Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Analysis of Apoptosis on the Somatic Cell Nuclear Transfer embryos in porcine

돼지 체세포 복제 수정란의 자가 사멸 분석

  • Ryu, Ji-Eun (Major in the Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University) ;
  • Yoon, Jong-Taek (Major in the Animal Biotechnology, Graduate School of Future Convergence Technology, Hankyong National University)
  • 류지은 (한경대학교 미래융합기술대학원 동물생명공학과) ;
  • 윤종택 (한경대학교 미래융합기술대학원 동물생명공학과)
  • Received : 2018.07.19
  • Accepted : 2018.09.20
  • Published : 2018.09.30
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Abstract

The purpose of this study is to examined the electrofusion and activation conditions for the production of porcine somatic cell nuclear transfer (SCNT) embryos. In this study, immature oocytes were cultured in TCM-199 with and without hormones for 22 hours. Skin fibroblasts cells of porcine were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion was performed with two different pulses that each one pulse (DC) of 1.1 kV/cm or 1.5 kV/cm for $30{\mu}sec$. After fusion subsequent activation were divided into three groups; non-treatment (control) and treatment with 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B for 4 hours. Transferred embryos were cultured in PZM-3 (Porcine Zygote Medium-3) in $5%\;CO_2$ and 95% air at $39^{\circ}C$ for 7 day. Apoptosis-related genes (Caspase-3, BCL-2, mTOR, and MMP-2) were analyzed by immunofluorescence staining. There was no significant difference between two different electrofusion stimuli in the cleavage rate; $64.9{\pm}4.8%$ in 1.1 kV/cm and $62.7{\pm}4.0%$ in 1.5 kV/cm. However, blastocyst formation rate (%) was significantly different among three different activation groups (no treatment, 2 mM 6-DMAP or $7.5{\mu}g/ml$ cytochalasin B) combined with electrofusion of 1.1 kV/cm. The blastocyst formation rate was $12.6{\pm}2.5$, $20.0{\pm}5.0$, and $34.9{\pm}4.3%$ in control, 2 mM 6-DMAP, and $7.5{\mu}g/ml$ cytochalasin B, respectively. Immunofluorescence data showed that expression levels of caspase-3 in SCNT embryos undeveloped to blastocyst stage were higher than those in the blastocyst stage embryos. Expression levels of Bcl-2 in blastocyst stage embryos were higher than those in the arrested SCNT embryos. These results showed that the combination of an electric pulse (1.1 kV/cm for $30{\mu}sec$) and $7.5{\mu}g/ml$ cytochalasin B treatment was effective for production of the porcine SCNT embryos.

