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.

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.

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.

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 was investigated the quality characteristics and antioxidant of dried Dioscorea bulbifera with various pre-soaking concentrations of oligosaccharide. Dioscorea bulbifera are prepared by additions of 0, 4, 6, 8 and 10% oligosaccharide solution, and dried at 50℃. The effects of pre-soaking percent of Dioscorea bulbifera slices were evaluated by the moisture, soluble solid, pH, titratable acidity, color, browning degree, texture, antioxidant activities and sensory test. According to the percent of pre-soaking oligosaccharide solution was increased, the moisture was increased but soluble solids and titratable acidity were decreased. With respect to the result of colors, Dioscorea bulbifera slices that underwent the 10% pre-soaked process (85.86%) were lighter than control (73.88%). However, the redness and yellowness scores were the lowest than control. The springiness and cohesiveness of texture showed no significant differences among all groups. Gumminess and chewiness of texture results were increased according to per-soaking concentration increase. Also the polyphenol, flavonoid and DPPH (α, α-diphenyl-β-picrylhydrazyl) and ABTS [2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] radical scavenging activities were significantly increased with increasing immersion concentration. The Dioscorea bulbifera slices supplemented with 6% pre-soaking oligosaccharide solution treatment showed the highest total sensory score. The results of our study indicated that when pre-soaking oligosaccharide solution is used to immerse the Dioscorea bulbifera slice, it has browning inhibition and antioxidant effect.

본 연구에서는 강화되는 황산화물 및 입자상물질의 배출규제를 만족시키기 위한 후처리장치인 스크러버(scrubber)에 대한 수치 해석적 연구를 수행하였다. 먼저 수치 해석을 통하여 기존 스크러버의 문제점을 파악하고, 이러한 문제점들을 개선할 수 있는 새로운 형 태의 와류형 스크러버를 설계하여 분석하였다. 그 결과 와류형 스크러버에서 배기가스는 하부에서 와류를 형성하고 그 중 일부는 바닥면 을 통과하여 가이드 베인을 따라 배출되는데, 이 때 수직 방향의 압력구배는 크지 않으나 배플의 내·외부에는 압력차가 발생하는 것으로 나타났다. 배기가스 유선의 형태를 확인한 결과, 물이 분사되지 않는 경우에는 물이 분사되는 경우에 비해 가이드 베인을 따라 출구까지 일정하게 유동하였으며, 유동의 형태에 가이드 베인과 노즐의 배열 및 수압 등이 영향을 미치는 것으로 확인되었다. 와류형 스크러버의 경우 입·출구의 차압이 기존의 스크러버 대비 절반 이하로서 기관의 배압에 미치는 영향이 훨씬 적은 것으로 나타났다.

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.

To evaluate the effect of Jingansikpungtang water extract on cultured mouse spinal sensory neuron which was inhibited by glucose oxidase(GO)-induced cytotoxicity, NR assay and TBARS assay for lipid peroxidation were carried out after the cultured mouse spinal sensory neuron were pre-incubated with various concentrations of Jingansikpungtang water extract for 3 hours prior to exposure of GO. GO, a oxygen radical, decreased the survival rate of the cultured mouse spinal sensory neuron cells on NR assay. Jingansikpungtang water extract have efficacy of decreasing lipid peroxidation increasing by GO in cultured mouse spinal sensory neuron. From above the results, it is concluded that Jingansikpungtang has marked efficacy as a treatment for the damages caused in the GO-mediated oxidative process..