a Immunofluorescent labeling of 0-, 10-, 30- and 50-day cultured gonadal tissues co-labeled for PLZF (red) and Ki67 (green). PLZF+Ki67+ cells (white triangles). Scale bar = 10 µm. b Percentage of PLZF-positive KI67-positive cells from three biological replicates. (Each dot represents the percentage in the cross section of a seminiferous tubule, different colors of the dots represent different specimens). c Immunostaining of cultured tissues for SYCP3 (green), γH2AX (red), MLH1 (red) and SYCP1 (green). DNA was counterstained with Hoechst 33342 (blue). Scale bar = 10 µm. d Transmission electron microscopy shows ultrastructural findings of seminiferous epithelium. Spermatids: white stars; spermatogonia: yellow stars; primary spermatocytes with nuclear synaptonemal complexes: blue stars; spermatids with acrosome vesicles: yellow triangle; and early tail structure: white triangles. Scale bars, 10 µm (a); 2 µm (b, d); 200 nm (c); 1 µm (e). e DNA content distribution of cells from 0- to 50-day cultured tissues by FACS. Haploid (1C), diploid (2C), and tetraploid (4C) cells are indicated. f Percentages of haploid cells in FACS analysis of 11 cultured samples.