Fig. 4: 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.