Fig. 5
Forced expression of Eomes in the germline impairs spermatogenesis and disrupts the homeostasis of undifferentiated spermatogonia. (A) Representative image of testes from control and Eomes conditional overexpression (Eomes-cOE) animals at PD90. Scale bar = 5 mm, n = 3. (B) Ratios of testes (mg) to body weight (g) of control and Eomes-cOE mice at PD90; **p value < 0.01, n = 3. (C) Sperm concentrations of control and Eomes-cOE mice at PD90; *p value < 0.05, n = 3. (D) Fertility test of control and Eomes-cOE mice after pairing with wild-type females; ****p value < 0.0001, n = 3. (E) H&E-stained testicular cross-sections of control and Eomes-cOE mice. The black asterisks indicate seminiferous tubules with impaired spermatogenesis. Scale bars = 100 μm, n = 3. (F) Immunofluorescence staining of testicular cross-sections from control and Eomes-cOE mice. Germ cells, Sertoli cells and undifferentiated spermatogonia were stained with GCNA1, SOX9, and LIN28A, respectively. White asterisks indicate seminiferous tubules devoid of undifferentiated spermatogonia. Scale bars = 50 μm, n = 3. (G) Quantitative analyses of seminiferous tubules with impaired spermatogenesis in control and Eomes-cOE animals. A total of 1000 spermatogenic tubules were quantified; **p value < 0.01, n = 3. (H) Quantification of seminiferous tubules devoid of undifferentiated spermatogonia in control and Eomes-cOE animals. A total of 1000 spermatogenic tubules were quantified; *p value < 0.05, n = 3. (I) The number of germ cells and undifferentiated spermatogonia per 500 Sertoli cells in testicular cross-sections of control and Eomes-cOE animals. At least 500 Sox9 + cells were counted for each sample; **p value < 0.01, n = 3. (J) Whole-mount immunostaining of LIN28A, Ki67 and EdU in the seminiferous tubules of control and Eomes-cOE mice. Spermatogonia are shown by white dotted lines. Scale bars = 50 μm/100 µm, n = 3. (K) Quantitative comparisons of the proportions of As, Apr and Aal spermatogonial cohorts in control and Eomes-cOE mice. At least 1500 LIN28A + cells were counted for each sample; **p value < 0.01, n = 3. (L) Percentage of As spermatogonia among LIN28A+ cells colocalized with EdU and (M) Ki67 from control and Eomes-cOE mice. The data are presented as the mean ± s.e.m. for at least 3 independent experiments. At least 1500 LIN28A + cells were counted for each sample; *p value < 0.05 and **p value < 0.01