Background: Post-ovulatory aging (POA) of oocytes is related to a decrease in the quality and quantity of oocytes caused by aging. Previous studies on the characteristics of POA have investigated injury to early embryonic developmental ability, but no information is available on its effects on mitochondrial fission and mitophagy-related responses. In this study, we aimed to elucidate the molecular mechanisms underlying mitochondrial fission and mitophagy in in vitro maturation (IVM) oocytes and a POA model based on RNA sequencing analysis. Methods: The POA model was obtained through an additional 24 h culture following the IVM of matured oocytes. NMN treatment was administered at a concentration of 25 μM during the oocyte culture process. We conducted MitoTracker staining and Western blot experiments to confirm changes in mitochondrial function between the IVM and POA groups. Additionally, comparative transcriptome analysis was performed to identify differentially expressed genes and associated changes in mitochondrial dynamics between porcine IVM and POA model oocytes. Results: In total, 32 common genes of apoptosis and 42 mitochondrial fission and function uniquely expressed genes were detected (≥ 1.5-fold change) in POA and porcine metaphase II oocytes, respectively. Functional analyses of mitochondrial fission, oxidative stress, mitophagy, autophagy, and cellular apoptosis were observed as the major changes in regulated biological processes for oocyte quality and maturation ability compared with the POA model. Additionally, we revealed that the activation of NAD+ by nicotinamide mononucleotide not only partly improved oocyte quality but also mitochondrial fission and mitophagy activation in the POA porcine model. Conclusions: In summary, our data indicate that mitochondrial fission and function play roles in controlling oxidative stress, mitophagy, and apoptosis during maturation in POA porcine oocytes. Additionally, we found that NAD+ biosynthesis is an important pathway that mediates the effects of DRP1-derived mitochondrial morphology, dynamic balance, and mitophagy in the POA model.

Reactive oxygen species (ROS) production and F-actin cytoskeleton dynamics play important roles in the survival rate of blastocysts after the vitrifiedwarming process. However, the protective effects of Mito-TEMPO against cryo-injury and viability through F-actin aggregation and mitochondrial-specific ROS production in vitrificated-warmed bovine embryos have not been investigated. The aims of the present study were to: (1) determine the effects of Mito-TEMPO on embryonic developmental competence and quality by F-actin stabilization during in vitro culturing (IVC), and (2) confirm the effects of Mito-TEMPO through F-actin structure on the cryotolerance of vitrification-warming in Mito-TEMPO exposed in vitro production (IVP) of bovine blastocysts. Bovine zygotes were cultured with 0.1 μM Mito-TEMPO treatment for 2 days of IVC. Mito-TEMPO (0.1 μM) exposed bovine embryos slightly improved in blastocyst developmental rates compared to the non-treated group. Moreover, the viability of vitrified-warmed blastocysts from Mito-TEMPO treated embryos significantly increased (p < 0.05, non-treated group: 66.7 ± 3.2% vs Mito-TEMPO treated group: 79.2 ± 5.9%; re-expanded at 24 hours). Mito-TEMPO exposed embryos strengthened the F-actin structure and arrangement in the blastocyst after vitrification-warming. Furthermore, the addition of Mito-TEMPO into the IVC medium enhanced embryonic survival and quality through F-actin stabilization after the vitrification-warming procedure. Overall, our results suggest that supplementing the culture with 0.1 μM Mito- TEMPO improves the embryonic quality and cryo-survival of IVP bovine blastocysts.

