Korean Journal of Animal Reproduction (한국가축번식학회지)

The Korean Society of Animal Reproduction (한국동물번식학회)
권호 표지

Apoptosis in the Bovine Blastocyst following Nnclear Transfer and In Vitro Fertilization

핵치환과 체외수정에 유래된 소의 배반포에서의 Apoptosis

  • Kim, . E.H (Animal Resources Research Center, Konkuk University) ;
  • D.W. Han (Animal Resources Research Center, Konkuk University) ;
  • K.S. Chung (Animal Resources Research Center, Konkuk University) ;
  • Lee, H.T. (Animal Resources Research Center, Konkuk University)
  • Published : 2002.06.01
Download PDF
⟨ Previous Next ⟩

Abstract

The mechanisms underlying of the visual assessment and resulting in optimum embryonic development following in vitro maturation, fertilization, and culture are unclear, It was known that in vitro produced embryos show more frequent occurrence of fragmentation, which resulted in poor developmental potential and decreased implantation rate. The objective of this study was to investigate the apoptotic rates in bovine blastocyst derived from in vitro fertilization (IVF) and nuclear transfer (NT). In addition, the expression levels of Bcl-2 and Bax gene were investigated in the blastocyst to confirm their potential roles in the regulation of apoptosis during preimplantation embryonic development. Analysis of apoptosis was carried out by using terminal deoxynucleotidyl transferase mediate dUTP nick end labeling (TUNEL) method. The levels of Bcl-2 and Bax gene in the blastocyst derived from IVF and NT were determined by RT-PCR. The proportion of TUNEL positive signal in blastocyst derived from NT was significantly higher than that in blastocyst derived from IVF (p<0.001). Bcl-2 expression level of blastocyst derived from IVF was higher than that of blstocyst derived from NT. However, high expression level of Bax was observed in the blastocyst derived from NT. These results indicates that apoptosis is more responsible for fiagmentation in bovine blastocyst derived from NT than IVF. These results suggested that the increase of developmental failure followed by NT could be caused by nuclear fragmentation as apoptosis.

포유동물의 체외수정란 치적발달조건을 수립하기 위하여 난자의 성숙, 수정 및 배양시 형태학적인 관찰만으로는 불충분하여 각 세포내 구성물 및 핵상 등의 변화를 조사하여 개별 수정란의 품질평가를 통하여 수정란 발달 능력을 향상시키는 방법의 개발이 필요하다. 최근 체외생산된 수정란이 발달율 및 착상율이 현저히 감소되는 원인을 조사한 결과 이러한 수정란에서 할구의 파편화가 보다 더 진행되는 것을 관찰하였다. 이에 본 실험의 목적은 체외수정과 핵치환 유래 소 배반포에서 세포사멸 기전으로 알려진 apoptosis 조절 유전자로써 Bcl-2와 Bax 유전자의 전사체 발현량을 비교 조사함으로써 착상전 수정란 발달과정에서의 역할을 확립하고자 하였다. Apoptosis의 분석은 TUNEL 방법으로 수행하였다 체외수정과 핵치환유래 배반포에서 Bcl-2와 Bax 유전자의 발현량은 RT-PCR로 확인하였다. 핵치환유래 배반포에 있어 TUNEL 표식으로 확인된 비율은 체외수정 된 배반포보다 유의하게 높다 (P<0.001). 체외수정된 배반포의 Bcl-2의 발현량은 핵치환유래 배반포보다 높다. 반대로 체외수정된 배반포의 Bax 발현량은 핵치환 유래 소 배반포보다 낮았다 (P<0.05). 이러한 결과는 체외수정보다 핵치환 유래 소의 배반포에서 좀더 많은 파편화를 초래함을 보여준다. 또한, 핵치환 유래 수정란의 발달 정지의 증가는 apoptosis와 같은 핵의 파편화로써 야기될 수 있다고 사료된다.

