Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지

Studies on the In Vitro Development of Cloned Embryos by Somatic Cell Nuclear Transfer in Korean Native Goats

재래산양의 체세포 핵이식에 의한 복제수정란의 체외발달에 관한 연구

  • Park H. S. (Department of Animal Science and Biotechnology & RAIRC, Jinju National University) ;
  • Kim T. S. (Department of Animal Science and Biotechnology & RAIRC, Jinju National University) ;
  • Jung S. Y. (Department of Animal Science and Biotechnology & RAIRC, Jinju National University) ;
  • Lee Y. H. (Department of Animal Science and Biotechnology & RAIRC, Jinju National University) ;
  • Jung J. Y. (Department of Animal Science and Biotechnology & RAIRC, Jinju National University)
  • 박희성 (진주산업대학교 동물생명과학과 동물생명산업지역협력연구센터) ;
  • 김태숙 (진주산업대학교 동물생명과학과 동물생명산업지역협력연구센터) ;
  • 정수영 (진주산업대학교 동물생명과학과 동물생명산업지역협력연구센터) ;
  • 이윤희 (진주산업대학교 동물생명과학과 동물생명산업지역협력연구센터) ;
  • 정장용 (진주산업대학교 동물생명과학과 동물생명산업지역협력연구센터)
  • Published : 2005.08.01
Download PDF
⟨ Previous Next ⟩

Abstract

The present study was conducted to examine some factors affecting in vitro development of oocytes from somatic cell nuclear transfer (SCNT) in Korean native goats. Recipient oocytes were surgically collected after superovulation by using CIDR and FSH, PMSG, hCG and estrous synchronization in Korean Native goats. For nuclear transfer, the fibroblasts from caprine ear cells and fetal fibroblasts were surgically harvested and were cultured in vitro until cell confluency in serum-starvation condition (TCM-199 + $0.5\%$ FBS) for 3 to 5 days. The zona pellucidae of matured oocytes were partially drilled by laser irradiation. A single somatic cell was individually transferred into each enucleated oocyte. The reconstructed oocytes were then electrically fused and activated. Activated NT embryos were cultured in mSOF medium supplemented with $0.8\%\;BSA\;6\~7\;day\;at\;39^{\circ}C,\;5\%\;CO_2,\;5\%\;O_2,\;90\%\;N_2$ in air. There were no significant difference in the number of embryos cleaved and 4-cell development between the fibroblast nuclei from mature ear cells and fetal cells, but the rate of 8-cell development was higher (P<0.05) in ear cells $(40.5\%)$ than in fetal cells $(55.5\%)$. However, the embryo development to morula or blastocyst was not significantly different between both the groups$(6.7\%\;vs\;16.0\%)$, respectively. The number of embryo cleaved $(79.0\%)$ were higher (P<0.05) in the oocytes activated with ionomycin+6-DMAP than in the oocytes activated electrically $(9.5\%)$. The development of fused embryos to morula or blastocyst was found $15.6\%$ in ionomycin+6-DMAP, but no morula or blastocysts were developed in electrical stimulation. The development rate of SCNT embryos to morula or blastocyst was love. (P<0.05) in SCNT embryos $(19.0\%\;vs\;0.0\%)$ than that in parthenotes $(66.1\%\;vs\;59.1\%)$. In the parthenotes, the cleavage rate and development to morula or blastocyst were significantly higher (P<0.05) as $86.8\%\;and\;50.0\%$ in ovulated oocytes than in follicular oocytes $(69.0\%\;vs\;23.6\%)$, respectively. These results suggest that some factors Including superovulation treatment, oocyte source, maturation of follicular oocytes, activation method and culture condition may affect in vitro developmental capability of embryos produced by somatic cell nuclear transfer in Korean Native goats, and the fusion rate be greatly low compared with other species.

본 연구는 재래산양에서 복제 수정란의 생산효율을 향상시키기 위한 기초 자료를 제시하고자 체세포 핵이식을 실시하여 공핵세포의 종류, 핵이식란의 활성화 처리 방법 및 수핵난자의 조건이 체외발달율에 미치는 영향을 조사, 검토하여 핵이식란 생산을 위한 최적의 조건을 규명하고자 실시하였다. 공핵세포의 종류에 따른 핵이식란의 체외발달율은 융합이 이루어진 핵이식란의 활성화 처리 후 분할율은 귀 유래 섬유아세포를 공핵세포로 사용하였을 때가 $40.5\%$로서 태아 유래 섬유아세포를 공핵세포로 사용하였을 때의 $55.5\%$와 유의적인 차이가 없었다. 또한 상실배 또는 배반포기로의 발달율도 각각 $6.7\%$ 및 $16.0\%$로서 유의적인 차이가 없었다. 핵이식란의 활성화 방법에 따른 체외발달율은 ionomycin+6-DMAP 처리를 하였을 때 분할율은 $79.0\%$로서 전기자극을 주었을 때의 $9.5\%$보다는 유의적(P<0.05)으로 높았다. 상실배 또는 배반 포기로의 발달율도 ionomycin+6-DMAP 처리를 하였을 때는 $15.6\%$가 발달하였으나, 전기 자극을 주었을 때는 4-세포기 이후로의 발달이 전혀 이루어지지 않았다. 체세포 핵이식란은 단위발생란에 비하여 분할율$(66.1\%\;vs\;59.18\%)$ 및 상실배 또는 배반포배로의 발달율$(19.0\%\;vs\;0.0\%)$이 유의적 (P<0.05)으로 낮았다. 단위발생란의 분할율은 체내 성숙난자에서 $86.8\%$로서 난포란의 $69.0\%$보다는 유의적(P<0.05)으로 높았다 단위발생란의 상실배 또는 배반포기로의 발달율에 있어서도 체내 성숙난자$(50.0\%)$가 난포란$(23.6\%)$보다 유의적 (p.<0.05)으로 발달율이 높았다. 이상의 결과로 볼 때 재래산양의 체세포를 이용한 복제수정란의 생산효율을 향상시키기 위해서는 다수의 난자 확보를 위한 과배란처리 방법의 개선, 난포란의 이용효율 개선 및 활성화 처리방법 등이 확립되어야 하며, 후기배로의 발달율 향상을 위해서는 최적의 체외 배양조건 확립이 시급한 것으로 생각된다.

