78 Mito-TEMPO, a Scavenger for Mitochondria-Derived Reactive Oxygen Species, Enhances Porcine Pre-Implantation Embryo Development

Abstract

The production of reactive oxygen species (ROS) from mitochondria contributes to redox signalling, mitochondrial functions, and apoptosis. However, the specific effects of mitochondria target superoxide (O₂ ^•–) on porcine embryo development remain unclear. The objective of present study was to examine the differences of mitochondrial functions and dynamics in 2 subpopulations of porcine zygotes (G1 and G2), and to investigate the effects of Mito-TEMPO on porcine embryo development. Porcine embryos were visually classified in 2 groups [Grade (G)1: over 90%, and G2: below 90%] according to the lipid distribution at the zygote stage. The blastocyst development rate was greater in G1 than in G2 embryos (G1: 26.5 ± 5.9% v. G2: 16.2 ± 7.9%; P < 0.05). To evaluate blastocyst quality, we performed a 4′,6-diamidino-2-phenylindole (DAPI)-TUNEL assay. The proportion of TUNEL-positive cells was higher (P < 0.05) in G2 than G1 embryos. We measured superoxide production by MitoSOX staining as mitochondrial superoxide specific fluorescence dye by iRiS^TM Digital Cell Image System (Logos Biosystems Inc., Gyeonggi-do, South Korea). Red fluorescence intensity of superoxide in G2 embryos significantly increased (P < 0.05) compared with that in G1. We investigated changes in mitochondrial functions using a Mitotracker JC-1 mitochondrial membrane potential assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and ATP determination kit, respectively. Mitochondria membrane potential and ATP production were lower (P < 0.05) in G2 embryos than in G1 embryos. To confirm the protein levels of mitochondria fission protein DRP1, we performed Western blot analysis (per 40 embryos). Phosphorylation DRP1-Ser616 was increased (P < 0.05) in G1 embryos at cleavage stage compared with that in zygote, but not significantly different in G2 embryos. Thus, G2 embryos showed low development rate until blastocyst via mitochondrial dysfunction, increase in fission protein expression and mitochondrial aggregation according to the elevation of mito-ROS. Subsequently, the effect of the adding superoxide scavenger Mito-TEMPO was investigated in G2 embryos. Blastocyst formation (G2+MitoTempo: 28.8 ± 4.0% v. G2: 19.1 ± 5.1%; P < 0.05) and mitochondrial aggregation were recovered by mito-ROS reduction mediated by Mito-TEMPO. Our observations demonstrated that regulation of superoxide in mitochondria is important in pre-implantation development of porcine embryos.

This work was supported by grants from the Next-Generation BioGreen 21 Program (PJ01117604) and the Bio-industry Technology Development Program (316037-04-2-HD020) through the Rural Development Administration and the Ministry of Agriculture, Food and Rural Affairs, Republic of Korea.