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References

  1. Alexander B, Coppola G, Di Berardino D, Rho GJ, St John E, Betts DH. and King WA. 2006. The Effect of 6-Dimethylaminopurine (6-DMAP) and Cycloheximide (CHX) on the Development and Chromosomal Complement of Sheep Parthenogenetic and Nuclear Transfer Embryos. Mol. Reprod. Dev. 73:20-30. https://doi.org/10.1002/mrd.20372
  2. Beebe LF, McIlfatrick S.J. and Nottle MB. 2009. Cytochalasin B and Trichostatin A Treatment Postactivation Improves In Vitro Development of Porcine Somatic Cell Nuclear Transfer Embryos. Cloning and Stem Cells 11:477-482. https://doi.org/10.1089/clo.2009.0029
  3. Bondioli K, Ramsoondar J, Williams B, Costa C and Fodor W. 2001. Cloned pigs generated from cultured skin fibroblasts derived from a H-transferase transgenic boar. Molecular Reproduction and Development 60:189-195. https://doi.org/10.1002/mrd.1076
  4. Boquest AC, Grupen CG, Harrison SJ, McIlfatrick SM, Ashman RJ and d'Apice AJ. 2002. Production of cloned pigs from cultured fetal fibroblast cells. Biology of Reproduction 66:1283-1287. https://doi.org/10.1095/biolreprod66.5.1283
  5. Castedo M, Ferri KF and Kroemer G. 2002. Mammalian target of rapamycin (mTOR): pro- and anti-apoptotic. Cell Death Differ 9:99-100. https://doi.org/10.1038/sj.cdd.4400978
  6. Cha SK, Kim NH, Lee SM, Baik CS, Lee HT and Chung KS. 1997. Effect of Cytochalasin B and cycloheximide on the activation rate, chromosome constituent and in vitro development of porcine oocytes following parthenogenetic stimulation. Reprod. Fertil. 9:441-446. https://doi.org/10.1071/R96078
  7. Chae JI, Cho SK, Seo JW, Yoon TS, Lee KS, Kim JH, Lee KK, Han YM and Yu K. 2006. Proteomic Analysis of the Extraembryonic Tissue from Cloned Porcine Embryos. Mol. Cell Proteomics. 5:1559-1566. https://doi.org/10.1074/mcp.M500427-MCP200
  8. Cong PQ, Kim ES, Song ES, Yi YJ and Park CS. 2008. Effects of fusion/activation methods on development of embryos produced by nuclear transfer of porcine fetal fibroblast. Animal Reproduction Science 103:304-311. https://doi.org/10.1016/j.anireprosci.2006.12.012
  9. Feugang JM, Roover DE, Leonard S, Dessy F and Donnay I. 2002. Kinetics of Apoptosis in preimplantation bovine embryos produced in vitro and in vivo. Theriogenology 57:494.
  10. Gjorret J, Wengle J, King WA, Schellander K and Hyttel P. 2002. Occurrence of Apoptosis in bovine embryos reconstructed by nuclear transfer or derived in vivo. Theriogenology 57:495.
  11. Hao Y, Lai L, Mao J, Im GS, Bonk A and Prather RS. 2003. Apoptosis and In Vitro Development of Preimplantation Porcine Embryos Derived In Vitro or by Nuclear Transfer. Biol. Reprod. 69:501-507. https://doi.org/10.1095/biolreprod.103.016170
  12. Hardy K. 1999. Apoptosis in the human embryo. Rev. Reprod. 4:125-134. https://doi.org/10.1530/ror.0.0040125
  13. Hyun SH, Lee GS, Kim DY, Kim HS, Lee SH, Kim S, Lee ES, Lim JM, Kang SK, Lee BC and Hwang WS. 2003. Effect 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
  14. Jeon YB, Biswas D, Yoon KY and Hyun SH. 2009 Prediction of Developmental Ability of In Vitro Fertilized Porcine Embryos by Analysis of Early Cleavage Pattern. Journal of embryo transfer 24:65-69.
  15. Jeong YS, Oh KB, Park JS, Kim JS and Kang YK. 2009. Cytoplasmic localixation of oocyte-specific variant of porcine DNA methyltransferase-1 during early development. Dev. Dyn. 238:1666-1673 https://doi.org/10.1002/dvdy.21975
  16. Ji Q, Zhu K, Liu Z, Song Z, Huang Y, Zhao H, Chen Y, He Z, Mo D and Cong P. 2013. Improvement of porcine cloning efficiency by trichostain A through early-stage induction of embryo Apoptosis. Theriogenology 79:815-23. https://doi.org/10.1016/j.theriogenology.2012.12.010
  17. Jolliff WJ and Prather RS. 1997. Parthenogenic development of in vitro-matured, in vivo-cultured porcine oocytes beyond blastocyst. Biology of Reproduction 56:544-548. https://doi.org/10.1095/biolreprod56.2.544
