Fig 2: (a) Effect of H2O2 on cell apoptosis in the atretic follicles. A: H&E staining and TUNEL assay in the SWFs (treated with H2O2) and ASWFs. Scale bar: 100 μm for H&E staining and 50 μm for TUNEL assay. B-I: Western blot and gray analysis of occludin, BAX, and PCNA expression after H2O2 treatment. B-II: the expression of PCNA, CDK2, CCND1, BCL2, BAX, and P53 mRNAs in the SWFs (treated with H2O2) and ASWFs. (b) Effect of H2O2 on yolk deposition capacity and steroidogenesis in the atretic follicle model by H2O2. A: immunofluorescent labels with occludin (green), VLDLR (red), and DAPI (blue) in the histological sections of the SWFs with different concentrations of H2O2. Scale bar: 50 μm. B-I: the expression of FSHR, ER-α, CYP17A1, and CYP19A1 mRNAs. B-II: the expression of yolk deposition-related genes VLDLR, PPAR-γ, and occludin. C: Western blot and grey analysis of ER-α and FSHR after H2O2 treatment. Values represent the means ± SEM of three replicates in each group. Different lowercase letters indicate significant difference (P < 0.05).

Fig 3: (a) Mechanism of antiaging/atresia of Met via the calcium ion (Ca2+) transport signaling pathway in the H2O2-induced ASWFs. A: the effect of Met on the expression of GRP75, Cyt c, and MFN2 with IF and IHC analyses. Scale bar: 50 μm. B: Western blot and grey analysis of IP3R, caspase3, GRP75, CAMKII, MFN2, and Cyt c expression in ASWFs. C: effect of Met on expression of Ca2+ transport-related genes. (b) Mechanism of antiaging/atresia of Met via the insulin signaling pathway. A: the effect of Met on expression of PI3K (red), BAD (green), and DAPI (blue) with IF. Scale bar: 50 μm. B: Western blot and grey analysis of p-AKT/AKT and p-GSK-3β/GSK-3β expression in ASWFs. C: effect of Met on expression of PI3K, BCL2, and GLUT4 mRNAs in ASWFs. (c) Mechanism of antiaging/atresia of Met via the lipid metabolism signaling pathway. A: Western blot and grey analysis of PPAR-γ, occludin, and VLDLR expression in ASWFs. B: qRT-PCR analysis of PPAR-γ, occludin, and VLDLR expression in ASWFs. Values represent the means ± SEM of three replicates in each group. Different lowercase letters indicate significant difference (P < 0.05).

Fig 4: Brain ABCA1 levels were upregulated, while LDLR and VLDLR were slightly reduced by LXR agonist treatment. The brain levels of the LXR target gene ABCA1 and of the apoE receptors LDLR and VLDLR were evaluated in RIPA extracted brain homogenates by Western blot analysis as described in Methods. Equal amounts of supernatant proteins were loaded per lane and actin was used as an additional loading control. A: Representative blots showing ABCA1 levels in the brain of rats treated with vehicle or LXR agonists (left panel). Densitometric analysis of Western blots for ABCA1 (right panel). B: Representative blots showing LDLR and VLDLR steady-state levels in the brain of rats treated with vehicle, T1317, and GW3965 for 10 days (left panel). Densitometric analysis of Western blots for LDLR and VLDLR (right panel). * p < 0.05 compared to vehicle treated controls by ANOVA, N = 4 per group.

Fig 5: Met promoted yolk synthesis by stimulating estrogen secretion in D580 hens. (a) The number of ER-α-positive cells (brown) in the liver of D580 hens was increased by Met. Scale bar: 50 μm. (b) Effect of Met on levels of plasma HDL, LDL, FFA, Tchol, TG, and serum E2 in D580 hens. (c) Effect of Met on expression of yolk deposition-related genes (VTGII, OVR, PPAR-γ, and VLDLR) and BCL2 in D580 hens. Values represent the means ± SEM in each group (n = 15). Different lowercase letters indicate significant difference (P < 0.05).