Mito-TEMPO improves development competence by reducing superoxide in preimplantation porcine embryos

Abstract

Mito-TEMPO is a well-known mitochondria-specific superoxide scavenger. However, the effect of Mito-TEMPO on porcine embryo development, to our knowledge, has not been studied yet. In the present study, porcine embryos were classified into two groups (G1 and G2) based on the cytoplasm lipid contents at the zygote stage. The development of blastocysts derived from G2 zygotes was reduced (G2:16.2 ± 7.9% vs G1: 26.5 ± 5.9%; 1.6-fold, p < 0.05) compared to those from G1 zygotes. In G2 embryos, the proportion of TUNEL-positive cells was also higher than that of G1 embryos. Superoxide in G2 embryos was significantly increased compared to that in G1 embryos. Mitochondrial membrane potential and ATP production were lower in G2 embryos than in G1 embryos. Phosphorylation of Drp1 at Ser 616 increased in G1 embryos during the cleavage stages compared to that in the zygote but was not significantly different in G2 embryos. Then, the effects of Mito-TEMPO were investigated in G2 embryos. Blastocyst formation rate (G2: 19.1 ± 5.1% vs G2 + Mito-TEMPO: 28.8 ± 4.0%; 1.5-fold, p < 0.05) and mitochondrial aggregation were recovered after superoxide reduction by Mito-TEMPO treatment. Thus, we showed that Mito-TEMPO improves blastocyst development by superoxide reduction in porcine embryos in vitro.

Figure 1

Morphology of embryos according to the cytoplasm area. (a) Morphology at each embryo stage was visualized using an optical microscope (left panel). White lines represent the area of embryo cytoplasm and arrows indicate fragments. Quantitative analysis of cytoplasm and equal-sized blastomeres of cleavage stage in G1 and G2 embryos (right panel). (b) Stained lipid droplets of zygote were visualized using an optical microscope (left panel). Extracted Oil Red O reagents were measured at 500 nm wavelength using a microplate reader (right panel). Data in the bar graph represent the means ± SEM from three independent experiments. To analyze these data, a t-test was used. Differences were considered significant at *p < 0.05, ** < 0.01. Scale bar = 100 μm.

Figure 2

Changes in intracellular ROS, superoxide, and apoptosis according to the grade in preimplantation embryos. (a) Expression of intracellular ROS detected by DCF-DA (green) and analyzed using epifluorescence microscope. (b) MitoSOX (red) as superoxide indicator detected using a confocal microscope. (C) DAPI (blue) and TUNEL-positive cells (green) of blastocysts shown using a fluorescence microscope. The arrows indicate apoptotic cells in nuclei. Data in the bar graph represent the means ± SEM from three independent experiments. To analyze these data, a t-test was used. Differences were considered significant at *p < 0.05 and ** < 0.01. Scale bar = 100 μm.

Figure 3

Changes in mitochondrial function and dynamics in the G1 and G2 groups. (a) Expression of J-aggregate (red) and J-monomer (green) were determined by JC-1 using a confocal microscope. (b) ATP contents were measured by an ATP determination kit using a microplate reader. (c) Mitotracker (green) as the mitochondria-selective probe was measured under image system. N indicates the nucleus and arrows represent mitochondrial aggregation form. (d) Cropped blots are used in this figure, and the gels have been run under the same experimental conditions. Full-length blots are included for key data in the supplementary information. Western blot showing expression of mitochondrial fission proteins (Drp1 and phosphorylated Drp1-Ser616). Reactive protein levels were normalized to β-actin. Data in the bar graph represent means ± SEM from three independent experiments. To analyze these data, Dunnett’s multiple comparison test and t-test were used. Differences were considered significant at *p < 0.05 and ** < 0.01. Scale bar = 50 μm.

Figure 4

Effects of Mito-TEMPO on early embryo development in porcine. (a) Red stars indicate the number of expanded blastocysts. Morphologies of blastocyst stage in G1, G2 and G2 with Mito-TEMPO treatment. Detection of (b) superoxide (red fluorescence) and (c) mitochondrial distribution (green fluorescence) in G2 zygotes treated with Mito-TEMPO during embryo development. The arrows show aggregation of mitochondria and N represents the nucleus. Data in the bar graph represent the means ± SEM from three independent experiments. To analyze these data, Tukey’s multiple comparison test was used. Differences were considered significant at *p < 0.05 and ** < 0.01. Scale bar = 50 μm.

Figure 5

Schematic diagram illustrating the effect of Mito-TEMPO on porcine embryo development. Top panel; zygotes with low cytoplasm had increasing mitochondrial superoxide. In addition, high superoxide production in G2 embryos caused defective MMP and ATP production. Mitochondrial fission and aggregation increased in G2 embryos. Bottom panel; Zygotes of low cytoplasm cultured with Mito-TEMPO for early embryo development. Reduction of mitochondrial superoxide increases the blastocysts development rate and decreases mitochondrial aggregation in porcine.