Regulation of Mitochondrial Ca2+ by Ruthenium Red during in vitro Fertilization Improves Mitochondrial Functions and Blastocyst Development in Porcine Embryos

Ho-Guen Jegal,Hyo-Jin Park,Jin-Woo Kim,Seul-Gi Yang,Min-Ji Kim,Joung Jun Park,Deog-Bon Koo 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06

Mitochondria have vital functions that regulate Ca2+ homeostasis and ATP production in fertilized mammalian embryos. In addition, regulation of mitochondrial Ca2+ is related to the mitochondrial functions and mitochondria mediated apoptosis. However, early embryonic development according to the change of mitochondrial Ca2+ regulation during IVF of porcine oocyte has not been reported. In this study, we investigated the regulation of mitochondrial Ca2+ by ruthenium red (RR) during IVF affects blastocyst development in porcine embryos. Here, we investigated the changes of mitochondrial Ca2+ by using Rhod-2 staining in fertilized oocytes during IVF progression (0, 3 and 6 h). After IVF, Rhod-2 expression significantly increased in most of fertilized oocytes at 3 and 6 h. Therefore, we treated RR into IVF medium for reduction of mitochondrial Ca2+. As expected, expressions of rhod-2 and mito-sox significantly decreased in RR treated zygotes. To confirm the mitochondrial functions, we performed the JC-1 and ATP determination in porcine zygotes. As a result, MMP and ATP increased in fertilized oocytes after RR treatment. Interestingly, blastocyst development rate was significantly increased in 20 μM RR treated fertilizing oocytes compared to other groups (p <0.05; 20 μM RR: 33.2±5.0% vs. 10 μM RR: 22.4±5.0% and Con: 24.7±3.8%). In addition, we confirmed that apoptosis in porcine blastocysts was reduced according to RR treatment during IVF procedure by using a TUNEL assay. These results demonstrated that positive effects of RR for regulation of mitochondrial Ca2+ during IVF progression improve the mitochondrial functions and blastocyst developmental competence in porcine embryos.

Reduction of mitochondria derived superoxide by Mito-TEMPO improves the porcine oocyte maturation

Seul-Gi Yang,Hyo-Jin Park,Jin-Woo Kim,Min-Ji Kim,Ho-Guen Jegal,In-Su Kim,Min-Young Guk,Sun-Mi Park,Ji-Eun Lee,Deog-Bon Koo,Joung Jun Park 한국수정란이식학회 2018 한국수정란이식학회 학술대회 Vol.2018 No.11

In general, the shape of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage is important roles on meiotic maturation of porcine oocyte during in vitro maturation (IVM). Then, mitochondria produce reactive oxygen species (ROS) such as superoxide from electron transport system during oocyte maturation. ROS levels on oocytes are regulated by various antioxidant enzymes in cumulus cells (CCs). However, the effect of mitochondria derived superoxide production from CCs during IVM of porcine oocyte has not been reported. Firstly, we divided groups according to large number of CCs (Grade 1: G1) and small number of CCs (Grade 2: G2). Then, we counted cumulus cells of G1 and G2 oocyte by using haemocytometer. The oocyte maturation rate was significant decreased (p < 0.05) in G2 oocytes than that of G1 oocytes. We measured mitochondria derived superoxide in G1 and G2 COCs by using Mito-SOX staining. Mitochondrial superoxide was higher in G2 COCs than G1 COCs. Then, the mRNA expression levels of antioxidant enzymes (SOD1, SOD2 and PRDX3) in G2 COCs were decreased compared to G1 COCs. To reduce mitochondria derived superoxide, we used Mito-TEMPO as mitochondrial superoxide scavenger. Oocyte maturation rates in both G1 and G2 groups treated with Mito-TEMPO were increased than that of non-treated groups. Mitochondrial superoxide was lower in G1 and G2 groups treated with Mito-TEMPO than that of non-treatment groups. The mRNA expression levels of antioxidant enzymes in G1 and G2 COCs treated with Mito-TEMPO were increased compared to non-treated groups. Based on these findings, we suggest that reduction of mitochondria derived superoxide by Mito-TEMPO assists maturation competence in porcine oocytes.

