Figure 3.
Activation of Primordial Follicles after HucMSC-exo Treatment
Newborn ovaries were treated for 24 h with HucMSC-exos at 5, 10, or 20 μg/mL (3 × 108, 6 × 108, 1.2 × 109 particles/mL, respectively). (A) Dose-dependent activation of the PI3K-Akt-mTOR signaling pathway in ovaries with increased expression of p-AKT (Ser473), p-mTOR (Ser2448), and p-rpS6 (Ser235/236). The expression of rpS6, mTOR, and β-tubulin were used as internal controls. (B) Immunostaining of Foxo3a in primordial follicles after newborn ovaries were incubated with 20 μg/mL HucMSC-exos for 24 h. The lower panel represents the magnified images of black frames in the upper panel. (C) Percentage of activated primordial follicles with nucleus exclusion of Foxo3a in control and exo-treated ovaries. n = 3/group. (D) Blockage of the PI3K-Akt-mTOR signaling pathway by its specific inhibitors LY294002, Akt VIII, and rapamycin and the exosome internalization inhibitor heparin. LY, LY294002; AI, Akt VIII; Ra, rapamycin; Hep, heparin. (E) Histology of ovaries collected at 96 h in each group. After 24 h of treatment, ovaries in each group were further cultured in control media for 72 h. Arrowhead indicates primordial follicles, and arrow indicates activated follicles. Data are shown as mean ± SEM. *p < 0.05. Scale bars, 50 μm.