Korean Journal of Animal Reproduction (한국가축번식학회지)

The Korean Society of Animal Reproduction (한국동물번식학회)
권호 표지

Study of Efficient Production of Cloned Embryos in Porcine II. Establishment of Conditional Enucleation

효율적인 돼지 복제수정란 생산에 관한 연구 II. 탈핵 여건의 확립

  • 위갑인 (전남대학교 농업생명과학대학 동물자원학부, 농업과학기술연구소) ;
  • 강만종 (전남대학교 농업생명과학대학 동물자원학부, 농업과학기술연구소) ;
  • 문승주 (전남대학교 동물자원학부)
  • Published : 2003.09.01
Download PDF
⟨ Previous Next ⟩

Abstract

This study was carried out to improve of enucleation efficiency on porcine recipient oocytes preactivated. In ethanol or $Ca^{2+}$ ionophore, effect of repeating and combinational activation with 6-DMAP or cycloheximide compared with alone activated treatment. Recipient oocytes's activation by $Ca^{2+}$ ionophore combined with 6-DMAP or cycloheximide were significantly higher than alone treatment(P<0.05). Between repeating and alone treatments were not significantly different. In ethanol, repeating treatment was significantly lower than alone(P<0.05), and combination treatments were not significantly different. On the basis of these results, efficiency of enucleation, electrical fusion and in vitro development compared preactivated with non-preactivated recipient oocytes. Enucleation and fusion rates of preactivated oocytes were improved significantly compared with non-preactivated oocytes(90.7%, 71.8 vs 77.8%, 61.1%; P<0.05). Behind the back, cleavage and in vitro development rates were significantly lower than non-preactivated oocytes(38.7%, 19.3% vs 68.8%, 30.6%; P<0.05).

체외 성숙시킨 M II기 난자의 여러 화학물질간의 중복 및 병용처리를 실시하여 수핵란으로서 적합한 활성화 조건과 활성화 후 자체 탈핵을 유도함으로써 보다 효율적인 탈핵 여건을 확립하고자 본 연구를 실시하였다. 1. Ethanol을 사용하여 6-DMAP 또는 cycloheximide 간의 단독, 중복, 그리고 병용처리를 실시한 결과, 활성화율, 난할율 및 체외 발달율에서 단독처리와 병용처리간의 차이는 없었으나 중복처리시 유의적으로 저하되었다(P<0.05). 2. $Ca^{2+}$ ionophore를 가지고 활성화 조건별로 처리하였을 경우, 6-DMAP와 병용 처리가 활성화율, 난할율 및 체외 발달율에서 각각 80.8%, 78.3%, 40.6%로 단독처리시의 58.0%, 62.9%, 27.0%보다 유의적으로 높았고(P<0.05), 또한 cycloheximide와의 병용처리도 활성화 및 난할율에서 단독처리와의 유의적 차이를 보였다(P<0.05). 그러나 중복처리시에는 효과를 나타내지 못하였다. 3. 탈핵전 수핵란에 미리 활성화 처리를 한 후 핵이식란을 재구성 한 결과 탈핵율 및 세포 융합율에서 90.7%, 71.8%로 활성화 처리하지 않은 것 77.8%, 61.1% 보다 유의적으로 높았다(P<0.05). 그러나 난할율 및 체외 발달율에서는 유의적으로 저하되었다(38.7%, 19.3% vs 68.8%, 30.6%, P<0.05). 또한 변성율도 활성화 처리하지 않은 구에 비해 유의적으로 높게 나타났다(P<0.05). 이상의 결과로 미루어 탈핵전 활성화하여 수핵란으로 이용할 경우 $Ca^{2+}$ ionophore와 6-DMAP를 병용 처리가 가장 효과적이며, 주입되는 체세포와 수핵란 간의 적정한 세포주기의 조절이 효율적인 돼지 복제수정란 생산을 위해 필요할 것이다.

