Journal of agriculture & life science (농업생명과학연구)

Institute of Agriculture & Life Science, Gyeongsang National University (경상대학교 농업생명과학연구원)
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Selection and Synchronization of Recipient Cytopalsts for Improving the Efficiency of Porcine Somatic Cell Nuclear Transfer

돼지 복제수정란 생산 효율 향상을 위한 수핵난자의 선별 및 동기화

  • Kim, Tae-Suk (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University) ;
  • Park, Sang-Hoon (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University) ;
  • Lee, Mi-Ran (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University) ;
  • Kim, So-Young (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University) ;
  • Eun, Hye-Ju (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University) ;
  • Baek, Sang-Ki (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University) ;
  • Ko, Yeoung-Gyu (Animal Genetic Resources Station, National Institute of Animal Science, Rural Development Administration) ;
  • Kim, Sung-Woo (Animal Genetic Resources Station, National Institute of Animal Science, Rural Development Administration) ;
  • Sung, Hwan-Hwoo (Animal Genetic Resources Station, National Institute of Animal Science, Rural Development Administration) ;
  • Lee, Joon-Hee (Department of Animal Bioscience, College of Agriculture and Life Sciences, Gyeongsang National University)
  • 김태숙 (경상대학교 농업생명과학대학 동물생명과학과) ;
  • 박상훈 (경상대학교 농업생명과학대학 동물생명과학과) ;
  • 이미란 (경상대학교 농업생명과학대학 동물생명과학과) ;
  • 김소영 (경상대학교 농업생명과학대학 동물생명과학과) ;
  • 은혜주 (경상대학교 농업생명과학대학 동물생명과학과) ;
  • 백상기 (경상대학교 농업생명과학대학 동물생명과학과) ;
  • 고응규 (농촌진흥청 축산과학원 유전자원시험장) ;
  • 김성우 (농촌진흥청 축산과학원 유전자원시험장) ;
  • 성환후 (농촌진흥청 축산과학원 유전자원시험장) ;
  • 이준희 (경상대학교 농업생명과학대학 동물생명과학과)
  • Received : 2013.10.07
  • Accepted : 2013.11.28
  • Published : 2013.12.31
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Abstract

The development of embryos reconstructed by nuclear transfer (NT) is dependent upon numerous factors. Central to development is the quality and development of the recipient cytoplast. Typically oocytes at either AI/TI or MII are potential candidates used as recipient cytoplasts for NT, because they contain active MPF, which causes NEBD and PCC in the transferred nucleus and may be essential for nuclear reprogramming. In vitro maturation of porcine oocytes generally spend longer times (44-48h), progress asynchronously from immature stage and also present insufficient MPF for NEBD and PCC. Here, we have developed three stages establishment of in vitro porcine oocyte maturation to select good quality of oocytes used as recipient cytoplasts for NT. Firstly, oocytes were assessed by the activity of glucose-6-phosphate dehydrogenase (G6PD) before in vitro oocyte maturation. Positively BCB stained oocytes reached to MII (67.0% vs. 58.4%) and produced more parthenogenetic blastocysts (50.2% vs. 29.6%) as compared with negatively BCB stained oocytes (P < 0.05). Secondly, positively BCB stained oocytes were treated with $5{\mu}g/ml$ CHXM to block meiotic progression for synchronizing the cell cycle of oocytes for 16 hr. All of oocytes (130/130) were efficiently synchronized at GV stage by treatment with CHXM, whereas most of oocytes (w/o CHXM) were passed through GV stage (80/176). In addition, retrieval from treatment of CHXM could reversibly induce meiotic resumption and progresses synchronously in porcine oocytes. However, there was no difference between CHXM treated and non-treated group (21.9% vs. 22.3%) in the developmental rate to the blastocyst stage of parthenogenetic embryos. Following treatment of 5 mM caffeine (a phosphatase inhibitor) for 12 hr, all of oocytes were significantly increased the activity of MPF (P < 0.05). Finally, a piglet was produced from NT embryos reconstructed by using non-caffeine treated recipients.

핵이식(NT) 기술을 이용하여 여러 동물 종에서 성공적으로 복제산자가 보고되고 있지만, 아직까지 비효율적인 기술로 남아있다. 본 연구에서는 돼지 체세포 복제 생산 효율성을 증진시키기 위한 방안으로 수핵난자의 품질에 초점을 맞추어 Brilliant cresyl blue (BCB) 염색을 통하여 발육능이 우수한 미성숙 난자를 선발하고, 난자의 감수분열 재개에 관여하는 단백질 합성을 비특이적으로 억제하는 cycloheximide (CHXM)을 이용하여 돼지 난자의 감수분열 재개를 억제시켜 난자의 성숙 동기화를 유도하였다. 또한 핵초기화에 밀접한 영향을 주는 핵막붕괴(NEBD)와 조기염색체응축 (PCC)을 유도하는 MPF의 활성화를 높이기 위하여 단백질 phosphatase 억제제인 caffeine을 첨가하여 수핵난자의 품질을 향상시키고자 하였다. 실험 방법으로는 13 mM BCB 첨가된 배양액에 90분 동안 미성숙난자를 배양하여 BCB 용액의 착색 여부를 구분하여 선발하고, $5{\mu}g/ml$ CHXM를 체외 성숙액에 첨가하여 난자성숙 동기화를 유도하였다. 또한 탈핵 후 탈핵난자를 caffeine을 처리하여 세포주기 관련 단백질의 활성화를 인위적으로 조절하여 체세포복제 수핵난자로 사용하였다. 실험 결과로서 BCB 염색 돼지 미성숙 난자를 대조구와 비교할 때 제2 감수분열 중기(MII)에 도달하는 체외성숙율과 단위발생란의 배반포기까지의 체외 발육율이 유의적으로 증가하는 것이 관찰되었다. 또한 미성숙 돼지 난자의 초기 성숙 (12~16시간)에 CHXM를 처리하였더니 난자 감수분열 재개가 억제되어 GV기에 핵 성숙이 정지되어 동기화가 유도되었다. GV기에 세포주기 동기화된 난자들은 CHXM를 제거하였을 때 난자 성숙의 진행속도도 일치하는 것이 관찰되었다. 이런 결과는 가장 적합한 탈핵시기인 제1 감수분열 후/말기(AI/TI)에 난자들이 다수 분포하여 대조구에 비하여 높은 탈핵율 (87.9%)을 얻을 수 있었다 (P < 0.05). 덧붙여 5 mM의 caffeine을 돼지 난자에 12시간 처리하였을 때 난자 MPF의 활성화가 증가하는 것이 관찰되었지만 (P < 0.05), 10 mM caffeine 농도를 처리하였을 때 MPF의 활성화가 오히려 감소되어 단위발생란의 배반포기까지의 체외발육에도 악영향을 주는 것이 관찰되었다.

Keywords

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