Fig. 6: GnRHa induces autophagy to relieve ER stress by inhibiting mTOR signaling.

a Expression of proteins in autophagy was assessed by western blotting. Compared with CTX-treated cells, LC3B-II and Beclin-1 were higher, P62 were lower in cells co-treated with CTX and GnRHa. b Expressions of proteins in mTOR pathway were assessed by western blotting. c Typical immunofluorescence staining, indicating the localization of AMH and LC3B within KGN cells. Yellow in the merged images indicates the colocalization of AMH and LC3B. Nuclei were stained with DAPI. Scale bars represent 100 μm. d, g Representative transmission electron microscopy results are shown for KGN cells treated with 750 µg/mL CTX, with or without GnRHa, for 36 h. Red arrowheads represent autophagic vacuoles; the yellow oval marks an area of ER stress. When the cells were co-treated with CTX and GnRHa, the numbers of autophagosomes appeared to be considerably higher. e, f Co-treatment with CTX and GnRHa reduced mTOR and P70S6K phosphorylation compared with CTX only. Compared with untreated control cells, the phosphorylation of mTOR and P70S6K were higher in the CTX-treated KGN cells. Data are expressed as mean ± SD of three independent experiments. Error bars show the standard error of the mean. Two-tailed Student’s t test was used for (e, f), two-way ANOVA (Tukey’s test) was used for g, *P < 0.05, **P < 0.001, and ***P < 0.0001.