Fig 1.
Status of peritubular myoid cells in Wt1SC-cKO testis during fetal mouse testis development.
(A) Immunofluorescence analysis of peritubular myoid cell (PMC) marker α-SMA (FITC, green fluorescence), and proliferation marker PCNA (TRITC, red fluorescence) in cross-sections of control vs. Wt1SC-cKO mouse testes in E13.5 to E18.5. Insets are the corresponding magnified views of the boxed areas. In control testes, α-SMA stained spindle-shaped PMCs. In E13.5 to E15.5 fetal testes, interstitial cells including interstitial progenitor cells were positive for α-SMA. However, the α-SMA expression was strongly up-regulated in PMCs and down-regulated in the interstitial cells in E16.5 to E18.5 fetal testes. The number of PMCs increased during fetal testis cord assembly from E13.5 to E18.5, in addition, most of the α-SMA-positive (α-SMA+) cells were in active cell cycles which were positive for PCNA. In Wt1SC-cKO testes, the number of α-SMA+ cells were down-regulated from E16.5 to E18.5, however, the α-SMA+ cells remained highly proliferative which were positive for PCNA. (B) α-SMA relative fluorescence intensity was used to estimate PMC differentiation status in E13.5 to E18.5 control and Wt1SC-cKO testes. The fluorescence intensity increased in E15.5 to E18.5 in control testes but reduced in Wt1SC-cKO testes, illustrating Wt1 deletion led to a decrease in PMC differentiation. (C) qPCR analysis of PMC marker genes α-Sma, Myh11 and Des in Wt1SC-cKO mouse testes vs. controls in postnatal day 1 (P1). (D) Immunoblot analysis of the PMC-associated protein α-SMA in Wt1SC-cKO mouse testes vs. controls in P1. The steady-state level of α-SMA proteins in P1 mutant testes was significantly down-regulated. (E) Mitotic index expressed as the ratio of PCNA+ and α-SMA+ PMCs to total PMCs (PCNA+ α-SMA+ PMCs:α-SMA+ PMCs). PMC mitotic indexes did not change between control and Wt1SC-cKO testes from E13.5 to E18.5. (F) PMC marker α-SMA (red fluorescence) and apoptotic signals (TUNEL assay) in E14.5 to E18.5 control and Wt1SC-cKO testes were assessed. Insets are the corresponding magnified views of the colored boxed areas. TUNEL+ (green insets) did not overlay with α-SMA+ PMCs in all ages examined (red insets). (G) Effects of Sertoli cell-specific deletion of Wt1 on PMC apoptosis. E, embryonic day; scale bar = 50 μm, and 10 μm in inset, which applies to all micrographs. Each bar is a mean±SEM of n = 3 mice. *, P<0.05; **, P<0.01.
Fig 2.
Fetal Leydig cells (FLCs) differentiate, but having abnormal proliferation status in the interstitium of Wt1SC-cKO mouse testes during fetal testis development.