Melatonin Reduces the Bisphenol A-induced Superoxide and Improves Oocyte Maturation through Reducing of Mitochondrial Derived Apoptosis in Pigs

Hyo-Jin Park,Jin-Woo Kim,Seul-Gi Yang1,Min-Ji Kim,Ho-Guen Jegal,Joung Jun Park,Deog-Bon Koo 한국동물생명공학회(구 한국동물번식학회) 2018 발생공학 국제심포지엄 및 학술대회 Vol.2018 No.06

Bisphenol-A (BPA) as an organic synthetic compound of exhibiting estrogen-mimicking and hormone-like properties, which is commonly used to induce cellular stress or female reproductive toxicity. In addition, BPA induces the increasing of mitochondrial derived reactive oxygen species (ROS) such as superoxide, and production of these ROS affects to the meiotic maturation and cumulus cells expansion on in vitro maturation (IVM) of porcine cumulus-oocyte complexes (COCs). However, anti-oxidative effect of melatonin for reduction of BPA-induced superoxide on porcine oocyte maturation has not been reported. Therefore, in present study, we confirmed that the reduction of BPA-derived superoxide by melatonin related to the reducing of mitochondria mediated apoptosis on meiotic maturation and cumulus cells expansion of porcine COCs. Then, to investigate the effects of superoxide specific scavenger, Mito-TEMPO, during porcine oocyte maturation progression, COCs cultured in maturation medium with Mito-TEMPO (0.1 μM) after pre-treatment of BPA (75 μM) for 22 h. Reduced meiotic maturation rate and cumulus cells expansion of COCs in the BPA (75 μM) treated group were recovered (p<0.01) by Mito-TEMPO treatment. Also, increasing of mitochondria derived apoptotic factors (AIF, Cleaved Caspase 3 and Cleaved PARP 1) protein levels by BPA treatment were reduced by Mito-TEMPO treatment in porcine COCs maturation. Positive effects of Mito-TEMPO for superoxide reduction on oocyte maturation and reducing mitochondrial apoptosis showed the same pattern in melatonin (0.1 μM) treated COCs. In case of supplemented with BPA and melatonin, superoxide production in COCs was not changed compared to control or melatonin treated groups. Based on these results, we concluded that melatonin as a regulator of superoxide such as Mito-TEMPO improves oocyte maturation through reduction of mitochondria derived apoptosis during IVM of porcine COCs.

Efficient Production of Gene Edited Bovine Embryos

Min-Ji Kim,Hyo-Jin Park,Jin-Woo Kim,Seul-Gi Yang,Jae-Min Jung,In-Su Kim,Ho-Guen Jegal,Man-Jong Kang,Deog-Bon Koo 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10

Transgenic animals has been produced by using microinjection method for target gene editing. However, the producing efficiency of gene edited embryos in livestock is very low. Therefore, the objective of this study is production of high quality blastocyst and establishment of appropriate microinjection condition for gene editing embryos. Frist, we treated the cytochalasin B (CB) within injection medium for reduction of microinjection damage, and microinjected embryos were cultured in CR1aa medium supplemented with 0.1 μM melatonin. As a result, blastocysts development of microinjected embryos in CB treated group was significantly higher than that of non-treated group (p<0.05, CB+, 18.5±2.0% vs CB-, 11.2±3.5%). After microinjection under CB treated condition, blastocyst formation was significantly increased (p<0.05) in Mela treated group. Next, to investigate DNA damage in blastocyst stage embryos, fluorescence expressions of H2AX139ph (DNA damage protein) and RAD51 (DNA repairs protein) were observed by using immunofluorescence staining. DNA damage in developing blastocysts was reduced in the CB and Mela treated embryos. Finally, we identified the blastocyst development after microinjection by using CRISPR/Cas9 system according to protein or mRNA of Cas9. Green fluorescence expressions by GFP were increased in developing blastocysts derived from Cas9 protein microinjected embryos. Moreover, gene targeting rate in GFP expressed blastocysts was increased in Cas9 protein injected group (p<0.05; Cas9 protein, 30% vs Cas9 mRNA, 20%). These results demonstrated that positive effect of CB and Mela treatment improves the blastocysts qualities and their developmental competence via reduction of microinjection damage in gene editing bovine embryos. We also verified the improvement of knock-in efficiency in microinjected bovine embryos by using Cas9 protein.

Regulation of Mitochondrial Superoxide Improves Developmental Competence and Embryonic Quality through the Control of Mitochondrial Functions and Dynamics in Pigs

Seul-Gi Yang,Hyo-Jin Park,Jin-Woo Kim,Jae-Min Jung,Min-Ji Kim,Ho-Guen Jegal,In-Su Kim,Deog-Bon Koo 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2

