Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Effects of Electric Stimulation and Activation Conditions on the Fusion and Development of Porcine Somatic Cell Nuclear Transfer Embryos

전기적 융합과 활성화 방법이 돼지 체세포 복제수정란의 체외발달에 미치는 영향

  • 정기화 (진주산업대학교 동물소재공학과)
  • Published : 2004.04.01
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Abstract

The present study was conducted to investigate the effects of fusion and/or activation protocol on in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine fetal fibroblast cells were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion and activation were induced simultaneous fusion/activation (SA) or delayed activation (DA) with or without cytochalasin B (CB) treatment with electic pulses in 0.28 M mannitol-based medium. The SCNT embryos were cultured in vitro for 7 days and stained with Hoechst 33342 to determine the number of nuclei. After 7 days culture, cleavage and blastocyst formation rates were 72.4% and 7.6% in SCNT and 76.3% and 20.4% in parthenotes. To examine the effect of electric field strengths on development of SCNT embryos, oocytes were fused two pulses of 110 V/mm, 130 V/mm or 150 V/mm for 30 sec post-injection. The fusion and cleavage rates in 130 V/mm group (70.2% and 72.6%) and 150 V/mm group (72.6% and 70.5%) were higher (P<0.05) than 110 V/mm group (47.1% and 48.6%), respectively. However, the rate of embryos developing to the blastocyst stage (8.1%, 9.7% and 10.7%) were not different among three groups. The cleavage rates and the blastcyst formation rates were not different among three treatment groups (SA group, 71.4% and 9.7%; SA+CB treatment group, 74.7% and 8.0%; DA+CB treatment group, 70.8% and 11.2%, respectively). And, no different in the number of cells in blastocysts was observed among the three groups (22.5$\pm$12.8, 23.3$\pm$11.2 and 21.6$\pm$10.4, respectively). These result suggest that two pulses of 130 V/mm or 150 V/mm for 30 sec with SA treatment or DA treatment are enough for fusion/activation of porcine somatic cell nuclear transfer (SCNT) embryos to develop to the blastocyst stage.

본 연구에서는 체세포를 이용한 돼지 복제수정란의 생산효율을 높이는 최적의 방법을 구명코자 핵이식 수정란에 각기 다른 조건들의 전기적 자극에 의한 융합과 활성화를 유도하여 융합율, 분할율, 후기배로의 발달율 및 배반포기배의 할구수를 비교ㆍ조사하여 다음과 같은 결과를 얻었다. 핵이식 복제 수정란과 전기자극에 의한 단위발생란과의 체외배양후 분할율을 비교한 결과 두 처리간에 유의적인 차이를 나타내지 않았으나, 배양7일째 배반포기배로의 발달율에 있어서는 복제수정란이 7.6%로 나타나 단위발생란의 20.4%에 비해 낮은 후기배로의 발달율을 나타내었다. 핵이식 수정란의 전기적 융합에 있어서 각기 다른 전기자극의 조건에 따른 융합율과 분할율 그리고 후기배로의 발달율에 있어서 110 V/mm의 전기적 자극이 주어진 군에서는 47.1%로 나타나 130 V/mm의 자극과 150 V/mm의 자극이 주어진 두 군에서의 70.2%와 72.6%에 비해 낮은 융합율을 나타내었고, 분할율에 있어서도 각각 48.6%, 72.6%와 70.5%로 나타나 110 V/mm의 자극이 주어진 군에서 두 처리군보다 낮은 성적을 나타내었다. 그러나 배반포기배로의 발달율에 있어서는 각각의 처리군에 있어서 8.1%, 9.7% 및 10.7%로 나타나 유의적인 차이를 나타내지 않았다. 체세포를 이용한 핵이식수정란의 전기적 자극에 의한 융합과 활성화에 있어서 3가지의 각기 다른 처리군(A type, SA 방법; B type, SA 방법과 CB 처리군; C type, DA 방법과 CB 처리군)으로 나누어 조사한 결과 3가지의 처리군에 있어서의 분할율은 각각 71.4%, 74.7% 및 70.8%로 나타나 유의적인 차이를 나타내지 않았으며, 배반포기배로의 발달율에 있어서도 각각 9.7%, 8.0% 및 11.2%로 나타나 유의적인 차이를 나타내지 않았다. 또한 배반포기배의 할구수에 있어서도 3가지 처리군에서 각각 22.5$\pm$12.8, 23.3$\pm$11.2 및 21.6$\pm$10.4로 나타나 유의적인 차이를 나타내지 않았다. 이상의 실험 결과들을 종합해 보면, 본 연구에서는 돼지의 체세포를 이용한 핵이식 수정란의 융합시 130 V/mm 또는 150 V/mm, 50 ${\mu}\textrm{s}$ec, 2 pul-se의 전기적 강도를 이용하고, 활성화 방법으로는 SA 방법 또는 DA 방법을 병행한다면 복제수정란의 생산효율을 향상시킬 수 있음을 시사하였다. 따라서 돼지의 체세포를 이용한 복제수정란의 생산효율을 향상시키기 위해서는 핵이식 수정란의 전기적 자극에 의한 융합과 활성화에 관한 조건이 확립되어야 하며, 또한 후기배로의 발달율 향상을 위한 최적의 체외배양조건이 확립되어야 할 것으로 사료된다.

Keywords

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