Fig. 9: Melatonin reprograms ovarian granulosa cells and mouse glycolysis to inhibit ferroptosis-induced ovarian reproductive damage.

A CCK8 assay showing cell viability in granulosa cells co-cultured with DON and different concentrations of melatonin (0.1, 1, 10 µM) for 24 h (n = 6). B ELISA measurement of E2 levels in granulosa cells cultured with DON alone or co-cultured with 1 µM melatonin for 24 h (n = 6). C, D Representative images of Fe²⁺ and ROS levels and their relative fluorescence intensity in granulosa cells cultured with DON alone or co-cultured with 1 µM melatonin for 24 h (n = 3). Scale bars: 40 µm. E–G Representative images of lipid peroxidation and mitochondrial membrane potential levels and their relative fluorescence intensity in CK, DON, and DON + MEL granulosa cells observed by laser confocal microscopy (n = 3). Scale bars: 40 µm. H ELISA analysis showing changes of lactate levels (n = 6) in granulosa cells cultured with DON alone or co-cultured with 1 µM melatonin for 24 h. I Schematic diagram of melatonin treatment in mice for 28 days (n = 6). J, K ELISA measurement of changes in reproductive hormones (FSH) and oxidative stress markers (GSH) in the serum of mice treated with DON alone or co-treated with DON + melatonin (n = 6). L Immunofluorescence analysis of cell proliferation (Ki67) levels in ovarian tissues of different mouse groups. (Scale bar: 40 µm) M–O qPCR analysis of glycolysis-related gene expression in ovarian tissues of different mouse groups (n = 3). P ELISA measurement of lactate levels in ovarian tissues of different mouse groups (n = 6). Q Immunofluorescence analysis of H3K18la levels in ovarian tissues of different mouse groups. (Scale bar: 40 µm) R qPCR analysis of STEAP3 gene expression in ovarian tissues of different mouse groups (n = 3). S ELISA measurement of Fe²⁺ levels in ovarian tissues of different mouse groups (n = 6). Bars indicate the mean ± SD. Data were analyzed using one-way ANOVA and two-way ANOVA with Tukey’s post hoc test, with significance levels denoted as follows: ns, p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (I) was created with Biorender.