Additional file 1 of Androgen-induced exosomal miR-379-5p release determines granulosa cell fate: cellular mechanism involved in polycystic ovaries

Additional file 1: Supplementary Figure 1. Androgen excess in human PCOS subjects is associated with reduced follicular fluid-derived exosomal mir-379-5p content and granulosa cell proliferation. (A) Follicular fluids from the dominant follicles (≥ 20 mm; n = 25 Non-PCOS and 13 PCOS subjects) of PCOS subjects exhibited significantly higher free testosterone level and lower mir-379-5p contents (relative to mir-92a-3p, determined by Next Generation sequencing) in exosomes. mir-379-5p was detected in extracellular vesicle (EV)-depleted follicular fluid (FF) but its levels was not different between PCOS and Non-PCOS subjects. (B & C) Granulosa cells from PCOS subjects had significantly lower proliferation (n = 12 Non-PCOS and 11 PCOS subjects) than those of non-PCOS subjects. MiRNA expression was assessed by TaqMan Advanced miRNA Assays (Thermo Fisher). Results are expressed as means ± SEM. Data were analyzed by t-test and Pearson correlation. *P < 0.05, **P < 0.01 and ***P < 0.0001. Supplementary figure 2. Androgen does not influence cellular and exosomal contents of mir-24, mir-9 and let-7d in rat pre-antral follicle granulosa cells. Granulosa cells were isolated from preantral follicles (Diethylstilbestrol-primed 21-day old rats; 1 mg/d, subcutaneous injection for 3 consecutive days). Granulosa cells were cultured with DHT (0 and 1 μM, 24 h and 36 h). Exosomes were isolated from granulosa cell-conditioned medium by differential centrifugation and their size and concentrations were determined by nanoparticle tracking analysis. miRNA expression was assessed by TaqMan miRNA Assays (Thermo Fisher) and normalized to U6. Results are expressed as means ± SEM (n = 3 replicates, each from 2 rats). Data were analyzed by two-way ANOVA and tukey post hoc. Supplementary figure 3. DHT treatment did not affect granulosa cell TGFBR1 protein content in rat preantral and antral follicles 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; animal sacrificed 48 h post-treatment). Granulosa cells were cultured with DHT (0 and 1 μM) for 24 h and 36 h. Supplementary figure 4. Androgen increased the cellular content of pri-miR-379 in rat granulosa cells. Granulosa cells were isolated from preantral follicles (Diethylstilbestrol-primed 21-day old rats; 1 mg/d,