Fig. 3

Eomes-positive spermatogonia are a subpopulation of undifferentiated spermatogonia that function as SSCs. (A) Whole-mount immunostaining of PLZF (ZBTB16) (PD60), GFRA1 (PD6) and c-KIT (PD6) with EOMES in seminiferous tubules. Scale bars = 20 μm/50 µm, n = 3. (B) Coimmunostaining of EOMES and ID4 in testes from 6-day-old mice. Scale bar = 10 μm, n = 3. (C) Whole-mount immunostaining of EdU and LIN28A, Ki67 and LIN28A, EdU and EOMES, and Ki67 and EOMES in seminiferous tubules 2 h after EdU injection. Scale bars = 100 μm, n = 3. (D) Quantification of EdU⁺ and ^Ki67+ spermatogonia in Lin28A⁺ cells and EOMES⁺ cells of testes at PD6 and PD60. At least 1500 LIN28A⁺ cells and 500 EOMES⁺ cells were counted at each time point. n = 3. *p value < 0.05, **p value < 0.01, and ***p value < 0.001. (E) Whole-mount immunostaining of EOMES and GFP expression in seminiferous tubules from Eomes-GFP transgenic mice at PD60. Scale bar = 50 μm, n = 3. (F) Whole-mount image of live seminiferous tubules from Eomes-GFP transgenic mice at PD6 and PD60. Scale bars = 20 μm/50 µm, n = 6. (G) Percentages of A_s or A_pr spermatogonia in Eomes⁺ cells. At least 500 EOMES⁺ cells were counted at each time point. n = 6. (H) Schematic diagram of the SSC transplantation experiment. (I) Microscopic examination of testes after receiving Eomes⁺ cells and Eomes⁻ cells. Scale bars = 0.2 mm, n = 6. (J) Donor-derived spermatogenic clones of Eomes⁺, Lin28⁺ cells and Eomes⁻ cells. SSC numbers were derived from donor-derived colonies of spermatogenesis in recipient testes and normalized to the 10⁴ cells injected. * p value < 0.05 and **** p value < 0.0001. Each dot represents a biological repetition