Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
권호 표지

Development of Somatic Cell Nuclear Transfer Bovine Embryos following Activation Time of Recipient Cytoplasm

수핵란의 활성화 시간에 따른 소 체세포 핵이식란의 발육

  • Park, Sun-Young (College of Animal Life Sciences, Kangwon National University) ;
  • Kwon, Dae-Jin (College of Animal Life Sciences, Kangwon National University) ;
  • Park, Choon-Keun (College of Animal Life Sciences, Kangwon National University) ;
  • Yang, Boo-Keun (College of Animal Life Sciences, Kangwon National University) ;
  • Cheong, Hee-Tae (School of Veterinary Medicine, Kangwon National University)
  • 박선영 (강원대학교 동물생명과학대학) ;
  • 권대진 (강원대학교 동물생명과학대학) ;
  • 박춘근 (강원대학교 동물생명과학대학) ;
  • 양부근 (강원대학교 동물생명과학대학) ;
  • 정희태 (강원대학교 수의학부대학)
  • Published : 2006.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

This study was conducted to examine the effect of pre activation treatment and activation time of recipient cytoplasm on the development of bovine somatic cell nuclear transfer(NT) embryos. Donor cells were transferred and electrofused to enucleated oocytes before(pre-AC) or after activation(post-AC). Activation was induced with a combination of $Ca^{2+}$-ionophore(A23187) and DMAP. NT embryos were cultured in CR1aa containing 3 mg/ml BSA for 9 days. Some NT embryos were fixed at 0.5 to 2.5 hr after fusion(for post-AC) or activation(for pre-AC) for confocal microscopy. Developmental rate to the blastocyst stage was slightly high in the post-AC group(20.6%) compared to that of pre-AC group(15.3%). However, developmental speed of embryos in the pre-AC group was faster than that of embryos in the post-AC group. Development rates to the blastocyst stage were similar among different activation time before fusion(0.5,2 and 4 hr). The result of the present study suggests that development and nuclear morphology are affected f the activation status of the recipient cytoplasm before fusion.

본 연구는 융합 전 수핵란의 활성화 처리 유무와 활성화 처리 시간이 소 체세포유래 핵이식란의 체외 발육능에 미치는 영향을 검토하기 위해 수행하였다. 소 체외성숙란의 탈핵 후 체세포 핵을 이식하고 일부는 전기 융합 후 활성화를 유기하였고(pre-AC), 일부는 먼저 활성화 처리 후 융합을 실시(post-AC)하였다. 난자의 활성화는 $Ca^{2+}$-ionophore(A23187)로 처리 후 DMAP로 4시간 배양하는 방법으로 유기하였다. 핵이식란은 CR1aa액에서 9일간 배양하여 발육율을 검토하였으며, 활성화 후 30분${\sim}$2.5시간에 고정하여 confocal microscope하에서 핵형 변화를 검사하였다. 배반포기까지 발육율은 post-AC구(20.6%)가 pre-AC(15.3%)보다 다소 높게 나타났다. 또한 활성화 처리를 하여 핵이식란의 배 발달을 비교한 결과 post-AC구가 더 늦은 배 발달 속도를 나타내었다. Post-AC구를 활성화 후 30분, 2시간, 4시간으로 나누어 융합하여 발육율을 검토한 결과 발육율에 차이가 없었다. 본 연구의 결과는 수핵란의 활성화 시간에 따라 핵이식란의 발육 및 핵형이 영향을 받을 수 있음을 시사한다.

