Fig. 4

Androgen-induced exosomal mir-379-5p release as a determinant of rat granulosa cell mir-379-5p content and proliferation. A & B Irrespective of DHT treatment, GW4869 at 20 nM effectively inhibited exosome release, as evident by increased granulosa cellular content and reduced conditioned medium level of CD63 protein (exosome marker; Supplementary Fig. 3). The inhibition of exosome release increased granulosa cell mir-379-5p content (normalized to U6; DHT/control ratio) and down-regulate cell proliferation (MCM2) induced by DHT; C DHT increased granulosa cell cortactin protein content in granulosa cells from preantral, but not antral follicle; D cortactin overexpression in combination with DHT significantly increased exosome release in antral follicle granulosa cells and reduced granulosa cell mir-379-5p content (normalized to U6). Granulosa cells were isolated from preantral follicles (Diethylstilbestrol-primed 21-day old rats;1 mg/d, subcutaneous injection, for 3 consecutive days) or antral follicles (Equine chorionic gonadotropin–injected 21-day old rats; 10 IU, intraperitoneal injection, animals sacrificed 48 h post-injection). Exosome release was inhibited with GW4869 pre-treatment (0, 10 and 20 mM, 24 h) before DHT (0 to 1 µM, 24 h and 36 h). Exosomes were isolated from granulosa cell conditioned medium by differential centrifugations. To induce exosome release in antral follicle stage, granulosa cells were infected with adenoviral sense full-length cortactin cDNA or adenoviral control vector at 50 MOI (24 h), and then cultured with DHT (0 to 1 µM, 24 h and 36 h). Protein extraction and Western blotting were performed as described previously [15]. Signal intensities generated on the film were measured densitometrically using Image J and normalized over that of GAPDH or β-Actin. U6 RNA was used to normalize miR-379-5p content. Results are expressed as means ± SEM (n = 3 replicates, each from 2 rats). Data were analyzed by (A & B) two-way ANOVA and tukey post hoc; (C & D) three-way ANOVA and tukey post hoc. *P < 0.05; **P < 0.01, ***P < 0.001