Vitrification methods are commonly used for mammalian reproduction through the long-term storage of blastocyst produced in vitro. However, the survival and quality of embryos following vitrification are significantly low compared with blastocyst from in vitro production (IVP). This study evaluates that the survival of frozen-thawed bovine embryos was relevant to mitochondrial superoxide derived mitochondrial activity. Here we present supplementation of the cryopreservation medium with Mito- TEMPO (0.1 μM) induced a significant (p < 0.001; non-treated group: 56.8 ± 8.7%, reexpanded at 24 h vs Mito-TEMPO treated group: 77.5 ± 8.9%, re-expanded at 24 h) improvement in survival rate of cryopreserved-thawed bovine blastocyst. To confirm the quality of vitrified blastocyst after thawing, DNA fragmentation of survived embryos was examined by TUNEL assay. As a result, TUNEL positive cells rates of frozenthawed embryos were lower in the Mito-TEMPO treated group (4.2 ± 1.4%) than the non-treated group (7.1 ± 3.5%). In addition, we investigated the intracellular ROS and mitochondrial specific superoxide production using DCF-DA and Mito-SOX staining in survived bovine embryos following vitrification depending on Mito-TEMPO treatment. As expected, intracellular ROS levels and superoxide production of vitrified blastocysts after cryopreservation were significantly reduced (p < 0.05) according to Mito-TEMPO supplement in freezing medium. Also, mitochondrial activity measured by MitoTracker Orange staining increased in the frozen-thawed embryos with Mito-TEMPO compared with non-treated group. These results indicate that the treatment of Mito-TEMPO during cryopreservation might induce reduction in DNA fragmentation and apoptosis-related ROS production, consequently increasing mitochondrial activation for developmental capacity of frozen-thawed embryos.

Gangliosides are glycolipids in which oligosaccharide is combined with sialic acids. Our previous studies have suggested an interplay between ganglioside GD1a/GT1b and meiotic maturation capacity in porcine oocyte maturation. Furthermore, ganglioside GD1a and GT1b are known for its antioxidant activity, but it is still unclear whether possible antioxidant role of GD1a and GT1b is involved in porcine embryos development competence during in vitro culture (IVC). Here, the effects of ganglioside GD1a and GT1b on the embryonic developmental competence during in vitro culture of porcine were investigated. The effects of ganglioside GD1a and GT1b on the expression of ST3GAL2 were confirmed during embryos development (2-cell, 4-cell, 8-cell and blastocyst) using immunofluorescent staining (IF). As a result, the fluorescent expression of ST3GAl2 was higher in embryos at 4-8 cells stage than blastocysts. Blastocyst development rate significantly increased in only 0.1 μM GD1a and GT1b treated groups compared with control group. To investigate the cellular apoptosis, we analyzed TUNEL assay. In case of only 0.1 μM GD1a and GT1b treated groups, the total number of cells in blastocyst compared with control group, but there was no significant difference in the rate of apoptotic cells. We identified the intracellular ROS levels using DCF-DA staining. According to the result, ROS production significantly decreased in blastocysts derived from the 0.1 μM GD1a and GT1b treated groups. These results suggest that ganglioside GD1a and GT1b improve the developmental competence of porcine embryos via reduction of intracellular ROS during preimplantation stage.

Cryopreservation is used for blastocyst preservation of most mammalian embryos and is an important technique for breeding. We aimed to compare the efficiency of the cryopreservation method using the standard Cryotop device and the ReproCarrier device, a domestic product manufactured in Korea. The efficacy of the two devices was analyzed based on the survival rate, intracellular levels of reactive oxygen species (ROS), and apoptosis of the vitrified bovine blastocysts. The survival rates of the vitrified-warmed blastocysts were similar between the ReproCarrier group (58.4 ± 17.7%) and Cryotop group (59.9 ± 14.1%). Intracellular ROS levels and apoptotic index were determined by DCFDA staining and TUNEL assay. Changes in intracellular ROS levels, number of total nuclei, and cellular apoptosis of vitrified blastocysts after cryopreservation were not significantly different between the two groups. These results indicate that the ReproCarrier device method is as effective as the standard Cryotop method for vitrification of bovine blastocysts in vitro.

Sestrin-2 (SESN2) as a stress-metabolic protein is known for its anti-oxidative effects as a downstream factor of PERK pathways in mammalian cells. However, the expression patterns of SESN2 in conjunction with the UPR signaling against to ER stress on porcine oocyte maturation in vitro, have not been reported. Therefore, we confirmed the expression pattern of SESN2 protein, for which to examine the relationship between PERK signaling and SESN2 in porcine oocyte during IVM. We investigated the SESN2 expression patterns using Western blot analysis in denuded oocytes (DOs), cumulus cells (CCs), and cumulus-oocyte complexes (COCs) at 22 and 44 h of IVM. As expected, the SESN2 protein level significantly increased (p < 0.01) in porcine COCs during 44 h of IVM. We investigated the meiotic maturation after applying ER stress inhibitor in various concentration (50, 100 and 200 μM) of tauroursodeoxycholic acid (TUDCA). We confirmed significant increase (p < 0.05) of meiotic maturation rate in TUDCA 200 μM treated COCs for 44 h of IVM. Finally, we confirmed the protein level of SESN2 and meiotic maturation via regulating ER-stress by only tunicamycin (Tm), only TUDCA, and Tm + TUDCA treatment in porcine COCs. As a result, treatment of the TUDCA following Tm pre-treatment reduced SESN2 protein level in porcine COCs. In addition, SESN2 protein level significantly reduced in only TUDCA treated porcine COCs. Our results suggest that the SESN2 expression is related to the stress mediator response to ER stress through the PERK signaling pathways in porcine oocyte maturation.

