Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
권호 표지

Development of Porcine Parthenogenetic Oocytes Activated with Different Combination of Chemicals

Chemical Agent를 이용한 추가 활성화 처리가 돼지 단위발생란의 발달에 미치는 영향

  • 서진성 (농촌진흥청 축산연구소, 순천대학교 동물자원과학과) ;
  • 황인선 (농촌진흥청 축산연구소) ;
  • 김세웅 (농촌진흥청 축산연구소) ;
  • 박효숙 (농촌진흥청 축산연구소) ;
  • 김동훈 (농촌진흥청 축산연구소) ;
  • 양병철 (농촌진흥청 축산연구소) ;
  • 공일근 (순천대학교 동물자원과학과) ;
  • 양보석 (농촌진흥청 축산연구소) ;
  • 임기순 (농촌진흥청 축산연구소)
  • Published : 2006.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

Artificial activation of oocytes is a prerequisite for the successful cloning by nuclear transfer. This study investigated the effect of the different combination of activation agents such as electric pulse (E), thimerosal (Thi) + dithiothreitol (DTT), 6-dimethylaminopurine (6-DMAP) or cycloheximide (CH) on the developmental ability of porcine embryos derived from parthenogenetic activation (PA). PA embryos activated with chemicals showed significantly higher developmental rate to the blastocyst stage compared to the embryos activated with E alone ($21.5{\sim}28.1%$ vs. 18.0%, respectively). Of chemicals, Thi + DTT supported higher development to the blastoryst stage (28.1%). There was no significant difference in 1 pronucleus (PN) formation rate $(59.9{\sim}64.7%)$, but 2PN formation rate was significantly higher in PA embryos with additional activation using chemicals $(7.2{\sim}9.7%)$. In conclusion, this study shows that chemical activation after electric pulse can increase the development of porcine PA embryos.

핵이식 방법을 이용하여 성공적인 복제를 이루기 위해서 인위적인 활성화 처리는 필수적인 요소이다. 본 연구는 전기자극에 의해 활성화된 난자를 chemical agent를 이용하여 추가적인 활성화 처리를 하였을 때 돼지 단위발생란의 발달에 미치는 영향을 알아보고자 수행되었다. 체외에서 $40{\sim}44$시간 동안 배양된 난자를 전기자극(E)으로 활성화 처리한 후 Thimerasol + Dithiothreitol(Thi+DTT), 6-Dimethylaminopurine(6-DMAP) 및 Cycloheximide(CH)를 사용하여 추가 활성화 처리를 하였다. 활성화 방법(E, E+Thi+DTT, E+6-DMAP 및 E+CH)에 따른 단위발생란의 배반포까지의 발달율을 조사한 결과, chemical agent에 의해 추가 활성화된 단위발생란이 전기자극만으로 처리된 구의 단위발생란보다 유의적으로 높은 발달율을 보였다($21.5{\sim}28.1%$ vs. 18.0%, P<0.05). 특히, E+Thi+DTT를 이용하였을 때 발달율이 유의적으로 높게 나타났다(28.1%, P<0.05). 활성화 처리별 전핵 형성율을 조사한 결과, chemical agent에 의해 추가 활성화 처리된 구에서 하나의 극체(1PN) 형성률은 처리별로 차이를 보이지 않았으나$(59.9{\sim}64.7%)$, 2PN 형성율은 추가 활성화 처리구에서 전기자극만을 사용하였을 때보다 유의적으로 높게 나타났다($7.2{\sim}9.7%$ vs. 4.3%, P<0.05). 이상의 결과를 살펴볼 때, 전기자극 후 chemical agent를 이용한 추가 활성화는 단위발생란의 배반포까지의 발달능력을 증가시키는 것으로 생각된다.

