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.

ABSTRACT
 서 론
 재료 및 방법
  시약
  난구세포 정량
  난포란의 핵 성숙 확인
  Mito-SOX 염색
  RNA 추출 및 역전사 PCR (RT-PCR)
  통계 분석
 결 과
  난구세포의 수에 따른 돼지 난포란의 핵 성숙율 확인
  성숙된 난포란에서 초과산화물 생성과 항산화 효소의 발현 조사
  Mito-TEMPO 첨가에 따른 돼지 난포란의 핵 성숙율 변화 확인
 고 찰
 결 론
 CONFLICTS OF INTEREST
 ACKNOWLEDGEMENTS
 REFERENCES

• 양슬기(대구대학교 공과대학 생명공학과) | Seul-Gi Yang (Department of Biotechnology, College of Engineering, Daegu University)
• 박효진(대구대학교 공과대학 생명공학과) | Hyo-Jin Park (Department of Biotechnology, College of Engineering, Daegu University)
• 이상민(대구대학교 공과대학 생명공학과) | Sang-Min Lee (Department of Biotechnology, College of Engineering, Daegu University)
• 김진우(대구대학교 공과대학 생명공학과) | Jin-Woo Kim (Department of Biotechnology, College of Engineering, Daegu University)
• 김민지(대구대학교 공과대학 생명공학과) | Min-Ji Kim (Department of Biotechnology, College of Engineering, Daegu University)
• 김인수(대구대학교 공과대학 생명공학과) | In-Su Kim (Department of Biotechnology, College of Engineering, Daegu University)
• 제갈호근(대구대학교 공과대학 생명공학과) | Ho-Geun Jegal (Department of Biotechnology, College of Engineering, Daegu University)
• 구덕본(대구대학교 공과대학 생명공학과) | Deog-Bon Koo (Department of Biotechnology, College of Engineering, Daegu University) Correspondence