Keywords

References

  1. Anas MK, Shoho A, Shimada M, Terada T (2000): Effects of wortmannin on the kinetics of GVBD and the activities of the maturation-promoting factor and mitogen-activated protein kinase during bovine oocyte maturation in vitro. Thriogenology 53:1797-1806 https://doi.org/10.1016/S0093-691X(00)00315-0
  2. Baguisi A, Overstrom EW (2000): Induced enucleation in nuclear transfer procedures to produce cloned animals. Theriogenology 53:209 (Abstr)
  3. Bordignon V, Smith LC (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 KHS, Ritchie WA, Wilmut I (1993): Disappearance of maturation promoting factor and the formation of pronuclei in electrically activated in vitro matured bovine oocytes. Theriogenology 39: 199 (Abstr) https://doi.org/10.1016/0093-691X(93)90054-9
  5. Campbell KHS, Loi P, Otaegui PJ, Wilmut I (1996): Cell cycle co-ordination in embryo cloning by nuclear transfer. Rev Reprod 1:40-46 https://doi.org/10.1530/ror.0.0010040
  6. Cheong HT, Takahashi Y, Kanagawa H (1992): Development of mouse embryonic nuclei transferred to enucleated oocytes. Jpn J Vet Res 40:149-159
  7. Cheong HT, Takahashi Y, Kanagawa H (1994): Relationship between nuclear remodeling and subsequent development of mouse embryonic nuclei transferred to enucleated oocytes. Mol Reprod Dev 37: 138-145 https://doi.org/10.1002/mrd.1080370204
  8. Cheong HT, Park TM, Ikeda K, Takahashi Y (2003): Cell cycle analysis of bovine cultured somatic cells by flow cytometry. Jpn J Vet Res 51:95-103
  9. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Leon AP, Robl JM (1998): Cloned transgenic calves produced from non-quiescent fetal fibroblasts. Science 280:1256-1258 https://doi.org/10.1126/science.280.5367.1256
  10. Collas P, Robl JM (1990): Factors affecting efficiency of nuclear transplantation in the rabbit embryo. BioI Reprod 43:877-884 https://doi.org/10.1095/biolreprod43.5.877
  11. Galli C, Lagutina I, Crotti G, Colleoni S, Turini P, Ponderato N, Duchi R, Lazzari G (2003): A cloned foal born to its dam twin. Nature 424:635
  12. Lorca T, Cruzalegui FH, Fesquet D, Cavadore JC, Mery J Means A, Doree M (1993): Calmodulindependent protein kinase II mediates inactivation of MPF and CSF upon fertilization of Xenopus eggs. Nature 366:270-273 https://doi.org/10.1038/366270a0
  13. Nurse P (1990): Universal control mechanism regulating the onset of M-phase. Nature 344:503-507 https://doi.org/10.1038/344503a0
  14. Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H, Perry ACF (2002): Pig cloning by microinjection of fetal fibroblast nuclei. Science 289:1188-1190 https://doi.org/10.1126/science.289.5482.1188
  15. Satoshi K, Yasumitsu N, Naojiro M, Masayasu Y, Hiroshi I (2002): Dependence of DNA synthesis and in vitro development of bovine nuclear transfer embryos on the stage of the cell cycle of donor cells and recipient cytoplast. BioI Reprod 67:643-647 https://doi.org/10.1095/biolreprod67.2.643
  16. Takahashi S, Kubota C, Nakahara H, Shimizu M, Tokunaga T, Yamaguchi M, Imai H (1998): Importance of cytoplasmic factor after oocyte activation on development of somatic cell-nuclear transferred bovine embryos. In: Laruia A, Gandolfi F, Enne G, Gianaroli L (eds), Gametes: Development and Function. Serono Symposia 607(Abstr)
  17. Tani T, Kato Y, Tsunoda Y (2001): Direct exposure of chromosomes to nonactivated ovum cytoplasm is effective for bovine somatic cell nucleus reprogramming. BioI Reprod 64:324-330 https://doi.org/10.1095/biolreprod64.1.324
  18. Vignon X, Chesne P, Le Bourhis D, Flechon JE, Heyman Y, Renard JP (1998): Developmental potential of bovine embryos reconstructed from enucleated matured oocytes fused with cultured somatic cells. CR Acad Sci III 321:735-745 https://doi.org/10.1016/S0764-4469(98)80014-0
  19. Wakayama T, Perry ACF, Zuccoti M, Johnson KR, Yanagimach R (1998): Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394:369-394 https://doi.org/10.1038/28615
  20. Ware CB, Barnes FL, Maili-Laurila M, First NL (1989): Age dependence of bovine oocyte activation. Gamete Res 22:265- 275 https://doi.org/10.1002/mrd.1120220304
  21. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KHS (1997): Viable offspring derived from fetal and adult mammalian cells. Nature 385:810-813 https://doi.org/10.1038/385810a0
  22. Woods GL, White KL, Vanderwall DK, Li GP, Aston KI, Bunch TD, Meerdo LN, Pate BJ (2003): A mule cloned from fetal cells by nuclear transfer. Science 301:1063 https://doi.org/10.1126/science.1086743
  23. Yin XJ, Tani T, Yonemura I, Kawakami M, Miyamoto K, Hasegawa R, Kato Y, Tsunoda Y (2002): Production of cloned pigs from adult somatic cells by chemically assisted removal of maternal chromosomes. Biol Reprod 67:442-446 https://doi.org/10.1095/biolreprod67.2.442