| Autor: |
Salehi, Reza, Wyse, Brandon A., Asare-Werehene, Meshach, Esfandiarinezhad, Fereshteh, Abedini, Atefeh, Pan, Bo, Urata, Yoko, Gutsol, Alex, Vinas, Jose L., Jahangiri, Sahar, Xue, Kai, Xue, Yunping, Burns, Kevin D., Vanderhyden, Barbara, Li, Julang, Osuga, Yutaka, Burger, Dylan, Tan, Seang-Lin, Librach, Clifford L., Tsang, Benjamin K. |
| Rok vydání: |
2023 |
| DOI: |
10.6084/m9.figshare.22618388.v1 |
| Popis: |
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, 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. miRNA expression was assessed by TaqMan miRNA Assays (Thermo Fisher) and normalized to 𝛽-actin. 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 5. The median size of exosomes and microvesicles isolated from rat pre-antral and antral follicle granulosa cell-conditioned medium. 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. Exosomes and microvesicles were isolated from granulosacell-conditioned medium by differential centrifugation as described in Materials and Methods. Results are expressed as means ± SEM (n = 3 replicates, each from 2 rats). Data were analyzed by three-way ANOVA and tukey post hoc. Supplementary figure 6. Exosome release is inhibited by GW4869 at 20 nM. (A) CD63 protein is an exosome marker and is expressed only in exosome fraction of granulosa cell conditioned medium. (B) GW4869 (20 nM) effectively suppressed exosome release, as evident by reduced CD63 protein content in conditioned medium. Granulosa cells were isolated from preantral follicles (Diethylstilbestrol-primed 21-day old rats; 1 mg/d, subcutaneous injection for 3 consecutive days). Exosomes were isolated from granulosa cell-conditioned medium by differential centrifugation as described in Materials and Methods. Supplementary Figure 7. Reconstitution of DHT-treated (mir-379-5p down-regulated) rat granulosa cells with mir-379-5p-enriched exosomes attenuates the DHT-induced proliferative response in vitro. (A) Experimental design: Preantral granulosa cells (donor cells) were treated without (CTR-exo) or with DHT (DHT-exo), and exosome were isolated from spent culture medium 36 h post-treatment. To prepare recipient cells, granulosa cells from preantral follicles were pre-treated without or with DHT for 12 h. Exosomes were labeled with the red fluorescent dye PKH26 by incubating with granulosa cell (10 μg protein/100,000 granulosa cells) for 24 h. Granulosa cells (105 cells) were stained with viability dye eFluor 450. (B) Exosomal uptake was assessed in viable granulosa cells by flow cytometry in the following 4 experimental groups: (1) CTR recipient cells + CTR-exo, (2) DHT recipient cells + CTR-exo, (3) CTR recipient cells + DHT-exo, and (4) DHT recipient cells + DHT-exo; (C) While androgen excess failed to alter exosomal uptake, culture of DHT-treated granulosa cells (mir-379-5p down-regulated) with mir-379-5p-enriched exosomes (DHT-exo) attenuates the DHT-induced proliferative response in vitro. Granulosa cells were isolated from preantral follicles (Diethylstilbestrol-primed 21-day old rats; 1 mg/d, subcutaneous injection for 3 consecutive days). Results are expressed as means ± SEM (n = 3 replicates). Data were analyzed by two-way ANOVA and tukey post hoc. *P < 0.05. Supplementary Figure 8. A hypothetical model illustrating androgen-induced exosomal mir-379-5p release from granulosa cells removes its inhibitory action on PDK1, a proliferative mechanism specific for preantral follicle granulosa cells. Androgen excess reduces granulosa cell mir-379-5p content by increasing its exosomal release in preantral follicles, but not in antral follicles. Reduced granulosa cell mir-379-5p content increases PDK1-mediated cell proliferation. Consequently, androgen excess promotes preantral but suppresses antral follicular development, as observed in PCOS. (Created in BioRender.com). Supplementary figure 9. Guide for inclusion or rejection of follicular fluid samples for study. Supplementary figure 10. Validation of lentivirus transduction following intrabursal injection. (A) Red fluorescent protein (RFP) signal in rat ovarian sections was detectable 7 days following intrabursal injection of lentivirus-RFP and saline (control), demonstrating successful delivery and transduction; (B) RFP mRNA expression was detectable (normalized to β-actin) in granulosa cells isolated from rats injected with lentivirus. Granulosa cells isolated from untreated rats were considered as Control. Results are expressed as means ± SEM; n = 3 per group, each from 2 rats. Data were analyzed by one-way ANOVA and tukey post hoc. **P < 0.01, ***P < 0.001. Supplementary Table 1. List of antibodiesand their dilution used in this study. Supplementary Table 2. Patient characteristics. Supplementary Table 3. List of TaqMan Advanced miRNA Assays and Taqman miRNA assay (Life Technologies, Inc.) used in this study. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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