Keywords

References

  1. Ayogi, Y., Konishi, M., Wada, T. and Takedomi, T. 1994. Unaged bovine oocytes successfully develop to blastocystes after parthenogenetic activation of nuclear transfer. Theriogenology 41: 157(Abstract)
  2. Boquest, A. C., Day, B. N. and Prather, R. D. 1999. Flow cytometry cell cycle analysis of cultured porcine fetal fibroblast cells. BioI. Reprod. 60:1013-1019 https://doi.org/10.1095/biolreprod60.4.1013
  3. Bordignon, V. and Smith, L. C. 1998. Telophase enucleation: An improved method to prepare recipient cytoplasts for use in bovine nuclear transfer. Mol. Reprod. Dev. 49:29-36 https://doi.org/10.1002/(SICI)1098-2795(199801)49:1<29::AID-MRD4>3.0.CO;2-Q
  4. Campbell, K. H. S., Loi, P., Otaegui, P. J. and Wilmut, I. 1993. Cell cycle coordination in embryo cloning by nuclear transfer. BioI. Reprod. 49:40-46
  5. Collas, P., Sulluvian, E. J. and Barnes, F. L. 1993a. Histone HI kinase activity in bovine oocytes following calcium stimulation. Mol. Reprod. Dev. 34:224-231
  6. Fissore, R. A. and Robl, J. M. 1992. Intracellular $Ca^{2+}$ + response of rabbit oocytes to electircal stimulation. Mol. Reprod. Dev. 33:357-362 https://doi.org/10.1002/mrd.1080330318
  7. Fulka, Jr. J. and Moor, R. M. 1993. Noninvasive chmical enucleation of mouse oocytes. Mol. Reprod. Dev. 34:427-430 https://doi.org/10.1002/mrd.1080340412
  8. Kono, T., Sotomaru, Y., Apno, F., Takahashi, T., Ogiwara, I., Sekizawa, F., Arai, T. and Nakahara, T. 1994. Effect of ooplast activation on the development of oocytes following nucleus transfer in cattle. Theriogenology 47:23-45
  9. Liu, L., Ju, J. C. and Yang, X. 1998a. Parthenogenetic development and protein patterns of newly matured bovine oocytes after chemical activation. Mol. Reprod. Dev. 49:298-307
  10. Liu, L., Ju, J. C. and Yang, X. 1998b. Differential inactivation of maturation promoting factor and mitogen-activated protein kinase following parthenogenetic activation of bovine oocytes. BioI. Reprod. 59:537-545
  11. Prather, R. S., Sims, M. M. and First, N. L. 1989. Nuclear transplantation in early pig embryos. BioI. Reprod. 41:414-418 https://doi.org/10.1095/biolreprod41.3.414
  12. Prather, R. S. and First, N. L. 1990. Cloning of embryos. J. Reprod. Fertil. 40:227(suppl.)
  13. Presicce, G. A. and Yang, X. 1994a. Nuclear dynamics of parthenogenesis of bovine oocytes matured for 20 and 40 hours and activated with combined ethanol and cycloheximide treatment. Mol. Reprod. Dev. 37:61-68
  14. Presicce, G. A. and Yang, X. 1994b. Parthenogenetic development of bovine oocytes matured for 24h and activated by ethanol and cycloheximide. Mol. Reprod. Dev. 38:380-385
  15. Rho, G. J., Wu, B., Leibo, S. P. and Betteridge, K. J. 1996. Bovine parthenogenesis following various oocyte activation regimens. Biol. Reprod. 54(Suppl.1)
  16. Shi, Z., Jiang, S. and Yang, X. 1993. Synergistic effect of A23187 and cycloheximide allows effective activation of frshly matured bovie oocytes. Theriogenology 38:309(Abstract)
  17. Smith, L. C. and Wilmut, I. 1989. Infuence of nuclear and cytoplasmic activity on the development in vitro of sheep embryos after nuclear transplantation. Biol. Reprod. 40:1027-1035
  18. Stice, S. L., Keefer, C. L. and Mattews, L. 1994. Bovine nuclear transfer embryos: oocyte antivation prior to blastomere fusion. Mol. Reprod. Dev. 38:61-68
  19. Stice, S. L., Strelchenko, N. S., Keefer, C. L. and Mattews, L. 1996. Pluripotent bovine embryonic cell lines direct embryonic following. nuclear transfer. BioI. Reprod. 54:1000-110
  20. Susko-Parrish, J. L., Leibfried-Rutledge, M. L., Notthy, D. L., Schutzkus, V. and First, N. L. 1994. Inhibition of protein kinase after an induced calcium transient causes transition of bovine oocytes to embryonic cycles without meiotic completion. Dev. Biol. 166:729-738 https://doi.org/10.1006/dbio.1994.1351
  21. Takahashi, S., Kubota, C., Ogata, Y., Tokunaga, T. and Imai, H. 1996. Parthenogenetic activation and development of bovine oocytes treated with protein synthesis of protein phosphorylation inhibitors. Theriogenology 45:156(Abstract)
  22. Tanaka, H. and Kanakawa, H. 1997. Influence of combined activation treatments on the success of bovine nuclear trarnsfer using of aged oocytes. Anim. Reprod. Sci. 49:113-123 https://doi.org/10.1016/S0378-4320(97)00070-5
  23. Westinhusin, M. E., Pryor, J. H. and Bondioli, K. R. 1991. Nuclear transfer in the bovine embryo: A comparison of 5-day, 6-day, frozenthawed, and nuclear transfer donor embryos. Mol. Reprod. Dev. 28:119
  24. Westinhusin, M. E., Levanduski, M. J., Scarborough, Looney and Bondioli, K. R. 1992. Viable embryos and normal cleaves after nuclear transfer into Hoechst stained enuleated demioocytes of cows. J. Reprod. Fertil. 95:475
  25. Yang, X., Jiang, S. and Shi, Z. 1992. Impreoved activation by combined cycloheximide and electic pulse treatment of bovine follicular oocytes matured for 23-24hr. Biol, Reprod. 46(Suppl.): 117(Abstract)
  26. Yang, X., Presicce, A., Morghan, L., Jiang, S. and Foote, R. H. 1994. Synergistic effect of ethanol and cycloheximde on activation of freshly matured oocyte. Theriohenology 41:395-403 https://doi.org/10.1016/0093-691X(94)90075-T
  27. 윤희준, 이효종, 최상용, 박충새. 1998. 활성화된 수핵란을 이용한 핵이식기법의 개선. 한국수정란이식학회지 13(3):219-226