한국수정란이식학회지 (Journal of Embryo Transfer)

  • 제29권3호
  • /
  • Pages.241-248
  • /
  • 2014
  • /
  • 2508-755X(pISSN)
  • /
  • 2288-0178(eISSN)
한국수정란이식학회 (The Korean Society of Embryo Transfer)
권호 표지
DOI QR코드

DOI QR Code

포유류 초기 배아의 효율적인 동결 보존 방법에 관한 연구

Development of Effective Cryopreservation Method for Mammalian Embryo

  • 김현 (일본 동경대학교 수의과학대학 수의생리학 교실) ;
  • 조영무 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 고응규 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 김성우 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 성환후 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 야마노우치 케이타로 (일본 동경대학교 수의과학대학 수의생리학 교실)
  • Kim, Hyun (Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo) ;
  • Cho, Young Moo (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Ko, Yeoung-Gyu (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Kim, Sung Woo (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Seong, Hwan-Hoo (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Yamanouchi, Keitaro (Department of Veterinary Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo)
  • 투고 : 2014.09.16
  • 심사 : 2014.09.26
  • 발행 : 2014.09.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

본 연구에서 생쥐 초기 배아를 이용하여 동결 보존된 배아의 생존율 또는 발달율에 영향을 미치는 요인들의 상관관계를 알아본 결과, 배아의 발달 단계에 따른 동결 - 해동 후 생존율에 있어서는 2세포기 배아가 4~8세포기 배아보다 유의하게 높았으나(p<0.01), 배 발달율에 있어서는 4~8세포기 배아가 유의하게(p<0.01), 높아 생존율과 배 발달율과는 상관관계가 없는 것으로 사료된다. 또한, 동결 보호제에 따른 배아의 생존율에 있어서는 2, 4~8세포기 배아 모두 DMSO에서 유의하게 높았으나(p<0.01), 배 발달율에 있어서는 EG가 DMSO에 비해 더 유의하게 높은 성적을 나타내어(p<0.01, p<0.05) 동해로 인한 상해가 적은 것으로 생각되어 2, 4~8세포기 배아에서 EG가 더 효과적인 동결 보호제로 사료되며, 동결 프로그램으로는 완만 동결 - 급속 해동법이 더 우수한 프로그램으로 보인다.

This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Two to eight cell embryos were obtained from oviducts of mated $F_1$ hybrid female mice superovulated by PMSG and hCG. Two-step EG, DMSO and 4-step EG, DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow-cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in EG and DMSO was significantly higher than 4~8 cell (65.4% versus 61.2%, 81.1% versus 72.5%) (p<0.01, p<0.01), but the development rates of 4~8 cell embryos in EG and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4~8 cell embryos in EG were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in EG(77.0% versus 64.4%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The development rate from morular to hatching blastocyst, however, was sinificantly higher in EG than in DMSO during 48 hr (p<0.01). The survival rate of 4~8 cell embryo was 62.5% in EG and 73.3% in DMSO. The development rates of embryo in EG were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. The survival rate of embryo in 2 cell stage was higher than in 4~8 cell stage, and EG appears more effective cryoprotectant than DMSO because EG showed better development rates of embryos in 2 and 4~8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.