Keywords

References

  1. Betthauser J, Forsberg E, Augenstein M, Childs L, Eilertsen K, Enos J, Forsythe T, Golueke P, Jurgella G, Koppang R, Lesmeister T, Mallon K, Mell G, Misica P, Pace M, Pfister-Genskow M, Strelchenko N, Voelker G, Watt S, Tompson S, Bishop M (2002): Production of cloned pigs from in vitro systems. Nat Biotechnol 18:1055-1059
  2. Cheong HT, Ikeda K, Martinez Diaz MA, Katagiri S, Takahashi Y (2000): Development of reconstituted pig embryos by nuclear transfer of cultured cumulus cells. Reprod Fertil Dev 12:15-20 https://doi.org/10.1071/RD00051
  3. Cibelli JB, Stice SL, Golueke PJ, Kane JJ, Jerry J, Blackwell C, Poncede Leon A, Robl JM (1998): Cloned transgenic calves produced from nonquiescent fetal fibroblasts. Science 280:1256-1258 https://doi.org/10.1126/science.280.5367.1256
  4. De Sousa PA, Dobrinsky JR, Zhu J, Archibald AL, Ainslie A, Bosma W, Bowering J, Bracken J, Ferrier PM, Fletcher J, Gasparrini B, Harkness L, Johnston P, Ritchie M, Ritchie WA, Travers A, Albertini D, Dinnyes A, King TJ, Wilmut I (2002): Somatic cell nuclear transfer in the pig: Control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy. BioI Reprod 66:642-650 https://doi.org/10.1095/biolreprod66.3.642
  5. De Sousa PA, Winger Q, Hill JR, Jones K, Watson AJ, Westhusin ME (1999): Reprogramming of fibroblast nuclei after transfer into bovine oocytes. Cloning 1:63-69 https://doi.org/10.1089/15204559950020102
  6. Fissore RA, Pintos-Correia C, Robl JM (1995): Inositol triphosphate-induced calscium release in the generation of calcium ascillations in bovine eggs. BioI Reprod 53:766-744 https://doi.org/10.1095/biolreprod53.4.766
  7. Hyun SH, Lee GS, Kim DY, Kim HS, Lee SH, Kim S, Lee ES, Lim JM, Kang SK, Lee BC, Hwang WS (2003): Effects of maturation media and oocytes derived from sows or gilts on the development of cloned pig embryos. Theriogenology 59:1641-1649 https://doi.org/10.1016/S0093-691X(02)01211-6
  8. Ikeda K, Takahashi Y (2001): Effects of maturational age of porcine oocytes on the induction of activation and development in vitro following somatic cell nuclear transfer. J Vet Med Sci 63:1003-1008 https://doi.org/10.1292/jvms.63.1003
  9. Im GS, Lai L, Liu Z, Hao Y, Wax D, Bonk A, Prather RS (2004): In vitro development of preimplantation porcine nuclear transfer embryos cultured in different media and gas atmospheres. Theriogenology 61:1125-1135 https://doi.org/10.1016/j.theriogenology.2003.06.006
  10. Im GS, Yang BS, Lai L, Liu Z, Hao Y, Prather RS (2005): Fragmentation and development of preimplantation porcine embryos derived by parthenogenetic activation and nuclear transfer. Mol Reprod Dev 71:159-65 https://doi.org/10.1002/mrd.20258
  11. Koo DB, Kang YK, Choi YH, Park JS, Han Sk, Park IY, Kim SU, Lee KK, Son DS, Chang WK, Han YM (2000): In vitro development of reconstructed porcine oocytes after somatic cell nuclear transfer. BioI Reprod 63:986-992 https://doi.org/10.1095/biolreprod63.4.986
  12. Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS (2002): Production of alpha-1,3-galactosyl-transferase knockout pigs by nuclear transfer cloning. Science 295:1089-1092 https://doi.org/10.1126/science.1068228
  13. Machathy Z, Wang WH, Day BN, Prather RS (1997): Complete activation of porcine oocytes induced by the sulfhydryl reagent, thimerosal. BioI Reprod 57: 1123-1127 https://doi.org/10.1095/biolreprod57.5.1123
  14. Machaty Z, Prather RS (1998): Strategies for activating nuclear transfer oocytes. Reprod Fertil Dev 10:599-613 https://doi.org/10.1071/RD98048
  15. Onishi A, Iwamoto M, Akita T, Mikawa S, Takeda K, Awata T, Hanada H, Perry ACF (2000): Pig cloning by microinjection of fetal fibroblast nuclei. Science 289:1188-1190 https://doi.org/10.1126/science.289.5482.1188
  16. Parrington J, Swann K, Shevchenko VI, Sesay AK, Lai FA (1996): Calcium oscillations in mammalian eggs triggered by a soluble regulation to oocyte maturation. Nature 379:364-368 https://doi.org/10.1038/379364a0
  17. Pincus G, Enzmann H (1935): The comparative be haviour of mammalian eggs in vivo and in vitro. I. The activation of ovarian eggs. Journal of Experimental Medicine 62:665-675 https://doi.org/10.1084/jem.62.5.665
  18. Polejava lA, Chen SH, Vaught TD, Page RL, Mullins J, Dai RF, Boone J, Walker S,' Ayares DL, Colman A, Campbell KHS (2000): Cloned pigs produced by nuclear transfer from adult somatic cells. Nature 407:86-90 https://doi.org/10.1038/35024082
  19. Sun FZ, Hoyland J, Huang X, Mason W, Moor RM (1992): A comparison of intracellular changes in porcine eggs after fertilization and electroactivation. Development 115:947-956
  20. Swann K, Ozil J-P (1994): Dynamics of the calcium signal that triggers mammalian egg activation. Int Rev Cytol 152:183-222 https://doi.org/10.1016/S0074-7696(08)62557-7
  21. Tao T, Machaty Z, Boquest AC, Day BN, Prather RS (1999): Development of pig embryos reconstructed by microinjection of cultured fetal fibroblast cells into in vitro matured oocytes. Anim Reprod Sci 56:133-141 https://doi.org/10.1016/S0378-4320(99)00037-8
  22. Wang WH, Abeydeera LR, Prather RS, Day BN (1998): Functional analysis of activation of porcine oocytes by spermatozoa, calcium ionophore, and electrical pulse. Mol Reprod Dev 51, 346-353 https://doi.org/10.1002/(SICI)1098-2795(199811)51:3<346::AID-MRD15>3.0.CO;2-0
  23. Wells DN, Misica PM, Tervit HR (1999): Production of cloned calves following nuclear transfer with cultured adult mural granulosa cells. BioI Reprod 60:996-1005 https://doi.org/10.1095/biolreprod60.4.996
  24. 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
  25. Zakhartchenko V, Durcova-Hills G, Stojkovic M, Schernthaner W, Prelle K, Steinborn R, Muller M, Brem G, Wolf E (1999): Effects of serum starvation and re-cloning on the efficiency of nuclear transfer using bovine fetal fibroblasts. J Reprod Fertil 114: 325-331
  26. Zernicka-Goetz M, Ciemerych MA, Kuibiak JZ, Tarkowiski AK, Maro B (1995): Cytostatic factor inactivation is induced by a calcium-dependent mechanism present until the second cell cycle in fertilized but not in parthenogenetically activated mouse eggs. J Cell Sci 108:469-474
  27. Zhu J, Telfer EE, Fletcher J, Springbett A, Dobrinsky JR, De Sousa PA, Wilmut I (2002). Improvement of an electrical activation protocol for porcine oocytes. BioI Reprod 66:635-641 https://doi.org/10.1095/biolreprod66.3.635