Melatonin protects porcine oocyte from copper exposure potentially by reducing oxidative stress potentially through the Nrf2 pathway

Modern animal feeds often include nutritional mineral supplements, especially elements such as Cu, P, Se, and Zn [1]. Copper (Cu) is an essential component and cofactor of many enzymes in the body, which directly participates in the metabolism and growth of pigs [2,3]. Studies have shown that rational use of a high copper diet can improve intestinal flora, intestinal structure, and function [4]. And adding copper during in vitro maturation (IVM) significantly increased both intracellular GSH content and DNA integrity of cumulus cells in bovine oocytes [5].

Although a high level of dietary copper (125 mg/kg copper sulfates or 125 mg/kg copper methionine) increased pigs’ growth performance and feed efficiency [4], copper is highly toxic at exceeded normal levels [6,7]. Copper concentration at 0.16 mg L−1 led to low hatchability, delayed hatching, high mortality, morphological abnormalities, and increased egg size in the embryos of marine medaka [8]. And the popular use of Cu as additives in animal feed could result in their excessive accumulation in manure to a toxic level, and thus, possibly threaten the environment when the manure is applied to farm land [9]. Heavy metal would enter soil and water through sewage systems, direct excretion, and land application of biosolids [10]. A recent study shows a high concentration of copper in the soil even causes antimicrobial resistance potentially [11].To summarize, although Cu is an essential nutrient for animals, excessive Cu supplementation may be harmful.

Heavy metals can increase the production of reactive oxygen species (ROS) [12], which is an important factor affecting the reproductive system of females. And female fertility is mainly determined by the quality of oocytes. Melatonin occurs naturally in plants and animals and is a ubiquitous antioxidant that scavenges radicals [13], it and its derivatives stimulate the expression of antioxidative enzymes [14]. Several studies showed that the existence of melatonin in the follicular fluid and its contents positively correlates with oocyte quality and maturation [15,16]. Human preovulatory follicles contain a high amount of melatonin compared with small immature follicles [17]. The endogenous melatonin concentration in bovine follicular fluid is approximately 10−11 M [18]. Melatonin was also detected in porcine follicular fluid [19]. Moreover, the study also shows the insufficiency of melatonin in follicular fluid is a reversible cause of advanced maternal age-related aneuploidy in oocytes [20]. All of these suggest that melatonin is directly involved in ovarian function in mammals. Previous reports have indicated that melatonin can promote the maturation of oocytes, it has been successfully used to protect oocytes from ROS exposure during IVM in sheep [21], rodents [22], pigs [23,24], and cattle [25,26].

When ROS ascends and oxidative stress occurs, the body initiates antioxidant mechanisms. As the Kelch-like erythroid cell-derived protein with CNC homology-associated protein 1 (Keap1)–Nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway, it is a critical regulator between cellular antioxidant response and aerobiotic metabolism [27]. Studies have shown that the Keap1–Nrf2 signaling pathway is an important regulatory component of cells in a steady-state environment, protecting cells from oxidative and aerobiotic stresses [28]. Under cellular oxidative stress conditions, Nrf2 detaches from Keap1 through the breakage of disulfide bonds, stabilizes itself, and translocates into the nucleus to facilitate antioxidant response elements (ARE) activation [29,30]. Then, the Nrf2-mediated transcription process is initiated to regulate the downstream gene expression; activate a series of antioxidant enzymes and phase II antioxidant enzymes, such as Heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase (NQO1), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px); remove ROS and other harmful substances; and facilitate antioxidative stress, anti-inflammatory, anti-apoptosis, and other cell protection mechanisms [31].

However, the effects and underlying mechanisms of melatonin that protect porcine oocytes from oxidative stress have not been clearly defined. In this study, we treated porcine oocytes with copper and melatonin to explore the effects and the molecular mechanisms underlying the damage and preventive effects during oocyte maturation. This study aimed to elucidate the effects of copper exposure on porcine reproduction, which will aid in furthering our understanding of the molecular mechanisms of reproductive damage due to environmental pollutants.

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