www.nature.com/scientificreports OPEN Mito-TEMPO improves development competence by reducing superoxide in Received: 19 January 2018 Accepted: 22 May 2018 preimplantation porcine embryos Published: xx xx xxxx Seul-Gi Yang1, Hyo-Jin Park1, Jin-Woo Kim1, Jae-Min Jung1, Min-Ji Kim1, Ho-Guen Jegal1, In-Su Kim1, Man-Jong Kang2, Gabbine Wee3, Hee-Young Yang3, Yun-Han Lee4, Ji-Hae Seo5, Sun-Uk Kim6 & Deog-Bon Koo1 Mito-TEMPO is a well-known mitochondria-specifc superoxide scavenger. However, the efect of Mito- TEMPO on porcine embryo development, to our knowledge, has not been studied yet. In the present study, porcine embryos were classifed 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 signifcantly 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 signifcantly diferent in G2 embryos. Then, the efects 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. Mitochondria are well-known organelles for adenosine triphosphate (ATP) production, which is important for controlling cell growth, signaling, dynamic response, and apoptosis in most mammalian cells. In porcine oocytes and/or embryos, a high level of ATP production in the cytoplasm is necessary for oocyte maturation, fertilization, and early embryo development in vivo and in vitro1. Particularly, afer in vitro fertilization (IVF), zygote stage porcine embryos contain high levels of lipids consisting of triglycerides for energy storage2. Many studies have shown that ATP production-related lipid metabolism is important for early embryo development3,4. During ATP production, reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, and hydroxyl radicals are generated by oxidative phosphorylation in mitochondria5. Tis production of ROS is linked to oocyte maturation, fertilization, and embryo development in pigs6. Many studies have shown that ROS accumulation reduces embryonic developmental competence and blastocyst quality in pigs7, cattle8 and mice9. In addition, an imbalance between free radical formation and removal can lead to oxidative stress, which can induce DNA dam- age and increase the expression of pro-apoptotic genes leading to cell death during oocyte maturation and early embryonic development10,11. 1Department of Biotechnology, College of Engineering, Daegu University, 201 Daegudae-ro, Jillyang, Gyeongsan, Gyeongbuk, 38453, Republic of Korea. 2Department of Animal Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, 61186, Republic of Korea. 3Laboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF), 80 Cheombok-ro, Dong-gu, Daegu, 41061, Republic of Korea. 4Department of Molecular Medicine, Keimyung University School of Medicine, Daegu, 42601, Republic of Korea. 5Department of Biochemistry, Keimyung University School of Medicine, Daegu, 42601, Republic of Korea. 6National Primate Research Center & Futuristic Animal Resource and Research Center, Korea Research Institute of Bioscience and Biotechnology, Ochang, Chungbuk, 28116, Republic of Korea. Seul-Gi Yang and Hyo-Jin Park contributed equally to this work. Correspondence and requests for materials should be addressed to D.-B.K. (email: [email protected]) SCIENTIFIC REPORTS | (2018) 8:10130 | DOI:10.1038/s41598-018-28497-5 1 www.nature.com/scientificreports/ Figure 1. Morphology of embryos according to the cytoplasm area. (a) Morphology at each embryo stage was visualized using an optical microscope (lef 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 (lef 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. Diferences were considered signifcant at *p < 0.05, ** < 0.01. Scale bar = 100 μm. Moreover, severe oxidative stress resulting from increasing ROS is known to induce mitochondrial fssion that elicits mitochondria’s dynamic response12, aggregation13 and dysfunctions14. Mitochondrial fssion, one of the mitochondrial dynamic responses, is regulated by the fssion protein, dynamin-related protein 1 (Drp1). Increasing mitochondrial fssion by the accumulation of ROS reduced the ATP production and mitochondrial membrane potential (MMP)15. Tis, in turn, induced mitochondrial-derived apoptosis as a result of dysfunction of MMP under conditions