The effect of melatonin on intracellular ROS production. Immature (3 wks) ICR mice were given an injection of 20 units of pregnant mare serum gonadotropin (PMSG) to stimulate the development of multiple follicles. Oocytes were collected by puncturing ovarian follicles after 48 hrs PMSG injection, and then the surrounding cumulus cells were removed. Oocytes were incubated with 1 mM H2O2 for 10 min in the presence of melatonin (0, 1 μg/ml, 100 μg/ml). Intracellular ROS were detected using an intracellular dye (dichlorofuorescin:DCF-DA). The nonfluorescent DCF-DA is oxidized by intracellular ROS to form the highly fluorescent DCF, intracellular ROS formation was visualized by fluorescence image and fluorescence intensity was analyzed using MetaMorph software. (A) H2O2 (1 mM); (B) H2O2 (1 mM)+ melatonin (1 μg/ml); (C) H2O2 (1 mM)+ melatonin (100 μg/ml); (D) Fluorescence intensity in the oocytes. Data are shown as the mean ± SEM for (6-9 oocytes). *: p < 0.05 versus H2O2 (1 mM).