Recent studies suggest that endogenous and exogenous free fatty acids play various important roles in mammalian oocyte and pre-implantation embryo development. Among fatty acids, linoleic acid (LA) has been reported to affect the apoptosis pathway via nuclear transcription factor-kappa B (NF-κB). The transcription factor NF-κB is a key modulator of apoptosis in a variety of cell types, but to date, this specific function of NF-κB has not been demonstrated in porcine pre-implantation embryos. To examine the effect of linoleic acid on invitro-produced parthenogenetic pig embryos, we treated LA by concentration (0, 10, 25, 50, and 100 µM) to identify developmental rate, NF-κB expression, and mRNA level of apoptotic-related genes. In addition, the mechanism was confirmed by examining the protein and mRNA expression of NF-kb and c-jun by immunostaining and quantitative PCR at the blastocyst stage. Linoleic acid had a positive effect on embryo development without toxicity at a certain concentration (25 µM), but toxicity was confirmed at higher (50-100μM) concentrations. Furthermore, it was confirmed that the concentration of NF-κB increased as the treatment concentration of LA increased, which was found to increase even at the concentration at which embryo development decreased. Previous studies have shown that the NF-κB pathway is involved in regulating anti- and pro-apoptotic gene expression. We also investigated the effects of LA on anti- (Bcl-xL, Mcl-1) and pro- (BAX1, TP53, Caspase3) apoptotic genes and NF-κB activation-related genes (RelA, JNK1, JNK2, IL-6) in porcine embryos. We have found that down-regulation of pro-apoptotic gene expression occurs in the LA-treated group. It was also found that Bcl-xL, one of the anti-apoptotic genes, was not affected by LA, which appears to be an effect of IL-6. In contrast, Mcl-1, an anti-apoptotic gene known not to be affected by IL-6, was found to have increased expression mRNA level in LA-treated pig embryos. Furthermore, through double-staining of apoptosis and immunocytochemistry, as the concentration of NF-kB level increases, the nuclear translocation of c-jun, the protein of which was also related with apoptosis, increased gradually depending on the LA concentration. These data could support that porcine embryo can use exogenous LA as a metabolic energy source. The data also demonstrate the important role of NF-kB in porcine early embryo development.
Support was provided by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Development of High Value-Added Food Technology program funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA, 118042-03-1-HD020).
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