Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Caffeine treatment during in vitro maturation improves developmental competence of morphologically poor oocytes after somatic cell nuclear transfer in pigs

돼지 난자의 체외성숙에서 Caffeine 처리가 난자 성숙과 체세포 핵이식 배아의 체외발육에 미치는 영향

  • Lee, Joohyeong (Institute of Veterinary Science, Kangwon National University) ;
  • You, Jinyoung (College of Veterinary Medicine, Kangwon National University) ;
  • Lee, Hanna (College of Veterinary Medicine, Kangwon National University) ;
  • Shin, Hyeji (College of Veterinary Medicine, Kangwon National University) ;
  • Lee, Geun-Shik (College of Veterinary Medicine, Kangwon National University) ;
  • Lee, Seung Tae (Division of Applied Animal Science, College of Animal Life Science, Kangwon National University) ;
  • Lee, Eunsong (Institute of Veterinary Science, Kangwon National University)
  • 이주형 (강원대학교 동물의학종합연구소) ;
  • 유진영 (강원대학교 수의과대학) ;
  • 이한나 (강원대학교 수의과대학) ;
  • 신혜지 (강원대학교 수의과대학) ;
  • 이근식 (강원대학교 수의과대학) ;
  • 이승태 (강원대학교 동물생명과학대학) ;
  • 이은송 (강원대학교 동물의학종합연구소)
  • Received : 2017.09.18
  • Accepted : 2017.09.25
  • Published : 2017.09.30
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Abstract

In most mammals, metaphase II (MII) oocytes having high maturation promoting factor (MPF) activity have been considered as good oocytes and then used for assisted reproductive technologies including somatic cell nuclear transfer (SCNT). Caffeine increases MPF activity in mammalian oocytes by inhibiting p34cdc2 phosphorylation. The objective of this study was to investigate the effects of caffeine treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after SCNT in pigs. To this end, morphologically good (MGCOCs) and poor oocytes (MPCOCs) based on the thickness of cumulus cell layer were untreated or treated with 2.5 mM caffeine during 22-42, 34-42, or 38-42 h of IVM according to the experimental design. Caffeine treatment for 20 h during 22-42 h of IVM significantly inhibited nuclear maturation compared to no treatment. Blastocyst formation of SCNT embryos was not influenced by the caffeine treatment during 38-42 h of IVM in MGCOCs (41.1-42.1%) but was significantly improved in MPCOCs compared to no treatment (43.4 vs. 30.1%, P<0.05). No significant effects of caffeine treatment was observed in embryo cleavage (78.7-88.0%) and mean cell number in blastocyst (38.7-43.5 cells). The MPF activity of MII oocytes in terms of p34cdc2 kinase activity was not influenced by the caffeine treatment in MGCOCs (160.4 vs. 194.3 pg/ml) but significantly increased in MPCOCs (133.9 vs. 204.8 pg/ml). Our results demonstrate that caffeine treatment during 38-42 h of IVM improves developmental competence of SCNT embryos derived from MPCOCs by influencing cytoplasmic maturation including increased MPF activity in IVM oocytes in pigs.

본 연구에서는 체외성숙 배양액 내 caffeine 첨가가 돼지 난자의 성숙과 단위발생 및 체세포 핵이식 후 배 발육에 미치는 영향을 조사하였다. 난세포질 및 난구세포 부착정도의 형태학적 특징에 따라 MGCOCs와 MPCOCs로 구분된 미성숙난자를 각각 무처리군(대조군)과 2.5 mM caffeine이 첨가된 배양액에서 체외성숙 22-42(20시간), 34-42(8시간), 38-42(4시간)동안 처리하는 군으로 나누어 체외성숙을 유도하였다. 또한 체외성숙 난자를 단위발생 및 체세포 핵이식에 공여하여 배아를 생산한 후 7일 동안 체외배양하여 체외성숙 동안 caffeine 처리가 분할률, 배반포 형성률 및 배반포의 세포수에 미치는 영향을 조사하였다. 연구 결과, 분할률 및 배반포 세포 수는 caffeine의 처리 시간에 따라 유의적인 영향을 받지 않았다. 그러나 MPCOCs 유래 난자에서 체외성숙 후기 4시간 동안 caffeine 처리는 체세포 핵이식 배아의 배반포 형성률을 유의적으로 증가시켰다. 이 결과는 caffeine 처리가 체외성숙 동안 난자의 MPF 수준의 감소를 억제시킴으로써 체세포 핵의 리모델링이나 리프로그래밍에 영향을 미쳐 핵이식 배아의 발육능에 영향을 미친 것으로 사료된다.

Keywords

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