Fig 1: Restoring CARF in Sertoli cells promotes GDNF expression and SSC self-renewal.a The GFP expression in seminiferous tubule driven by the Sox9 promoter from freezing sections of testis were infected with lentivirus (Scale bars, 50 μm). The outlines of seminiferous tubules are indicated by dashed lines. Of note, Leydig cells have autofluorescence which is defined as variable, background fluorescence that can be detected in multiple channels. b Schematic strategy for rescuing CARF expression in Sertoli cells by lentivirus. c Western blot analysis for CARF and GDNF expression in the testes from 8-week-old WT control, CARF−/− mice, and CARF−/− mice were infected with lentivirus. Quantification of blotting intensity for indicated proteins is shown (The WT sample is set as 1.0 after normalization with β-actin blotting). d H&E staining of representative testis sections from 8-week-old WT and CARF−/− mice after lentivirus infection (Scale bars, 200 μm). The white asterisk indicates an SCO seminiferous tubule. Dot graph shows the percentage of SCO seminiferous tubules from 8-week-old WT and CARF−/− mice testis sectional after lentivirus infection (mice, n = 10; sections of one mouse, n > 5). Each dot in the graphs represents an individual testis from one mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by paired t-test, **P < 0.01. e Testis sectional images of PLZF (red) immunostaining and DAPI (blue) staining of WT and CARF−/− mice after lentivirus infection at 8 weeks of age (Scale bars, 50 μm). Dot graph shows the ratios between PZLF+ undifferentiated spermatogonia and Sertoli cells per seminiferous tubule in testis sections from lentivirus-infected WT and CARF−/− mice. Each dot in the graphs represents an individual testis from one mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by paired t-test, **P < 0.01. f The average weight of the testis from 8-week-old WT and CARF−/− mice after lentivirus infection (n = 12). Each dot in the graphs represents an individual testis from one mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by paired t-test, **P < 0.01. g Comparison of male fertility of WT and CARF−/− mice after lentivirus infection (8–16 weeks, n = 8). Each dot in the graphs represents an individual litter. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, *P < 0.05.
Fig 2: Ablation of CARF in undifferentiated spermatogonia results in reduced proliferation of undifferentiated spermatogonia.a Cross-sectional images of PLZF (green) immunostaining, BrdU (red) immunostaining and DAPI (blue) staining of WT control, CARF−/− and CARF−/− + SOX9-CARF mice (CARF−/− mice was infected with lentivirus expressing CARF specifically in Sertoli cells) at 8 weeks of age (Scale bars, 50 μm). The outlines of seminiferous tubules are indicated by dashed lines. Arrowheads indicate PLZF+&BrdU+ cells, and arrows indicate PLZF+ BrdU- cells. Dot graph shows the percentage of BrdU+&PLZF+ cells in total PLZF+ cells of testis sections from 8-week-old WT control, CARF−/− and CARF−/− + SOX9-CARF mice (mice, n = 10; tubules of one mouse, n ≥ 6). Each dot in the graphs represents an individual mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, n.s., not significant, **P < 0.01. b Cross-sectional images of differentiating spermatogonial cells marker c-Kit (green) immunostaining and DAPI (blue) staining of testis sections from 8-week-old WT control, CARF−/− and CARF−/− + SOX9-CARF mice (Scale bars, 50 μm). Dot graph shows the ratios between c-Kit+ cells and PLZF+ cells per seminiferous tubule in testis sections from 8-week-old WT control, CARF−/− and CARF−/− + SOX9-CARF mice (mice, n = 3; tubules of one mouse, n ≥ 10). Each dot in the graphs represents an individual mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, n.s., not significant, **P < 0.01. c Quantitative real-time PCR analysis of mRNA levels of SSC markers in testis and Wnt target genes in SSCs from WT control and CARF−/− mice. Data are expressed as means ± SEM. Fold changes were compared with WT control, and normalized to Rps2.
Fig 3: The depletion of CARF in male mice leads to SCO syndrome and impaired fertility.a Comparison of male fertility of WT control and CARF−/− mice (n = 5) at 8–16 weeks of age. Each dot in the graphs represents an individual litter. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, ***P < 0.001. b Gross morphology of representative testes, epididymides and seminal vesicles from 16-week-old WT control (left) and CARF−/− mice (right). c Average testes weight of WT control mice (n = 11) and CARF−/− (n = 8) at 8–16 weeks of age. Each dot in the graphs represents an individual mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, **P < 0.01. d H&E staining of representative testis sections from 16-week-old WT control and CARF−/− mice (Scale bars, 200 μm). The boxed area is magnified on the right side. The white asterisk indicates an SCO seminiferous tubule. Dot graph shows the ratios of SCO seminiferous tubules in the testis sections from 8- to 16-week-old WT control and CARF−/− mice (mice, n = 6; sections of one mouse, n > 5). Each dot in the graphs represents an individual mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, ***P < 0.001. e Immunostaining for undifferentiated spermatogonia (including SSCs) marker PLZF (red) in testis sections from 16-week-old WT control and CARF−/− mice, with co-staining for DAPI (blue) (Scale bars, 200 μm). The boxed area is magnified on the right side. Dot graph shows the ratios between PZLF+ undifferentiated spermatogonia and Sertoli cells per seminiferous tubule in testis sections from 8- to 16-week-old WT control and CARF−/− mice (mice, n = 5; tubules of one mouse, n ≥ 6). Each dot in the graphs represents an individual mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, **P < 0.01. f Cross-sectional images of PLZF (red) immunostaining, BrdU (green) immunostaining and DAPI (blue) staining of testis sections from 16-week-old WT control and CARF−/− mice (Scale bars, 50 μm). Dot graph shows the percentage of BrdU+&PLZF+ cells in total PLZF+ cells of testis sections from 8- to 16-week-old WT control and CARF−/− mice (mice, n = 8; tubules of one mouse, n ≥ 6). Each dot in the graphs represents an individual mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, *P < 0.05.
