Figure 4. Effects of melatonin on ROS levels and total glutathione in aged oocytes. (A) Fresh oocytes. (B) Oocytes aged for 24 hr. (C) IVA 24 hr oocytes treated with 2 mM melatonin. (D) Oocytes aged for 48 hr. (E) IVA 48 hr oocytes treated with 2 mM melatonin. (F) ROS fluorescence pixel intensity in IVA oocytes. (G) ROS level (counts of photons) detection of single cell by optical nanoprobes. (H) Total glutathione (T-GSH) content in fresh and IVA oocytes. (I) The expression of anti-oxidative stress genes (SOD1, GPx4 and CAT) in fresh and aged oocytes. All graphs show mean ± s.e.m. Abbreviations used in this and all subsequent figures: ROS, reactive oxygen species. Independent replicates were conducted with a minimum of 30 oocytes/replicate, at least 3 stable replicates were obtained. **P <0.01, ***P <0.001. Bar = 50 μm.