at least three independent experiments and error bars represent ± SD. Asterisks (*) mark samples significantly different with P <0.05.
Figure 4.
SOX9 is required for androgen/AR to repress AMH expression.
C3H10T1/2 cells stably transfected with SOX9 RNAi and treated with or without DHT for 24~48 hours and the expression of SOX9 and AMH were analyzed by Western blot (A) and quantitative real-time RT-PCR (B). The optical densities obtained for protein and mRNA expression from vector with vehicle treatment were normalized using GAPDH or β-actin expression levels and set as 1. All data are representative of at least three independent experiments and error bars represent ± SD. Asterisks (*) mark samples significantly different with P <0.05.