(A) Immunofluorescence analysis of FLC marker HSD3B1 (FITC, green fluorescence) and proliferation marker Ki67 (TRITC, red fluorescence) in cross-sections of control vs. Wt1SC-cKO mouse testes in E13.5 to E18.5. Insets are the corresponding magnified views of the boxed areas. FLCs found in the testicular interstitium were rarely mitotically active in control testes, while in mutant testes FLC density was gradually increased and some FLCs remained mitotically active (white arrowheads) from E16.5 to E18.5. (B) HSD3B1 relative fluorescence intensity was used to estimate FLC differentiation status in E13.5 to E18.5 control and Wt1SC-cKO testes. The fluorescence intensity increased in E13.5 to E16.5 and maintained in E16.5 to E18.5 in control testes but increased in E13.5 to E18.5 in Wt1SC-cKO testes, illustrating Wt1 deletion led to an increase in FLC differentiation. (C) qPCR analysis of FLC marker genes StAR, Cyp11a1, Cyp17a1, Hsd3b1 in Wt1SC-cKO mouse testes vs. controls in P1. (D) Immunoblot analysis of the FLC-associated protein CYP11A1 and HSD3B1 in Wt1SC-cKO mouse testes vs. controls in P1. The steady-state level of CYP11A1 and HSD3B1 proteins in P1 Wt1SC-cKO testes was considerably up-regulated. (E) Mitotic index expressed as the ratio of HSD3B1-positive and Ki67-positive cells to HSD3B1-positive cells (HSD3B1+ Ki67+ cells:HSD3B1+ cells) was used to estimate mitotic activity of FLC in E13.5 to E18.5. FLCs in control testes were less proliferative, however, the mitotic index of FLC in mutant testes was significantly up-regulated from E16.5 to E18.5. (F) FLC marker HSD3B1 (red fluorescence) and apoptotic signals (TUNEL assay) in E14.5 to E18.5 control and Wt1SC-cKO testes were assessed. Insets are the corresponding magnified views of the colored boxed areas. TUNEL+ (green insets) did not overlay with HSD3B1+ cells in all ages examined (red insets). (G) Effects of Sertoli cell-specific deletion of Wt1 on FLC apoptosis. E, embryonic day. Scale bar = 50 μm, and 10 μm in inset, which applies to all micrographs. Each bar is a mean±SEM of n = 3 mice. **, P<0.01.
Fig 3.
Disruption on the differentiation status of PMC and FLC in Wt1SC-cKO mice that leads to a reduced ratio of PMC:FLC during fetal testis development.
(A) Immunofluorescence analysis of PMC marker α-SMA (TRITC, red fluorescence) and HSD3B1 (FITC, green fluorescence) in cross-sections of control vs. Wt1SC-cKO mouse testes from E13.5 to E18.5. Insets are the corresponding magnified views of the boxed areas. In control testes, the number of α-SMA+ PMCs increased considerably from E13.5 to E18.5 during fetal testis assembly. HSD3B1-stained FLCs were found between testis cords during the development of interstitium. In Wt1SC-cKO testes, deletion of Wt1 in Sertoli cells led to a considerable reduction in PMC number from E15.5 to E18.5. HSD3B1-positive (HSD3B1+) FLCs were found to be differentiated, forming cell clusters from E16.5 to E18.5 in Wt1SC-cKO testes. The α-SMA+ PMCs were found in and around the remnant tubules, and normal testis architecture was not established. (B) The ratio of α-SMA to HSD3B1 relative fluorescence intensity was obtained by measuring the relative fluorescence intensity, which increased in E15.5 to E18.5 in control testes but reduced in Wt1SC-cKO testes, illustrating Wt1 deletion led to changes in the differentiation status of PMC vs. FLC. (C) Consistent with findings shown in (B), the ratio of α-SMA+ PMCs:HSD3B1+ FLCs (α-SMA+ PMCs:HSD3B1+ FLCs) which obtained by scoring these two cell types increased in E15.5 to E18.5 in control testes but reduced in Wt1SC-cKO testes.
Fig 4.
Uncharacterized interstitial progenitor cells are considerably differentiated to FLCs in Wt1SC-cKO vs. control testes during fetal testis development.
