Fig 2: FSH upregulates beclin1 expression in porcine GC mass via the PI3K/JNK/c-Jun pathway. (A) GC mass were cultured in DMEM/F12 medium containing 0.01 IU/mL FSH for 24 h, beclin1 and ß-actin levels were detected by western blotting of the whole cell lysates. (B) GC mass were cultured in DMEM/F12 medium containing 0.01 IU/mL FSH for the indicated hours, and total RNA was collected for cDNA synthesis. Expression of beclin1 and ß-actin mRNA was measured by PCR assay. (C) GC mass were pre-treated with LY294002 (10 µM, inhibitor of PI3K/AKT signaling pathway) for 1 h before stimulation with 0.01 IU/mL FSH for 24 h, p-AKT, AKT, p-JNK, JNK, p-c-Jun, Beclin1, and ß-actin levels were detected in whole cell lysates. (D) GC mass were pre-treated with SP600125 (10 µM, inhibitor of SAPK/JNK signaling pathway) for 1 h before stimulation with 0.01 IU/mL FSH for 24 h, p-AKT, AKT, p-JNK, JNK, p-c-Jun, Beclin1, and ß-actin levels were detected in the whole cell lysates. **, P < 0.01, and *, P < 0.05 compared to the control group.

Fig 3: Schematic illustration of autophagy regulation by FSH stimulation in porcine GCs. FSH promotes progesterone secretion by accentuating autophagy through upregulation of Beclin1 via the PI3K/JNK/c-Jun pathway to accelerate LD degradation and increase the expressions of steroidogenesis enzyme genes in porcine GCs.

Fig 4: FSH degrades lipid droplets by enhancing autophagy to promote progesterone secretion in porcine follicle GCs. (A) Porcine adherent granulosa cells were transfected with Atg5 siRNAs (siAtg5 1-3#), NC siRNA, and blank control (Mock) for 24 h and then harvested and analyzed by immunoblotting with antibodies against Atg5, LC3, and actin. (B) Porcine adherent granulosa cells were transfected with Beclin1 siRNAs (siBeclin1 1-3#), NC siRNA, and blank control (Mock) for 24 h, harvested, and analyzed by immunoblotting with antibodies against Beclin1, LC3, and Histone 3. (C) BODIPY 493/503 LD staining of adherent granulosa cells, pre-treated with OA for 12 h, after treatment with NC siRNA, siAtg5 2#, and siBeclin1 3#, and then treated with FSH for 24 h; Hoechst 33342 staining of the cell nuclei, Bars: 25 µm. (D) The average number of LDs per granulosa cell in the Mock, NC, Mock + FSH, siAtg5, siAtg5 + FSH, siBeclin1, and siBeclin1 + FSH groups. (E) The average size of lipid droplets per granulosa cell in Mock, NC, Mock + FSH, siAtg5, siAtg5 + FSH, siBeclin1, and siBeclin1 + FSH groups. NC, negative control; NS, no significant. (F) The relative progesterone level in the culture medium from mock, NC, mock + FSH, siAtg5, siAtg5 + FSH, siBeclin1, and siBeclin1 + FSH groups was calculated as pg/µg protein. *, P < 0.05, **, P < 0.01.

Fig 5: 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. ß-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.