Keywords

References

  1. Andrew, J., Watson, P. D. and Sousa, A. C. 2000. Impact of bovine oocyte maturation media on oocyte transcript levels, blastocyst development, cell number, and apoptosis. Biol. Reprod., 62:355-364 https://doi.org/10.1095/biolreprod62.2.355
  2. Brison, D. R. and Schultz, R. M. 1997. Apoptosis during mouse blstocyst formation: evidence for a role for survival factors including transforming growth factora. Biol. Reprod., 59: 1088-1096
  3. Brison, D. R. and Schultz, R. M. 1998. Increased incidence of apoptosis in transforming growth factor a-deficient mouse blastocysts. Biol. Reprod., 59:136-144 https://doi.org/10.1095/biolreprod59.1.136
  4. Byrne, A. T., Southgate, J., Brison, D. R. and Leese H. J. 1999. Analysis of apoptosis in the preimplantation bovine embryo using TUNEL. J. Reprod. Fertil., 117:97-105
  5. Cibelli, J. B., Stice, S. L., Gilueke, P. J., Kane, J. J., Jerry, J., Blackwell, C., Poncede, L. F. A. P. and Robi, J. M. 1998. Cloned transgenic calves produced from non quiescent fetal fibroblast. Science, 280:1256-1258 https://doi.org/10.1126/science.280.5367.1256
  6. Chao, D. T. and Korsmeyer, S. J. 1998. Bel-2 family: regulators of cell death. Annu. Rev. Immunol., 16:395-419 https://doi.org/10.1146/annurev.immunol.16.1.395
  7. Cory, S. and Adams, J. M. 1998. Matters of life and death: programmed cell death at cold spring harbor. Biochim. Biophys. Acta., 1377: R25-R44
  8. Ginger, E., Exley, C. T., Abigail, S. McElhinny, and Carol, M. W. 1999. Expression of caspase and Bel-2 apoptotic family members in mouse preimplantation embryos. Biol. Reprod., 61:231-239 https://doi.org/10.1095/biolreprod61.1.231
  9. Handyside, A. H. and Hunter, S. 1986. Cell devision in the mouse blastocyst before implantation. Rouxs Archi. Dev. Biol., 195:519-526
  10. Hardy, K.. Handyside, A. H. and Winston. 1989. The human blastocyst: cell number, death and allocation during late pre-implantation development in vitro. Development, 107:597604
  11. Hardy, K. 1997. Cell death in the mammalian blastocyst. Mol. Reprod. Dev., 3:919-925 https://doi.org/10.1093/molehr/3.10.919
  12. Heyman, Y., Chavatte-Palmer, P., Lebourhis, D. and Camous, S. 2002. Frequency and Occurrence of Late-Gestation Losses from Cattle Cloned Embryos. Bio. Reprod., 66:6-13
  13. Jurisicova, A., Varmuza, S. and Casper, R. F. 1996. Programed cell death and human embryo fragmentation. Mol. Hum. Reprod., 2:93-98
  14. Jurisicova, A., Rogers, I., Fasciani, A., Casper, R. F. and Varmuza, S. 1998. Effects of maternal age and conditions of fertilization on programmed cell death during murine preinplantation embryo development. Mol. Hum. Reprod., 4:139-257 https://doi.org/10.1093/molehr/4.2.139
  15. Kato, Y., Tani, T., Sotomaru. Y., Kurokawa, K., Kato, J., Doguchi, H. Yasue, H. and Tsunoda, Y. 1998. Eight calves cloned from somatic cells of a single adult. Science, 282: 2095-2098
  16. Keskintepe, L. and Brackett, B. G. 1996. In vitro developmental competence of in vitro -matured bovine oocytes fertilized and cultured in completely defined media. Biol. Reprod., 55:333-9 https://doi.org/10.1095/biolreprod55.2.333