키워드

참고문헌

  1. Al-Hasani S, Diedrich K, van der Van H, Reinecke A, Hartje M and Krebs D. 1987. Cryopreservation of human oocytes. Hum. Reprod. 2:695-700. https://doi.org/10.1093/oxfordjournals.humrep.a136616
  2. Cohen J, Somons FR, Edwards RG, Fehilly CB and Fishel SB. 1985. Pregnancies following the frozen storage of expanding human blastocytes. J. IVF ET. 2:59-64.
  3. Emiliani S, Van den Bergh M, Vannin AS, Biramane J and Englert Y. 2000. Comparison of ethylene glycol, 1,2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts. Hum. Reprod. 15:905-910. https://doi.org/10.1093/humrep/15.4.905
  4. Fahy LE. 1986. The relevance of cryoprotectant "Toxicity" to cryobiology. Cryobiology. 23:1-13. https://doi.org/10.1016/0011-2240(86)90013-1
  5. Fehill CB, Cohen J, Simons RF, Fishel SB and Edwards RG. 1985. Cryopreservation of cleaving embryos and expanded blastocysts in the human ; A comparative study. Fertil. Steril. 44:638-644. https://doi.org/10.1016/S0015-0282(16)48980-6
  6. Friedler S, Giudice LC and Lamb EJ. 1988. Cryopreservation of embryos and ova. Fertil. Steril. 49:743-764. https://doi.org/10.1016/S0015-0282(16)59879-3
  7. Fuller BJ and Bernard A. 1986. The relationship between intracellular glycerol permeation and survival following cryopreservation of in vitro fertilized 2-cell murine embryo. Cryo-Letters. 7:257-259.
  8. Gook DA, Osborn SM and Johbston WIH. 1993. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Hum. Reprod. 8:1101-1109. https://doi.org/10.1093/oxfordjournals.humrep.a138201
  9. Kasai M, Komi JH, Takakamo A, Tsudera T, Sakurai T and Machida T. 1990. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J. Reprod. Fertil. 89:91-97. https://doi.org/10.1530/jrf.0.0890091
  10. Kasai M, Hamaguchi Y, Zhu SE, Miyake T, Sakurai T and Machida T. 1992. High survival of rabbit morulae after vitrification in an ethylene glycol- based solution by a simple method. Biol. Reprod. 46:1042-1046. https://doi.org/10.1095/biolreprod46.6.1042
  11. Kasai M, Ito M and Edashige K. 2002. Morphological appearance of the cryopreserved mouse blastocyst as a tool to identify the type of cryoinjury. Hum. Reprod. 17:1863-1874. https://doi.org/10.1093/humrep/17.7.1863
  12. Kim MK, Lee SJ, Uhm EY, Yoon SH, Park SP, Chung KS and Lim JH. 1996. Cryopreservation of mouse IVF zygotes by vitrification. J. Animal. Reprod. 20:119-126.
  13. Liu J, Van den Abbeel E and Van Steirteghem AC. 1993. Assessment of ultrarapid and slow freezing procedures for 1-cell and 4-cell mouse embryos. Hum. Reprod. 8:1115-1119. https://doi.org/10.1093/oxfordjournals.humrep.a138203
  14. Hamberger L and Hazekamp J. 2002. Towards single embryo transfer in IVF. J. Reprod. Immunol. 55:141-148. https://doi.org/10.1016/S0165-0378(01)00135-8
  15. Junca AM, Mandelbaum J, Alnot MO, Plachot M, Cohen J and Salat-Baroux J. 1988. Factors involved in the success of human embryo freezing. Does cryopreservation really improve the IVF results? Ann. N Y Acad. Sci. 541:575-582. https://doi.org/10.1111/j.1749-6632.1988.tb22294.x
  16. Lassalle B, Testart J and Renard JP. 1985. Human embryo features that influence the success of cryopreservation with the use of 1,2 propanediol. Fertil. Steril. 44:645-651. https://doi.org/10.1016/S0015-0282(16)48981-8
  17. Macas E, Xie M, Keller PJ, Imthurn B and Rulicke T. 1991. Developmental capacities of two-cell mouse embryos frozen by three methods. J. IVF ET. 8:208-212.
  18. Martino A, Songsasen N and Leibo SP. 1996. Development into blastocysts of bovine oocytes cryopreserved by ultrarapid cooling. Biol. Reprod. 54:1059-1069. https://doi.org/10.1095/biolreprod54.5.1059
  19. Mazur P. 1963. Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J. Gen. Physiol. 47:347-369. https://doi.org/10.1085/jgp.47.2.347
  20. Nowshari MA, Nayudu PL and Hodges JK. 1995. Effect of cryoprotectants and their concentration on post-thaw survival and development of rapid frozen-thawed pronuclear stage mouse embryos. Hum. Reprod. 10:3237-3242. https://doi.org/10.1093/oxfordjournals.humrep.a135895
  21. Polge C, Smith AU and Parkes AS. 1949. Retrieval of spermatozoa after vitrification and dehydration at low temperatures. Nature 164:666-676. https://doi.org/10.1038/164666a0
  22. Polge C and Willadsen SM. 1978. Freezing eggs and embryos of farm animals. Cryobiology 15:370-373. https://doi.org/10.1016/0011-2240(78)90054-8
  23. Rayos AA, Takahashi Y, Hishinuma M and Kanagawa H. 1994. Quick freezing of unfertilized mouse oocytes using ethylene glycol with sucrose or trehalose. J. Reprod. Fertil. 24:100-123.
  24. Renard JP, Bui-Xuan Nguyen and Garnier V. 1984. Two-step freezing of two cell rabbit embryos after partial dehydration at room temperature. J. Reprod. Fertil. 71:573-580. https://doi.org/10.1530/jrf.0.0710573
  25. Schneider U. 1986. Cryobiological principles of embryo freezing. J. IVF ET. 3:3-9.
  26. Siebzehnruebl ER, Todorow S, van Uem J, Koch R, Wildt L and Lang N. 1989. Cryopreservation of human and rabbit oocytes and one-cell embryos : a comparison of DMSO and propanediol. Human Reprod. 4:312-317. https://doi.org/10.1093/oxfordjournals.humrep.a136895
  27. Szell A, Zhang J and Hudson R. 1990. Rapid cryopreservation of sheep embryos by direct transfer into liquid nitrogen vapour at-180 degrees C. Reprod. Fertil. Dev. 2:613-618. https://doi.org/10.1071/RD9900613
  28. Testart J, Lassalle B, Belaisch-Allart J, