Fig. 1

DHT reduces mir-379-5p content, increases its exosomal release and PDK1 content in granulosa cells from rat preantral but not antral follicle in vitro. A Reduced granulosa cell mir-379-5p content was associated with increased PDK1 and P-PDK1 protein levels and activation of Akt-mediated cell proliferation in preantral (increased P-AKT/GAPDH, P-AKT/AKT ratio and MCM2/GAPDH protein levels) but not antral follicle stage in vitro. B DHT treatment reduced microvesicles concentration at 36 h in both pre- and antral follicular stages. Exosomes concentration was reduced at 36 h post-DHT treatment in granulosa cells from antral follicles, but not in that of preantral follicle. Microvesicles/exosomes ratio was reduced at 36 h post-DHT treatment in granulosa cells from preantral follicles, but not in that of antral follicles. C DHT increased exosomal mir-379-5p level (normalized to U6), but not that of microvesicles in the conditioned medium in vitro. Granulosa cells were isolated from preantral follicles (Diethylstilbestrol-primed 21 day old rats; 1 mg/d, subcutaneous injection for 3 consecutive days) and antral follicles (Equine chorionic gonadotropin–injected 22-day old rats; 10 IU intraperitoneal injection, animals sacrificed 48 h post-injection) and cultured without or with ± DHT (1 µM, 24 h and 36 h). Exosomes and microvesicles were isolated from granulosa cell-conditioned medium by differential centrifugations, and sizing and concentration of extracellular vesicles were determined by nanoparticle tracking analysis. 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. 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 three-way ANOVA and tukey post hoc. *P < 0.05; **P < 0.01, ***P < 0.001