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ISSN 2052-1472 (online)

Reproduction Abstracts (2014) 1 P192 | DOI: 10.1530/repabs.1.P192

Effect of melatonin supplementation during in vitro maturation on intracellular ROS levels in porcine oocytes

Maria Helena Coelho Cruz1, Jurandir Ferreira Cruz2, Russel J Reiter3, Katia Lancellotti Schwarz1, Fernanda Cavallari Castro1, Maria Carolina R V Cunha1 & Claudia Lima Verde Leal1

1University of São Paulo, Pirassununga, Brazil; 2Southwest Bahia State University, Vitória da Conquista/Bahia, Brazil; 3University Texas Health Science Center at San Antonio, San Antonio, Texas, USA.

Oocytes of domestic mammals present high concentrations of lipids, and this is particularly evident in porcine oocytes that exhibit higher levels than other species, becoming more susceptible to oxidative stress and lipid peroxidation. Considering that melatonin is an effective antioxidant for protecting macromolecules against oxidative stress caused by reactive oxygen species (ROS), the purpose of the present study was to examine the effect of melatonin supplementation during in vitro maturation on intracellular ROS levels in porcine oocytes. Oocytes were in vitro matured in TCM199 medium containing different concentrations (0, 10−6 and 10−9 M) of melatonin (Melatonin, Sigma–Aldrich). As a positive control (induction of oxidative stress), oocytes were incubated with 1 mM H2O2 for 10 min. Intracellular ROS levels were detected using the intracellular dye 2,7-dichlorofluorescein diacetate (DCF-DA, Sigma–Aldrich) and assessed by fluorescence intensity using the ImageJ software (NIH, USA). Melatonin supplementation contributed to reduce intracellular ROS (P<0.01), and values were 43.79±14.9, 23.47±12.2, 17.76±7.5 in medium containing H2O2, 0 and 10−6 M melatonin, respectively. However, melatonin effectiveness was more expressive in the lowest concentration (12.83±6.5 in 10−9 M; P<0.05). The substantial intracellular ROS decrease may be attributed to the powerful ability of melatonin to scavenge ROS and its indirect antioxidant properties, which stimulate anti-oxidative enzymes and inhibits pro-oxidative enzymes. Even though ROS play important roles as second messengers in cellular functions through activation of cell signaling cascade in the oocyte, high concentrations may promote imbalance between the oxidation–reduction reactions, negatively interfering on cell function. Thus, melatonin supplementation during in vitro maturation reduces intracellular ROS levels and may improve porcine oocyte viability.

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