Mitochondrial functions are required for early embryonic development in pigs. Reactive oxygen species (ROS) can produce from the maintenance of mitochondrial functions during in vitro culture (IVC) of embryos. However, the role of mitochondria specific ROS (mito-ROS) in porcine early embryonic development remains unknown. Therefore, the objective of present study was to confirm the changes in functions, aggregation and dynamics of mitochondria by the regulation of mito-ROS in porcine embryo development. Here, we performed the IVC from classified two groups (G1; high lipid and G2; low lipid contents) at the zygote stage. Blastocyst developmental rate and total nuclei numbers in G2 embryos were lower (p<0.05) than that of G1 embryos. To investigate the superoxide as mitochondrial-target ROS on preimplantation development of G1 and G2 embryos, we performed the MitoSOX staining. Superoxide levels significantly increased (p<0.05) in G2 embryos. And, changes of mitochondrial membrane potential (MMP), ATP levels and expression of mitochondria fission protein were investigated by using JC-1 kit, ATP determination kit and Western blot analysis. MMP and ATP levels were significantly decreased (p<0.05) in G2 embryos compared with G1 embryos. Protein level of mitochondrial fission protein Drp1 was increased (p<0.05) in the cleavage stage of G2 embryos. To confirm the effects of reducing mito-ROS on the porcine embryo development, we treated with the Mito-TEMPO, mitochondria specific ROS scavenger, for G2 embryos. Mito-TEMPO improved preimplantation development to the blastocyst stage and reduced mito-ROS level in the G2 embryos (p<0.05). These results demonstrated that regulation of mito-ROS improves the blastocysts development and their qualities through the control of mitochondrial functions and dynamics of porcine embryos.

Exogenous Ganglioside GT1b Enhances Oocytes Maturation, Cumulus Cells Expansion and Further Preimplantation Developmental Competence in Pigs

Jin-Woo Kim,Hyo-Jin Park,Seul-Gi Yang,Jae-Min Jung,Min-Ji Kim,In-Su Kim,Ho-Guen Jegal,Deog-Bon Koo 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05

Ganglioside GT1b, glycosphigolipids with three sialic acid, is known to play an important role in signal transduction such as epidermal growth factor receptor (EGFR). EGF is also known to induce resumption of meiosis and cumulus cells expansion during porcine oocyte maturation. Therefore, this study was conducted to evaluate the effects of ganglioside GT1b on resumption of meiosis and cumulus cells expansion in porcine oocyte maturation. First, porcine cumulus-oocyte complexes were cultured in NCSU-23 medium supplemented with GT1b (0, 1, 2 and 4 μM) at 44 h. We observed that the proportion of the metaphase II (M II) stage was significantly increased in the 2 μM GT1b (78.0 ± 2.3) treated group than in the other groups. Furthermore, expression of cumulus cells expansion factor genes (Has2, TNFAIP6, Ptx3) were significantly increased in the 2 μM GT1b treated group than in the other groups. Next, we investigated the meiotic maturation and the expressions of cumulus cells expansion factor genes after GT1b and/or EGF treatment. The proportion of the M II stage was significantly higher in the GT1b+EGF (90.1 ± 2.3) treated group than in the other groups. Moreover, expressions of cumulus cells expansion factor genes were significantly increased in the GT1b+EGF treated group than in the control group. After in vitro fertilization, fertilization rate, preimplantation development competence and quality of blastocyst were improved in oocytes derived from GT1b+EGF treated group. Taken together, these results suggest that exogenous ganglioside GT1b improving the developmental competence of porcine embryos via increase of resumption of meiosis and cumulus cells expansion during in vitro maturation of porcine oocytes.

Melatonin Improves Oocyte Maturation and Mitochondrial Functions by Reducing Bisphenol A-Derived Superoxide in Porcine Oocytes In Vitro

Park, Hyo-Jin,Park, Soo-Yong,Kim, Jin-Woo,Yang, Seul-Gi,Kim, Min-Ji,Jegal, Ho-Guen,Kim, In-Su,Choo, Young-Kug,Koo, Deog-Bon MDPI 2018 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.19 No.11

<P>Bisphenol A (BPA) is synthetic organic compound that exhibits estrogen-like properties and it induces mitochondrial superoxide production. Melatonin (Mela) protects against BPA-mediated cell damage and apoptosis. However, the antioxidative effects of Mela against BPA-induced superoxide production in porcine oocytes are still not known. In this study, we investigated the antioxidative effects of Mela against BPA-derived superoxide on oocyte maturation in pigs. To investigate the effects of the superoxide specific scavenger, Mito-TEMPO, on porcine oocyte maturation in response to BPA exposure apoptosis proteins, we treated the oocytes with Mito-TEMPO (0.1 µM) after pre-treating them with BPA (75 µM) for 22 h. As expected, the reduction in meiotic maturation and cumulus cell expansion of cumulus-oocyte-complexes (COCs) in the BPA (75 µM) treated group was recovered (<I>p</I> < 0.01) by treatment with Mito-TEMPO (0.1 µM). An increase in the levels of mitochondrial apoptotic proteins (AIF, cleaved Cas 3 and cleaved Parp1) in response to BPA-induced damage was also reduced by Mito-TEMPO treatment in porcine COCs. Interestingly, we confirmed the positive effects of Mela with respect to superoxide production upon BPA exposure during oocyte maturation and also confirmed the reduction in mitochondrial apoptosis in Mela (0.1 µM)-treated porcine COCs. These results provide evidence for the first time that antioxidative effects of Mela on BPA-derived superoxide improve porcine oocyte maturation.</P>