Fig. 4

Culture of isolated Thy1 positive spermatogonia and treatment with retinoic acid (RA). a The cell morphology of Thy1 positive spermatogonia cultured for 15 days, showed single, paired and aligned cells. Arrows indicate the intercellular bridges. Bar: 100 μm and 50 μm. b The mRNA levels of TFEB and spermatogonial differentiation markers, Kit, Sohlh2 and Stra8, in cultured spermatogonia with RA treatment, showed that expression levels of TFEB and spermatogonial differentiation markers were maximized after RA treatment for 24 h. Error bars represent SD (n = 6). **p < 0.01. c Immunofluorescence detection of TFEB (green fluorescence) and GFRA1 (red fluorescence) in cultured spermatogonia showed greatly reduced GFRA1 in cell surface and nuclear translocation of TFEB after RA treatment. Arrows indicated the nucleus. Bar: 5 μm. d The mRNA levels of TFEB and spermatogonial markers in cultured spermatogonia with RNAi and RA treatment. TFEB expression was significantly increased after RA treatment for 24 h, Error bars represent SD (n = 6). **p < 0.01. and significantly decreased after shRNA lentiviral transduction. Error bars represent SD (n = 6). * p < 0.05