The Transgenic livestock can be useful for the production of disease-resistant animals, pigs for xenotranplantation, animal bioreactor for therapeutic recombinant proteins and disease model animals. Previously, conventional methods without using artificial nuclease-dependent DNA cleavage system were used to produce such transgenic livestock, but their efficiency is known to be low. In the last decade, the development of artificial nucleases such as zinc-finger necleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas has led to more efficient production of knock-out and knock-in transgenic livestock. However, production of knock-in livestock is poor. In mouse, genetically modified mice are produced by co-injecting a pair of knock-in vector, which is a donor DNA, with a artificial nuclease in a pronuclear fertilized egg, but not in livestock. Gene targeting efficiency has been increased with the use of artificial nucleases, but the knock-in efficiency is still low in livestock. In many research now, somatic cell nuclear transfer (SCNT) methods used after selection of cell transfected with artificial nuclease for production of transgenic livestock. In particular, it is necessary to develop a system capable of producing transgenic livestock more efficiently by co-injection of artificial nuclease and knock-in vectors into fertilized eggs.

Morphology of cumulus-oocyte-complexes (COCs) at germinal vesicle (GV) stage as one of the evaluation criteria for oocyte maturation quality after in vitro maturation (IVM) plays important roles on the meiotic maturation, fertilization and early embryonic development in pigs. When cumulus cells of COCs are insufficient, which is induced the low oocyte maturation rate by the increasing of reactive oxygen species (ROS) in porcine oocyte during IVM. The ROS are known to generate including superoxide and hydrogen peroxide from electron transport system of mitochondria during oocyte maturation in pigs. To regulate the ROS production, the cumulus cells is secreted the various antioxidant enzymes during IVM of porcine oocyte. Our previous study showed that Mito-TEMPO, superoxide specific scavenger, improves the embryonic developmental competence and blastocyst formation rate by regulating of mitochondria functions in pigs. However, the effects of Mito-TEMPO as a superoxide scavenger to help the anti-oxidant functions from cumulus cells of COCs on meiotic maturation during porcine oocyte IVM has not been reported. Here, we categorized experimental groups into two groups (Grade 1: G1; high cumulus cells and Grade 2: G2; low cumulus cells) by using hemocytometer. The meiotic maturation rate from G2 was significantly (p < 0.05) decreased (G1: 79.9 ± 3.8% vs G2: 57.5 ± 4.6%) compared to G1. To investigate the production of mitochondria derived superoxide, we used the mitochondrial superoxide dye, Mito-SOX. Red fluorescence of Mito-SOX detected superoxide was significantly (p < 0.05) increased in COCs of G2 compared with G1. And, we examined expression levels of genes associated with mitochondrial antioxidant such as SOD1, SOD2 and PRDX3 using a RT-PCR in porcine COCs at 44 h of IVM. The mRNA levels of three antioxidant enzymes expression in COCs from G2 were significantly (p < 0.05) lower than COCs of G1. In addition, we investigated the anti-oxidative effects of Mito-TEMPO on meiotic maturation of porcine oocyte from G1 and G2. Meiotic maturation and mRNA levels of antioxidant enzymes were significantly (p < 0.05) recovered in G2 by Mito-TEMPO (0.1 μM, MT) treatment (G2: 68.4 ± 3.2% vs G2 + MT: 73.9 ± 1.4%). Therefore, our results suggest that reduction of mitochondria derived superoxide by Mito-TEMPO may improves the meiotic maturation in IVM of porcine oocyte.

