FIGURE 4.

Autophagy participates in progesterone production and lipid droplet metabolism upon FSH treatment in porcine GC mass. (A) Specific inhibitors of autophagy (chloroquine; 10 μM), PI3K/AKT (LY294002; 10 μM), and SAPK/JNK (SP600125; 10 μM) were added 1 h before treatment with 0.01 IU/mL FSH for 24 h in GC mass. The cells and culture medium were collected separately. The total proteins were extracted from the cells and quantified using the BCA method. The concentration of progesterone in the culture medium was analyzed using radioimmunoassay. The relative progesterone level in the culture medium was calculated as ng/μg protein. Different superscripts (a, b, c) indicate significant differences (P < 0.05) (B) Specific inhibitors of autophagy (chloroquine; 10 μM), PI3K/AKT (LY294002; 10 μM), and SAPK/JNK (SP600125; 10 μM) were added 1 h before treatment with 0.01 IU/ml FSH for 24 h in GC mass. Total RNA was extracted from the cells, reverse transcribed to cDNA, and analyzed by real-time PCR with primers against beclin1, StAR, and P450scc. (C) Adherent granulosa cells of the porcine ovary were cultured in DMEM/F12 serum-free medium with or without oleic acid (0.5 mM) for 12 h, and then cultured in DMEM/F12 serum-free medium with FSH (0.01 IU/mL) or with FSH and OA (0.5 mM) for 24 h. β-actin, LC3-II, and Beclin 1 levels were detected by western blotting. (D) BODIPY 493/503 LD staining of adherent granulosa cells after treatment with OA and FSH; Hoechst 33342 staining of the cell nuclei, Bars: 30 μm. (E) The average number of LDs per granulosa cell in the OA and OA + FSH groups. (F) The average size of LDs per granulosa cell in the OA and OA + FSH groups. Five visual regions were used for statistics. ^∗, P < 0.05, ^∗∗, P < 0.01.