Fig 4: Restoration of Wnt signaling substantially rescues fertility defects in CARF−/− mice.a Schematic illustration of the drug administration scheme. b Total testis lysates from CARF−/− mice after drug treatment for 4 weeks were analyzed for GDNF expression by western blotting, with β-actin as a loading control. Quantification of blotting intensity for indicated proteins is shown (The WT sample is set as 1.0 after normalization with β-actin blotting). c H&E staining of representative testis sections from 4-week-old WT control and CARF−/− mice after drug treatment (Scale bars, 200 μm). The white asterisk indicates an SCO seminiferous tubule. Dot graph shows the percentage of SCO seminiferous tubules in testis sections from 4-week-old WT control and CARF−/− mice (mice, n = 12; sections of one mouse, n > 5) after drug treatment. Each dot in the graphs represents an individual mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, **P < 0.01. d Cross-sectional images of PLZF (green) immunostaining, BrdU (red) immunostaining and DAPI (blue) staining of testis sections from 4-week-old WT control and CARF−/− mice after drug treatment (Scale bars, 50 μm). The outlines of seminiferous tubules are indicated by dashed lines. Dot graph shows the percentage of BrdU+&PLZF+ cells in total PLZF+ cells of testis sections from 4-week-old WT control and CARF−/− mice after drug treatment (mice, n = 5; tubules of one mouse, n ≥ 6). Each dot in the graphs represents an individual mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, *P < 0.05. e Ratios between PZLF+ undifferentiated spermatogonia and Sertoli cells per seminiferous tubule in testis sections from 4-week-old WT control and CARF−/− mice after drug treatment (mice, n = 5; tubules of one mouse, n ≥ 6). Each dot in the graphs represents an individual mouse. Bar graphs represent means ± SEM. f Comparison of male fertility in WT control and CARF−/− mice after drug treatment (8–16 weeks, n = 6). Each dot in the graphs represents an individual litter. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, **P < 0.01. g H&E staining of representative testis sections from 16-week-old WT, CARF−/−, APCmin/+ and CARF−/−; APCmin/+ mice (Scale bars, 200 μm). Percentage of SCO seminiferous tubules from 8- to 16-week-old WT, CARF−/−, APCmin/+ and CARF−/−; APCmin/+ testis sectional (mice, n = 8; sections of one mouse, n > 5). Each dot in the graphs represents an individual mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, **P < 0.01. h The average weight of testes from 8- to 16-week-old WT, CARF−/−, APCmin/+, and CARF−/−; APCmin/+ mice (n = 3). Each dot in the graphs represents an individual mouse. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, **P < 0.01.
Fig 5: Decreased GDNF expression in Sertoli cells causes defective SSC self-renewal.a Quantitative real-time PCR analysis of mRNA levels of Wnts and Wnt target genes in Sertoli cells from 10-day-old WT control and CARF−/− mice. Data are presented as means ± SEM. Fold changes were compared with WT controls, and are normalized to β-actin. b Western blot analysis of GDNF in Sertoli cells from WT control and CARF−/− mice. Quantification of blotting intensity for indicated proteins is shown on the top (The first WT sample is set as 1.0 after normalization with β-actin blotting). c Immunostaining for GDNF (red) in testis sections from 10-day-old WT control and CARF−/− mice, with co-staining for undifferentiated spermatogonial marker PLZF (green) and DAPI (blue) (Scale bars, 20 μm). The outlines of seminiferous tubules are indicated by dashed lines. Dot graph shows the relative intensities of immunostaining for GDNF, data are presented as means ± SEM. Statistical analysis was performed by two-tailed t-test, **P < 0.01. d Testis cross-sectional images of β-catenin (green) immunostaining, CyclinD1 (red) immunostaining and DAPI (blue) staining of WT control and CARF−/− mice at 10 days of age (Scale bars, 20 μm). The outlines of seminiferous tubules are indicated by dashed lines. Dot graph shows the relative intensities of immunostaining for CyclinD1, data are presented as means ± SEM. Statistical analysis was performed by two-tailed t-test, **P < 0.01. e The donor-derived GFP-positive colonies within seminiferous tubule of recipient testis 2 months after transplantation. f GFP-positive pups were produced by donor cell-derived sperm. Comparison of the number of GFP-positive pups of WT and CARF−/− recipient (n = 3) that was transplanted with WT GFP-positive germ cells and mated with a WT female mouse within 2 months. Each dot in the graphs represents an individual litter. Bar graphs represent means ± SEM. Statistical analysis was performed by two-tailed t-test, **P < 0.01.
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