미토콘드리아는 세포질 칼슘 항상성 및 ATP 생산에 중요한 역할을 하는 세포 소기관으로 이러한 미토콘드리아의 기능은 성숙과 수정 그리고 배 발달에 매우 중요한 역할을 한다. 미토콘드리아 칼슘 축적은 기능장애를 일으킨다. 그러나 돼지 체외성숙란 및 수정란에서 미토콘드리아 칼슘 변화의 관련성에 관한 연구는 보고된 적이 없다. 본 연구의 목적은 미토콘드리아 칼슘 지시자로 알려진 Rhod-2 염색을 이용하여 성숙란 및 수정란에서 미토콘드리아 칼슘 축적의 변화를 확인하였다. 형태학적 모습의 기준을 통해 난구세포의 세포층과 세포질의 균질도를 바탕으로 G1과 G2로 나누어서 체외성숙을 진행하였다. 이후 두 그룹에서 핵 성숙율을 비교하였을 때, G2가 G1에 비해 낮게 나타났다(p<0.001). 돼지 체외성숙란 및 수정란에서 평균적인 Rhod-2 spot 의 수는 G1보다 G2에서 더 많이 나타났다(6시간째 체외수정란: p<0.05). 다음으로 Rhod-2 spot 수에 따른 난모세포의 비율을 확인하기 위해 Rhod-2 spot 의 수를 4개의 군(n<10, 10≤n<20, 20≤n<30, 그리고 30≤n)으로 나누어 해당 난모세포의 비율을 확인하였다. 체외성숙란 및 체외수정란 모두 G1이 G2에 비해 10개 미만(n<10)인 Rhod-2 spot 의 수를 가지는 난모세포가 많았으며, 체외수정란에서는 유의적으로 높았다(p<0.05). 마지막으로 체외성숙란 및 수정란에서 Rhod-2 intensity 값을 측정하여 두 그룹을 비교하였을 때, G2가 G1에 비해 유의적으로 높은 것을 확인 할 수 있었다(성숙란; p<0.001 그리고 수정란; p<0.05). 본 연구의 결과를 토대로 돼지에서 미성숙 난포란의 형태학적인 품질은 체외성숙 및 체외수정 과정 동안 미토콘드리아 내 칼슘 축적과 관련이 있음을 확인하였다.

Ganglioside GM3 is known as an inhibition factor of cell differentiation and proliferation via inhibition of epidermal growth factor receptor (EGFR) phosphorylation. Our previous study showed that the exogenous ganglioside GM3 reduced the meiotic maturation of porcine oocytes and induced apoptosis at 44 h of in vitro maturation (IVM). However, the role of ganglioside GM3 in the relationship between EGFR signaling and apoptosis during porcine oocyte maturation has not yet been studied. First, porcine cumulus-oocyte complexes (COCs) were cultured in the NCSU-23 medium with exogenous ganglioside GM3 according to maturation periods (non-treated, only IVM I: 0 - 22 h, only IVM II: 22 - 44 h and IVM I & II: 0 - 44 h). We confirmed that the proportion of germinal vesicle breakdown (GVBD) increased significantly in the IVM I treated group than in the control group. We also confirmed that the meiotic maturation until M II stage and polar body formation decreased significantly in the only IVM I treated group. Cumulus cell expansion and mRNA levels of the expansion-related factors (HAS2, TNFAIP6 and PTX3) decreased significantly in the IVM I treated group than in the control group. Protein levels of EGFR, p-EGFR, ERK1/2, and p-ERK1/2 decreased significantly in the GM3-treated groups, during the IVM I period. In addition, cellular apoptosis, determined using TUNEL assay, and protein levels of Cleaved caspase 3, were increased significantly in the GM3-treated COCs during the IVM I period. Based on these results, ganglioside GM3 exposure of porcine COCs during the IVM I period reduced meiotic maturation and cumulus cell expansion via inhibition of EGFR activity in pigs.