Keywords

References

  1. Apimeteetumrong M, Thuangsanthia A, Leingcharoen N, Yiengvisavakul V, Harintharanon A, Kunavongkrit A, Sumretprasong J, Vignon X and Techakumphu M. 2004. The effect of activation protocols on the development of cloned goat embryos. Theriogenology, 66:1529-1534
  2. Bagugisi A, Behboodi E, Melican DT, Pollock JS, Destrempes MM, Cammuso C, Williams JL, Nims SD, Porter CA, Midura P, Palacios MJ, Ayres SL, Denniston RS, Hayes ML, Ziomek CA, Meade HM, Godke RA, Gavin WG, Overstrom EW and Echelard Y. 1999. Production of goats by somatic cell nuclear transfer. Nature Biotehnology, 17:456-461 https://doi.org/10.1038/8632
  3. Butler RE, Melican D, Hawkins N, Jellerette T, Nims S, Graslie K and Gavin WG. 2004. Effects of cycloheximide on caprine somatic cell nuclear transfer embryo and fetal development. Reprod. Fert. Dev. Abst., 16:138
  4. Echelard Y, Memili E, Ayres SL, O'Coin M, Chen LH, Meade HM and Behboodi E. 2004. Comparison of the developmental potential of caprine nuclear transfer embryos derived from in vitro and in vivo matured oocytes. Reprod. Fert. Dev. Abst., 16:140 https://doi.org/10.1071/RDv16n1Ab36
  5. Hill JR, Winger QA, Long CR, Looney CR, Thompson JA and Westhusin ME. 2000. Development rates of male bovine nuclear transfer embryos derived from adult and fetal cells. Biol. Reprod., 62:1135-1140 https://doi.org/10.1095/biolreprod62.5.1135
  6. Keefer CL, Baldassarre H, Keyston R, Wang B, Bhatia B, Bilodeau AS, Zhou JF, Leduc M, Downey BR, Lazaris A and Karatzas CN. 2001. Generation of dwarf goat(Capra hircus) clones following nuclear transfer with transfected and nontransfected fetal fibroblasts and in vitro-matured oocytes. Biol. Reprod., 64: 849-856 https://doi.org/10.1095/biolreprod64.3.849
  7. Keefer CL, Keystone R, Lazaris A, Bhatia B, Begin I, Bilodeau AS, Zhou FJ, Kafidi N, Wang B, Baldassarre H and Karatzas CN. 2002. Production of cloned goats after nuclear transfer using adult somatic cells. Biol, Reprod., 66:199-203 https://doi.org/10.1095/biolreprod66.1.199
  8. McGrath J and Solter D. 1983. Nuclear transplantation in the mouse embryo on micromanipulation an cell fusion. Science, 220:1300-1302 https://doi.org/10.1126/science.6857250
  9. Melican D, Butler R, Hawkins N, Nims S, Buzzel N, Jellerette T and Gavin WG. 2004. Estrus synchronization of dairy goats utilized as recipients for caprine nuclear transfer embryos. Reprod. Fert. Dev., 16:151(abstr) https://doi.org/10.1071/RDv16n1Ab58
  10. Ohkoshi K, Takahashi S, Koyama S, Akagi S, Adachi N, Furusawa T, Fujimoto J, Takeda K, Kubo M, Izaike Y and Tokunaga T. 2003. In vitro oocyte culture and somatic cell nuclear transfer used to produce a live-born cloned goat. Cloning Stem Cells, 5:109-115 https://doi.org/10.1089/153623003322234704
  11. Reggio BC, James AN, Green HL, Gavin WG, Behboodi E, Echelard Y and Godke R. A. 2001. Cloned transgenic offspring resulting from somatic cell nuclear transfer in the goats: oocytes derived from both follicle-stimulating hormone-stimulated and nonstimulated abattoirderived ovaries. Biol, Reprod., 65:1528-1533 https://doi.org/10.1095/biolreprod65.5.1528
  12. Reggio BC, Green HL, Sansinena M, Chen LH, Behboodi E, Denniston RS, Echelard Y and Godke RA. 2002. Production of cloned transgenic goats as a potential source for human pharmaceuticals. Theriogenology, 57:445(abstr.)
  13. Wilmut I, Schnieke AE, McWhir I, Kind AJ and Campbell KHS. 1997. Viable offspring derived from fetal and adult mammalian cells. Nature, 385:810-813 https://doi.org/10.1038/385810a0
  14. Zou XG, Chen Y, Wang Y, Luo JP, Zhang QB, Zhang XC, Yang Y, Ju HM, Shen Y, Lao W, Xu S and Du MA. 2001. Production of cloned goats from enucleated oocytes injected with cumulus cell nuclei or fused with cumulus cells. Cloning, 3:31-37 https://doi.org/10.1089/152045501300189312
  15. Zou XG, Wang UG, Cheng Y, Yang YE, Ju HM, Tang HL, Shen Y, Mu ZY, Xu SF and Du M. 2002. Generation of cloned goats(Capra hircus) from transfected foetal fibroblast cells, the effect of donor cell cycle. Mol. Rerod. Dev., 61:164-172 https://doi.org/10.1002/mrd.1143