Fig. 4

Dihydrotestosterone promotes generation of ROS and TGF-β in GCs. a The expression of FSHR and LHR in GCs and TCs was measured by immunofluorescence staining (Alexa Fluor 488) and the nuclei were stained with DAPI (40×). Images are representative of three independent experiments with similar results. b GCs treated with various concentrations of DHT for 24 h followed by immunoblot analysis of the AR and TGF-β proteins. c GCs were pre-treated with DHT (500 nM) for 24 h, followed by treatment with flutamide (Flu) at various concentrations (0, 20, 50, 100, 200, 500 μM) for 24 h. The AR and TGF-β protein levels were measured by western blot. d ROS generation in GCs following various treatments was measured using the DCF-DA probe. DCF-DA fluorescence (green fluorescence) was measured by confocal microscopy (40×). Images are representative of three independent experiments with similar results. Quantification of the fluorescence is shown. Three independent experiments were performed with similar results. Data are shown as the mean ± SD. *p ≤ 0.05. DHT dihydrotestosterone, AR androgen receptor, TGF-β transforming growth factor-beta, FSHR follicle-stimulating hormone receptor, LHR luteinizing hormone receptor, GCs granulosa cells, TCs theca cells, ROS reactive oxygen species