  17. Kuhholzer, B. and Prather, R. S. 2000. Ad- vances in livestock nuclear transfer. Proc. Soc, Exp. Bio. Med., 224:240-245
  18. Levy, R, Benchaib, M., Cordonier, H., Souchier, C. and Guerin, J. F. 1997. Annexin V labelling and terminal transgerase-mediated DNA end labelling (TUNEL) assay in human arrested embryos. Mol. Hum. Reprod., 4:775-783
  19. Lindner, G. M. and Wright, R. W. Jr. 1983. Bovine embryo morphology and evaluation. Theriogenology, 20:407-415 https://doi.org/10.1016/0093-691X(83)90201-7
  20. Ming-Yuan, Y. and Rajadurai, R. 2002. Expression of Bel-2 and Box proteins in relation to quality of bovine oocytes and embryos produced in vitro. Anim. Reprod. Science, 70: 159-169
  21. MsConkey, D. J., Zhivotovsky, B. and Orrenius, S. 1996. Apoptosis: molecular mechaism and biomedical implications. Mol. Aspects. Med., 17:1-110
  22. Negoescu, A., Guillermet, C., Lorimier, P., Brambill, E. and Labet-Moleur F. 1998. Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity. Biomed. Pharmacother, 52:252-258
  23. Oltvai, Z. N., Milliman, C. I., and Korsmeyer, S. J. 1993. Bel-2 hetherodimerized in vivo with a connserved homolog, Box, that accekerates programmed cell death. Cell., 74:609-619 https://doi.org/10.1016/0092-8674(93)90509-O
  24. O'Neill, C. 1998. Autocrin mediators are required or act on the embryo by the 2-cell stage to promote normal development and survival of mouse preimplantation embryos in vitro. Biol. Reprod., 58:1303-1309
  25. Parrish, J. J., Susko-Parrish, J. L. and First, N. L. 1985. Effect of heparin and chondroitin sulfate on the acrosome reaction and fertility of bovine sperm in vitro. Theriogenology, 24:537549
  26. Plante, L. and King, W. A. 1994. Lignt and electron microscopic analysis of bovine embryos derived by in vitro and in vivo fertilization. J. Assisrt. Reprod. Dev., 8:835-841
  27. Polejaeva, I. A. Chen, S., Vaught, T., Page, R., Mullins, J., Ball, S., Dal, Y., Boone, J., Walker, S. Ayatres, D., Colman, A. and Campbell, K. 2000. Cloned pigs produced by nuclear transfer from adult somatic cells. Nature, 407:86-90
  28. Rosenkrans, C. F., Zeng, G. Q., McNamara, G. T., ScPlante, L. and King, W. A. 1994. Light and electron microscopic analysis of bovine embryos derived by in vitro and in vivo fertilization. J. Assist. Reprod. Genet, 11:515 -529 https://doi.org/10.1007/BF02216032
  29. Savill, J. 1994. Apoptosis in disease. Eur. J. Clin. Invest., 24:715-723 https://doi.org/10.1111/j.1365-2362.1994.tb01067.x
  30. Schultz, R. M. 1993. Regulation of zygotic gene activation in the mouse. Biocssays, 15:531-538
  31. Thornberry, N. A. and Lazebnik, Y. 1998. Caspase: enemies within. Science, 281:1312-1316
  32. Wakayama, T. and Yanagimachi, R. 1999. Cloning of male mice from adult tip tail cells. Nat. Genet., 22:127-128 https://doi.org/10.1038/9632
  33. Warner, C. M. and Cao, W. Exley, G. E. cElhinny AS. 1998. Genetic regulation of egg and embryo survival. Hum. Reprod.13 Supplement, 3:178-190
  34. Wilmut, I., Schnieke, A., McWhir, J., Kind, A. J. and Campbell, K. H. S. 1997. Viable dffspring derived from fetal and adult mammalian cells. Nature, 385:810-813 https://doi.org/10.1038/385810a0