The knock-in efficiency is very important to manipulate gene editing in the transgenic domestic animal. Recently, it is reported that transiently loosen nucleosome folding of transcriptionally inactive chromatin might have potential tp enhance the homologouse recombination efficiency. Histone deacetylases (HDAC) are a class of enzymes that remove acetyl groups from an amino acid on a histone. This is important because DNA is wrapped around histones, and DNA expression is regulated by acetylation and de-acetylation. In this study, Mac-T cell were treated with 10uM VPA (valproic acid, HDAC inhibitor) for 24 h and transfected with Knock-in vector and TALEN at targeting of β-casein gene. After 3 day of transfection, knock-in efficiency was confirmed by PCR. The level of HDAC2 protein in Mac-T cells was decreased by VPA treatment. The knock-in efficiency in the Mac-T cell with treated HDAC inhibitor was higher than cell not treated HDAC inhibitor. These results indicated that chromatin modification by HDAC inhibitor enhances homologous recombination efficiency in the Mac-T cell.

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.

Mitochondrion is an organelle for regulating calcium (Ca2+) homeostasis. Mitochondrial Ca2+ plays important roles on oocyte maturation, fertilization and embryonic development for ATP production. Low quality oocytes have mitochondrial dysfunction, which lead to overloaded Ca2+ in mitochondria. Recently, Rhod-2 is well known as a mitochondrial derived Ca2+ indicator. However, the changes of Rhod-2 in matured or fertilized porcine oocytes have not been reported. Therefore, the aim of study was to identify the effects of mitochondrial Ca2+ using Rhod-2 on quality assessment of matured oocyte and zygotes in pigs. Thus, we classified two groups (group 1: G1, compact COCs and group 2: G2, uncompact COCs) according to differences of cumulus cells amount and cytoplasm morphology in germinal vesicle (GV) stage of porcine COCs. Therefore, we investigated number of Rhod-2 spots in matured and fertilized oocytes from G1 and G2 groups. The Rhod-2 spot numbers were separated into four parts; n<10, 10≤ n < 20, 20 ≤ n < 30, and 30 < n. The Rhod-2 spots number of G2 group had greater than G1 group in part of 20 ≤ n. Additionally, we investigate mean number of Rhod-2 spots from G1 and G2 groups in matured and fertilized oocytes. As a result, we confirmed that average number of Rhod-2 spots in G2 group increased than that of G2 group. Finally, we also measured the Rhod-2 intensity in matured and fertilized oocytes of G1 and G2 groups. Interestingly, the Rhod-2 intensity in G2 group was higher than that of G1 group. (oocyte: p < 0.001 and fertilized oocyte: p < 0.05). These results demonstrated that changes in Rhod-2 spots and intensity were increased in low quality of matured and fertilized oocytes. Therefore, our results suggest that the differences in mitochondrial calcium level are associated with morphological quality of porcine COCs.

This study aimed to produce high-quality blastocysts and establish appropriate microinjection conditions for the introduction of target gene. First, we identified embryo development to the blastocyst stage after microinjection using the CRISPR/Cas9 system on the Cas9 protein or mRNA. As a result, we confirmed that blastocyst development in the Cas9 mRNA injected group significantly increased when compared to the Cas9 protein injected group (p<0.05). However, the blastocyst gene targeting rate increased in the Cas9 protein injected group when compared to the Cas9 mRNA injected group (p<0.05). Next, we treated the injection medium with 10 μg/ml of cytochalasin B (CB), and the microinjected embryos were cultured in CR1-aa medium supplemented with 0.1 μM of melatonin (Mela). Consequently, the blastocyst formation rate significantly increased in the CB treated group (p<0.05). After microinjecting embryos with the CB treated injection medium, we investigated blastocyst formation and quality via Mela treatment. Consequently, the Mela treated group demonstrated significantly increased blastocyst formation rates when compared to the non-treated group (p<0.05). Furthermore, immunofluorescence assay using RAD51 (DNA repair detection protein) and H2AX139ph (DNA damage detection protein) showed an increase in RAD51 positive cells in Mela treated embryos. Therefore, we verified the improvement in knock-in efficiency in microinjected bovine embryos using Cas9 protein. These results also demonstrated that the positive effect of the CB and Mela treatments improved the embryonic developmental competence and blastocyst qualities in genetically-edited bovine embryos.

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.