  18. Kim SH, Kang HA, Kim DS, Lee MS, Seo KS, Min KS and Yoon JT. 2010. Expression Analysis of Matrix Metalloproteinases and Tissue Inhibitor of Matrix Metalloproteinases from In Vitro Maturation Oocytes Complexes in Porcine. Reproductive & developmental biology 34:55-62.
  19. Ko YG, Im GS, Lee HC, Cho SR, Choi SH, Choi CY, Lee PY, Cho JH and Yoo YH. 2011. Non-CPC methylation of Pre-1 sequence in pig SCNT blastocyst. Reprod. Dev. Biol. 35:93-97.
  20. Kubota C, Yamakuchi H, Todoroki J, Mizoshita K, Tabara N, Barber M and Yang X. 2000. Six cloned calves produced from adult fibroblast cells after long-term culture. Proceedings of the National Academy of Sciences of the United States of America 97:990-995.
  21. Lai L and Prather RS. 2003. Production of cloned pigs by using somatic cells as donors. Cloning Stem Cells 5:233-241. https://doi.org/10.1089/153623003772032754
  22. Lee GS, Kim HS, Hyun SH, Lee SH, Jeon HY, Nam DH, Jeong YW, Kim S, Kim JH, Han JY, Ahn C, Kang SK, Lee BC and Hwang WS. 2005. Production of transgenic cloned piglets from genetically transformed fetal fibroblasts selected by green fluorescent protein. Theriogenology 63:973-991. https://doi.org/10.1016/j.theriogenology.2004.04.017
  23. Levy R, Benchaib M, Cordonier H, Souchier C and Guerin JF. 1998. Annexin V labelling and terminal transferase-mediated DNA end labelling (TUNEL) assay in human arrested embryos. Molecular Human Reproduction 4:775-783. https://doi.org/10.1093/molehr/4.8.775
  24. Pang YW, An L, Wang P, Yu Y, Yin QD, Wang XH, Xin-Zhang, Qian-Zhang, Yang ML, Min-Guo, Wu ZH and Tian JH. 2013. Treatment of porcine donor cells and reconstructed embryos with the antioxidant melatonin enhances cloning efficiency. J. Pineal. Res. 54:389-397. https://doi.org/10.1111/jpi.12024
  25. Park CH, Uh KJ, Mulligan BP, Jeung EB, Hyun SH, Shin T, Ka H and Lee CK. 2001 Analysis of imprinted gene expression in nomal fertilized and uniparental preimplantation porcine embryos. PLoS One 6:e22216.
  26. Park JY, Park MR, Hwang KC, Chung JS, Bui HT, Kim T, Cho SK, Kim JH, Hwang S, Park SB, Nguyen VT and Kim JH. 2011. Comparative gene expression analysis of somatic cell nuclear transfer-derived cloned pigs with normal and abnomal umbilical cords. Biol. Reprod. 84:189-199. https://doi.org/10.1095/biolreprod.110.085779
  27. Park MR, Kim BK, Lee HC, Lee P, Hwang S, Im GS, Woo JS, Cho C, Choi SH, Kim SW and Ko YG. 2010. The imprinted messenger TNA expression in cloned porcine pre-implantation embryos. J. Emb. Trans. 25:127-131.
  28. Park YG, Lee SE, Kim EY, Hyun H, Shin MY, Son YJ, Kim SY and Park SP. 2015. Effects of Feeder Cell Types on Culture of Mouse Embryonic Stem Cell In Vitro. Development & Reproduction 19:119-126. https://doi.org/10.12717/DR.2015.19.3.119
  29. Park, S.J, Ryu, J, Kim, I.H, Choi, Y.H, Nam, T.J. 2015. Activation of the mTOR signaling pathway in breast cancer MCF-7 cells by a peptide derived from porphyra yezoensis. Oncol Rep. 33:19-24. https://doi.org/10.3892/or.2014.3557
  30. Prather RS. 2000. Cloning. Pigs is pigs. Science. 289:1886-1887. https://doi.org/10.1126/science.289.5486.1886
  31. Ranganathan P and Rangnekar VM. 2005. Exploiting the TSA connections to overcome Apoptosis-resistance. Cancer Biol. Ther. 4:391-392.
  32. Walker SC, Shin TY, Zaunbrecher GM, Romano JE, Johnson GA and Bazer FW. 2002. A highly efficient method for porcine cloning by nuclear transfer using in vitro-matured oocytes. Cloning Stem Cells 4:105-112. https://doi.org/10.1089/153623002320253283
  33. Yang YH, Choi SY, Lee SY, Park CK, Yang BK, Kim CI and Cheong HT. 2003. Study on the improvement of bovine somatic cell nuclear transfer rechnique. Korean J. Animal Reprod. 27:233-240.
  34. Yin XJ, Cho SK, Park MR, Im YJ, Park JJ, Bhak JS, Kwon DN, Jun SH, Kim NH and Kim JH. 2003 Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions. Zygote 11:167-174. https://doi.org/10.1017/S096719940300220X