Melatonin and Glutathione Co-Supplementation Improves Redox Homeostasis and Developmental Competence in Sheep Oocytes

Oxidative stress is a major factor limiting oocyte quality during in vitro maturation (IVM) and subsequent embryonic developmental competence. This study investigated the effects of combined melatonin (MLT) and glutathione (GSH) supplementation on sheep oocyte quality and the potential regulatory mechanisms involved. Sheep oocytes were assigned to Control, MLT, GSH, and MLT+GSH groups, and oocyte maturation, redox homeostasis, and mitochondrial function were evaluated by assessing cumulus expansion, first polar body extrusion, F-actin organization, ROS- and GSH-associated fluorescence, JC-1 staining, and MitoTracker signals, while subsequent developmental competence was assessed following in vitro fertilization (IVF). SMART-seq2 transcriptomic analysis, Nrf2 immunofluorescence, antioxidant-related gene expression analysis, and pharmacological inhibition of Nrf2 with ML385 were further performed to explore the underlying mechanisms. MLT and GSH, either alone or in combination, improved multiple indices related to oocyte maturation, redox status, and mitochondrial function, with the MLT+GSH group generally showing the most favorable numerical changes. MLT+GSH treatment was associated with greater cumulus expansion, a higher first polar body extrusion rate, improved F-actin organization, reduced ROS accumulation, increased GSH-associated fluorescence, a higher JC-1 red-to-green fluorescence ratio, and enhanced mitochondrial-associated fluorescence. However, for some endpoints, the numerical improvement observed with MLT+GSH did not differ significantly from the corresponding single treatment. Following IVF, MLT+GSH treatment increased cleavage and blastocyst formation rates and improved subsequent developmental outcomes. Mechanistically, MLT+GSH treatment was accompanied by increased Nrf2 immunofluorescence intensity and altered expression of NFE2L2 and several Nrf2-related antioxidant genes, including NQO1, HMOX1, GCLC, GCLM, and GPX4, suggesting the involvement of an Nrf2-associated antioxidant regulatory network. Addition of the Nrf2 inhibitor ML385 to the MLT+GSH treatment increased ROS levels and reduced JC-1 and other redox- and mitochondria-related indices, while partially attenuating improvements in oocyte maturation and embryonic development. Overall, MLT and GSH co-supplementation was associated with improved redox homeostasis, antioxidant capacity, mitochondrial function, and subsequent developmental competence in sheep oocytes, with Nrf2-associated antioxidant regulation potentially contributing to these beneficial effects under standard IVM conditions.