70 Trolox during in vitro maturation of bovine oocytes protects developing embryos from palmitic acid-induced lipotoxicity

Abstract

Maternal metabolic disorders are associated with elevated concentrations of palmitic acid (PA), which is known to jeopardize bovine oocyte and embryo development and quality. Analyses of PA-exposed bovine oocytes and embryos point toward oxidative stress (OS)-related pathways. Previous research has shown that the detrimental effects of PA exposure during oocyte IVM cannot be alleviated by antioxidant supplementation, e.g. Trolox (TR, water-soluble vitamin E), during IVM or in vitro culture (IVC). In contrast, supplementing TR during IVM could protect developing zygotes from PA-induced lipotoxicity by increasing their development into 4-cell embryos and Day 8 blastocysts. In the present study, we examined the effects of TR during IVM or IVC on PA-exposed oocytes and embryos by evaluating OS and mitochondrial membrane potential (MMP) of the produced ≥2-cell Day 2 embryos (Table 1). Bovine cumulus-oocyte complexes (COCs) were matured, fertilised, and cultured in 2 experiments. In Experiment 1, COCs were exposed to pathophysiological follicular PA concentrations (150 µM), and subsequent embryos were cultured under solvent control (ethanol) conditions (PA-SC). The TR treatment was applied during IVM or IVC (100 µM; PATR-SC, PA-TR). In Experiment 2, COCs were matured under SC conditions, and subsequent embryos were exposed to pathophysiological oviductal PA concentrations (230 µM; SC-PA). The TR treatment was applied during IVM or IVC (100 µM; TR-PA, SC-PATR). In each experiment, a SC was included (SC-SC). A total of 126 and 137 Day 2 embryos were stained with JC-1 and CellRox Deep Red in Experiment 1 and Experiment 2 (3 repeats), respectively. The MMP was evaluated as active/total mitochondria and OS as OS/total mitochondria and analysed by one-way ANOVA. Exposure of oocytes and embryos to PA significantly increased OS and MMP in Day 2 embryos compared with controls. Regardless of the moment of PA exposure, TR treatment during IVM increased MMP even more. The increased MMP levels in PA-exposed oocytes and embryos were not influenced by TR treatment during IVC. Regardless of the antioxidant effect of TR, increased embryonic OS levels observed in PA-exposed oocytes and embryos could not be reversed to control levels by TR treatment during IVM or IVC. However, when OS levels were expressed on active mitochondria, we found that TR treatment before the PA insult (TR-PA) generated Day 2 embryos that displayed significantly lower OS/active mitochondria rates compared with SC-PA-treated embryos. Taken together, we conclude that the combination of relatively low OS levels with highly active mitochondria may be a mechanism implicated in the protective effect of TR before the PA insult on embryo developmental competence.

Table 1.  Effects of TR during IVM or IVC on PA-exposed oocytes and embryos in Experiments 1 and 2