of ROS accumulation16. Most of all, superoxide derived from mitochondrial ROS can act as signaling molecules to activate pro-growth responses in cancer cells17. It was recently proven that triphenylphosphonium chloride (Mito-TEMPO) is a superoxide scavenger and a physiochemical compound mimicking superoxide dismutase from mitochondria. It can easily pass through the lipid bilayers and accumulate in the mitochondria18. Mito-TEMPO has been impli- cated in the functions of mitochondria because it has the role of eliminating mitochondrial ROS. For instance, Mito-TEMPO prevents oxalate-induced injury by inhibiting mitochondrial dysfunctions and modulating oxida- tive stress in NRK-52E cells19 and inhibition of mitochondrial ROS with Mito-TEMPO reduced diabetic cardio- myopathy18. However, the efects of Mito-TEMPO on porcine embryo development has not been investigated yet. In order to understand the efects of Mito-TEMPO on porcine embryo development, we divided fertilized zygotes into two groups (high cytoplasm components: grade 1, G1 and low cytoplasm components: grade 2, G2) based on the cytoplasm components of the zygotes. Further, we investigated the changes in expression of the mitochondria-derived superoxide on porcine embryos preimplantation development in G1 and G2 groups using the mitochondrial superoxide-specifc staining indicator, Mito-SOX. Subsequently, we measured the maintenance of mitochondrial functions and regulation of dynamic response in the early development of porcine embryos. Subsequently, efects of Mito-TEMPO as a superoxide scavenger were assessed by examining the embryo develop- ment rate as well as the distribution of mitochondria. Here, we show how superoxide reduction is linked to better embryo development in porcine using Mito-TEMPO, which changes the mitochondrial ROS and distribution. Results Developmental competence until blastocyst stage for G1 and G2 porcine zygotes. As shown in Fig. 1a, we divided the zygotes into two groups (grade 1, G1: high cytoplasm components; grade 2, G2: low cyto- plasm components) according to the cytoplasm and morphology of the zygotes at stages described by Van Soom et al., Ajduk, and Zernicka-Goetz20,21. To confrm the diference in the cytoplasm components of the zygotes, we measured lipids as triglycerides using the Oil Red O stain. As expected, the values of isopropanol absorbance spectrum measurements in extracted lipid derived from zygote cytoplasm signifcantly increased (p < 0.05) in the SCIENTIFIC REPORTS | (2018) 8:10130 | DOI:10.1038/s41598-018-28497-5 2 www.nature.com/scientificreports/ No. of embryos % of embryos % of blastocysts Groups cultured cleaved (n) (n) Grade 1 290 78.4 ± 14.1 (227) 26.5 ± 5.9 (78)a Grade 2 268 67.5 ± 15.3 (192) 16.2 ± 7.9 (44)b Table 1. Development rate according to lipid of cytoplasm at zygote. Data are expressed as means ± SD of three independent experiments. Diferent superscript letters a and b denote signifcant diferences (p < 0.05). G1 zygote group compared to G2 zygotes (Fig. 1b). In addition, lipid droplet size in G1 zygote was smaller than G2 zygote. In the cleaved stage, we also observed the increase of large lipid droplets in G2 group embryos than that of G1 group embryos (data not shown). Moreover, the development rate of blastocysts from G2 zygotes was signifcantly reduced (1.8-fold, p < 0.05, G2: 16.2 ± 7.9% vs G1: 26.5 ± 5.9%) compared to G1 zygotes (Table 1). Terefore, embryos of G1 and G2 groups were classifed based on the cytoplasm lipid contents at the zygote stage, and embryos developed from two groups showed the diferent developmental potential until blastocyst stage. Based on these results, embryos of G1 and G2 groups could be used for subsequent experiments on porcine embryo development. Detection of intracellular ROS, superoxide, and cellular apoptosis during the preimplantation development of G1 and G2 embryos. We measured intracellular ROS and superoxide levels using dichlorofuorescin diacetate (DCF-DA) (green fuorescence) and MitoSOX (red fuorescence) as mitochon- drial superoxide indicators in G1 and G2 embryos, respectively. Intracellular ROS levels