Ganglioside GD1a is specifically formed by the addition of sialic acid to ganglioside GM1a by ST3 β- galactoside α -2,3-sialyltransferase 2 (ST3GAL2). Above all, GD1a are known to be related with the functional regulation of several growth factor receptors, including activation and dimerization of epidermal growth factor receptor (EGFR) in tumor cells. The activity of EGF and EGFR is known to be a very important factor for meiotic and cytoplasmic maturation during in vitro maturation (IVM) of mammalian oocytes. However, the role of gangliosides GD1a for EGFR-related signaling pathways in porcine oocyte is not yet clearly understood. Here, we investigated that the effect of ST3GAL2 as synthesizing enzyme GD1a for EGFR activation and phosphorylation during meiotic maturation. To investigate the expression of ST3GAL2 according to the EGF treatment (0, 10 and 50 ng/ml), we observed the patterns of ST3GAL2 genes expression by immunofluorescence staining in denuded oocyte (DO) and cumulus cell-oocyte-complex (COC) during IVM process (22 and 44 h), respectively. Expression levels of ST3GAL2 significantly decreased (p<0.01) in an EGF concentration (10 and 50 ng/ml) dependent manner. And fluorescence expression of ST3GAL2 increased (p<0.01) in the matured COCs for 44 h. Under high EGF concentration (50 ng/ml), ST3GAL2 protein levels was decreased (p<0.01), and their shown opposite expression pattern of phosphorylation-EGFR in COCs of 44 h. Phosphorylation of EGFR significantly increased (p<0.01) in matured COCs treated with GD1a for 44 h. In addition, ST3GAL2 protein levels significantly decreased (p<0.01) in GD1a (10 μM) treated COCs without reference to EGF pre-treatment. These results suggest that treatment of exogenous ganglioside GD1a may play an important role such as EGF in EGFR-related activation and phosphorylation in porcine oocyte maturation of in vitro.

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the β-casein gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5’ arm region and 1.8 kb or 0.64 kb of 3’ arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of endostatin gene and inserted into exon 7 of the β-casein gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3’ arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine β-casein gene in the mammary gland.

Mitochondria are well known to regulate the mammalian embryo development. Recent studies showed that the mitochondrial dynamics responses are mainly generated through mitochondrial membrane potential (MMP) and cellular ATP production, which is dependent on mitochondrial reactive oxygen species (ROS). However, these mechanisms are unclear on development process of preimplantation porcine embryos. The aim of this study was to evaluate that difference of the mitochondrial dynamics-derived various functions on the embryo development according to lipid composition of zygote. First, zygote was classify two groups (high lipid, grade 1: G1 and low lipid, grade 2: G2) by lipid composition of cytoplasm. And, we performed the in vitro culture (IVC) using zygote of divided groups. The nuclei numbers and developmental rates of blastocysts were lower in G2 than those of G1 embryos. Next, we investigated the intracellular ROS and mitochondrial derived superoxide production in porcine embryos by using DCF-DA and Mito-SOX staining. As expected, both intracellular ROS and mitochondrial derived superoxide were significantly increased (p<0.05) in the preimplantation stage embryos of G2 group compared with G1 group. In addition, to observe difference of the mitochondrial functions, we investigated the mitochondrial membrane potential (MMP, ΔΨ) and contents of ATP in the preimplantation stage embryos by using JC-1 kit and ATP determination kit. These functions of mitochondria were dramatically reduced in cleavage stage embryos or blastocysts of G2 group. Finally, to verify the difference of the mitochondrial dynamics-derived various functions, we investigated the expressions of mitochondrial fission (Drp1, pDrp1-616) and fusion (Mfn1, Mfn2) factors by Western blotting analysis. Interestingly, the protein levels of pDrp1-616 in embryos of G1 group were continuously increased until blastocyst stage. Whereas, the expression patterns of Mfn1/2 in embryos of G2 group were significantly reduced during IVC progression. The expression patterns of mitochondria dynamic between the two groups were shown opposite. These results demonstrated that the lipid contents of zygote were related the positive-correlation with mitochondrial dynamics-derived functions in porcine embryos. Moreover, we suggest that lipid of zygote is play a important role on mitochondrial functions and dynamics during preimplantation embryos development in pigs.

