Fig. 6

Androgen regulates Sertoli cell phagocytosis of apoptotic germ cells via miR-471-5p-Dock180/autophagy protein signaling pathway. a Western blot analysis on enriched Sertoli cells isolated from Sham, flutamide–acyline (Flu+Acy), and flutamide-acyline-testosterone (Flu+Acy+T)-treated mice testes using antibodies against Dock180, Atg12, Tecpr1, and Becn1. β-Actin was used as a loading control. Gel photograph is representative of two independent experiments; Sertoli cells were pooled from 7 mice per experiment. Bar graphs showing quantification of band intensities are shown in Supplementary Fig. 11a. b Immunofluorescence analysis showing number of pHrodo-labeled apoptotic germ cells engulfed by Sertoli cells in the presence (+) and absence (−) of testosterone. c Histogram showing number of Sertoli cells engulfing apoptotic germ cells in the absence (−T) and presence (+T) of testosterone as derived from the flow cytometry analysis. The data are presented as mean ± SEM of four independent experiments. ****p < 0.0001; two-tailed unpaired student t-test. d Immunofluorescence analysis showing engulfment of pHrodo-labeled apoptotic germ cells by primary Sertoli cells isolated from flutamide/acyline (Flut+Acy)-treated mice and cultured in charcoal-stripped medium and transfected with scramble (Scr) and miR-471-5p antagomiR (Anti-miR-471-5p). e Histogram showing number of primary Sertoli cells engulfing germ cells in experimental groups described in d. The data are presented as mean ± SEM of three independent experiments. ****p < 0.0001, two-tailed unpaired student t-test. f Immunofluorescence analysis showing in vitro phagocytosis by purified primary Sertoli cells from Sham, flutamide-acyline-treated (Flu+Acy), and flutamide-acyline-treated and supplemented with testosterone (Flut+Acy+T) mice. Purified Sertoli cells were cultured for 48 h in complete media, charcoal-stripped media, or charcoal-stripped media supplemented with testosterone before being subjected to phagocytosis assay. Sertoli cells are marked with white dotted line to clearly indicate outer border. g Histogram showing percentage of Sertoli cells from Sham, Flu+Acy, and Flu+Acy+T-treated mice engulfing apoptotic germ cells as derived from flow cytometry analysis. The data are presented as mean ± SEM of three independent experiments. *p < 0.01, one-way ANOVA followed by Tukey’s multiple comparisons test. Scale bar indicates 25 μm (b, f); 20 μm (d)