(A) Immunofluorescence analysis of uncharacterized interstitial progenitor cell marker VCAM1 (TRITC, red fluorescence), and FLC marker CYP11A1 (FITC, green fluorescence) in cross-sections of control vs. Wt1SC-cKO mouse testes in E14.5 to E18.5. Insets are the corresponding magnified views of the colored boxed areas. In control testes, VCAM1 stained testicular undifferentiated interstitial progenitor cells which contained FLC precursor cells (red box). CYP11A1 stained FLCs which were present between cords (green box). VCAM1 and CYP11A1 double stained early differentiated FLCs (orange box, arrow heads). In Wt1SC-cKO mouse testes, uncharacterized interstitial progenitor cells were found near the remnant cords (red box), and FLCs formed dense clusters in the testicular interstitium (green box). Clusters of early differentiated FLCs were found in E16.5 Wt1SC-cKO mouse testes (orange box, white arrow heads). (B) qPCR analysis of interstitial progenitor cell marker genes Arx, Lhx9, Patched1, Pdgfα, Vcam1, Nestin, Cdh5 in Wt1SC-cKO mouse testes vs. controls in P1. (C) Western blot analysis of the interstitial progenitor cell-associated protein VCAM1 in Wt1SC-cKO mouse testes vs. controls in P1. The steady-state levels of VCAM1 protein was significantly down-regulated. (D) Testicular interstitial progenitor cell relative index to FLC expressed as the ratio of VCAM1-positive (VCAM1+) interstitial cells to CYP11A1-positive (CYP11A1+) FLCs (VCAM1+ cells:CYP11A1+ cells) was used to estimate the balance of interstitial progenitor cell maintenance and FLC differentiation during fetal testis development. The ratio of interstitial progenitor cells to FLCs was down-regulated in Wt1SC-cKO mouse testes from E16.5 to E18.5. (E) FLC differentiation index, expressed as the ratio of VCAM1+ CYP11A1+ cells to VCAM1+ cells (VCAM1+ CYP11A1+ cells:VCAM1+ cells), was used to estimate the differentiation ratio of interstitial progenitor cells to FLCs. FLC differentiation index was increased in Wt1SC-cKO mouse testes from E16.5 to E18.5. E, embryonic day; scale bar = 50 μm, and 10 μm in inset, which applies to all micrographs. Each bar is a mean±SEM of n = 3 mice. *, P<0.05; **, P<0.01.
Fig 5.
Normal vasculature development but down-regulation of Notch signaling are found in Wt1SC-cKO vs. control testes during fetal testis development.
(A) Immunofluorescence analysis of vascular smooth muscle cell (VSMC) marker α-SMA (FITC, green fluorescence), and proliferation marker PCNA (TRITC, red fluorescence) in cross-sections of control vs. Wt1SC-cKO mouse testes in E14.5, E16.5 and E18.5. Insets are the corresponding magnified views of the boxed areas. VSMCs were normally differentiated and proper vasculature was established in both control and Wt1SC-cKO testes during fetal testis morphogenesis. (B) Mitotic index expressed as the ratio of PCNA+ α-SMA+ VSMCs to total VSMCs (PCNA+ α-SMA+ VSMCs:α-SMA+ VSMCs) was used to estimate mitotic activity of VSMC. VSMC mitotic index did not change between control and Wt1SC-cKO testes from E13.5 to E18.5. (C) VSMC marker α-SMA (red fluorescence) and apoptotic signals (TUNEL assay) in E14.5, E16.5 and E18.5 control and Wt1SC-cKO testes were assessed. Insets are the corresponding magnified views of the colored boxed areas. TUNEL+ (green insets) did not overlay with α-SMA+ VSMCs in all ages examined (red insets). (D) Effects of SC-specific deletion of Wt1 on VSMC apoptosis. (E) Immunohistochemistry analysis of Notch signaling ligand JAG1 in cross-sections of control vs. Wt1SC-cKO mouse testes in E14.5, E16.5 and E18.5. JAG1 was expressed in vasculature-associated interstitial cells in control testes. The JAG1 expression level was down-regulated comparing to corresponding controls in Wt1SC-cKO mouse testes in E14.5, E16.5 and E18.5. (F) qPCR analysis of Notch ligand gene Jag1, receptor genes Notch2, Notch3, target gene Hes1 in Wt1SC-cKO mouse testes vs. controls in P1. (G) Western blot analysis of Notch signaling ligand protein JAG1 in Wt1SC-cKO mouse testes vs. controls in P1. The steady-state levels of JAG1 protein was significantly down-regulated. SE, embryonic day; scale bar = 50 μm, and 10 μm in inset, which applies to all micrographs. Each bar is a mean±SEM of n = 3 mice. *, P<0.05; **, P<0.01.