The plastic monomer bisphenol A (BPA) is well known as a representative environmental hormones. Recent studies showed that the BPA exposure induced mitochondrial dysfunction and mitochondrial derived reactive oxygen species (mito-ROS). However, changes of antioxidant enzymes expression and ROS production from mitochondria according to the BPA exposure on in vitro maturation (IVM) of porcine oocytes have not been studied. We hypothesized that regulation of ROS production from mitochondria by BPA may play a critical role in meiotic maturation or expansion of cumulus cells in cumulus-oocyte complexes (COCs). To investigate the negative effects of BPA exposure on oocyte maturation, immature pig oocytes were matured in NCSU-23 medium supplemented with BPA (50, 75 and 100 μM) for 44 h. Expectedly, the rates of meiotic maturation and cumulus cell expansion of COCs in the BPA (75 μM) treated group was significantly lower than those of control group (p<0.01). Most of secretion factors expressions from COCs were significantly decreased (p<0.05) in the BPA treated COCs. Next, we investigated the intracellular ROS and mitochondrial specific superoxide production according to the BPA exposure using DCF-DA and mito-SOX staining, respectively. BPA exposure were showed that increasing of both intracellular ROS and mito-ROS, as well as mitochondrial related antioxidant enzymes (sod2, prdx3, prdx5) mRNA expression significantly increased (p<0.01) in COCs. And then, mitochondria membrane potential (MMP) dramatically reduced, and mitochondrial-derived apoptotic factors (bax, bcl-xl, caspase 3) mRNA expressions were increased (p<0.01) in BPA treated COCs. In additon, protein levels of mitochondrial-derived apoptosis genes (AIF, cleaved parp1 and caspase 3) were significantly increased (p<0.05) by BPA exposure. To confirm the reduction of BPA-induced mito-ROS, we used to the mitochondrial-targeted ROS scavenger, mito-TEMPO. Interestingly, addition of mito-TEMPO (0.1 μM) to the BPA pre-treated COCs recovered in meiotic maturation of porcine oocytes. These results demonstrated that BPA exposure was induced increasing of mitochondrial dysfunction, mito-ROS and mitochondrial-mediated apoptosis on pig oocyte maturation. Therefore, we suggest that controlling of mito-ROS plays a critical role in pig oocyte maturation in vitro. These findings will be helpful to solve causes of mitochondrial-related infertility.

Melatonin has an important role as anti-oxidative effect and reducing of endoplasmic reticulum(ER)-stress on oocyte maturation and embryo development. Under ER-stress condition, unfolding protein response (UPR) is a defence mechanism in mammalian cells. Recently, regulation of UPR signaling genes are involved in oocyte maturation, embryo development and female reproduction. However, there is no report on the role of melatonin for UPR signaling and ER-stress mediated apoptosis during pig oocyte maturation progression. Moreover, the changes of UPR genes expression according to the porcine oocyte maturation is not yet fully understood. Here, we investigated the changes of UPR signal (BIP/GRP78, ATF4, p90/p50ATF6, and XBP1) and ER-stress apoptotic factor CHOP genes expressions in porcine oocyte maturation by Western blot and RT-PCR analysis. During oocyte maturation, UPR marker and CHOP genes expressions were significantly increased in matured oocytes or cumulus-oocyte complexes (COCs). UPR markers expressions were significantly increased by ER-stress inducer, tunicamycin (Tm), treated (1, 5, 10 μg/ml) groups in a dose-dependent manner compared with control group. To confirm the reducing of ER-stress by melatonin (0.1 μM), we were compared to the effects of ER-stress inhibitor, TUDCA (200 μM), after pre-treated Tm (5 μg/ml) for 22 h maturation. Expressions of UPR markers and meiotic maturation were recovered by melatonin (0.1 μM) in COCs. And, we observed the role of Grp78/Bip as UPR signaling beginning marker using siRNA. In result, reduction of Grp78/Bip gene expression by siRNA was induced the inhibition of oocyte maturation (32.5±10.1 vs control; 77.8±5.3), and p50ATF6 protein level was significantly decreased (p<0.001) in cultured COCs for 44 h. In addition, these results were recovered through the addition of melatonin (0.1 μM) or TUDCA (200 μM) in maturation medium. These results demonstrated that the regulation of UPR signaling via Grp78/Bip gene induction plays a critical role in porcine oocyte maturation in vitro. Furthermore, this present study first confirmed a functional link between inhibition effect of ER-stress by melatonin and regulating of UPR signaling in porcine oocyte maturation. In conclusion, melatonin improves the oocyte maturation and cumulus cells expansion of COCs through the regulation of UPR signal pathway by BIP/GRP78 against the ER-stress during porcine oocyte maturation periods.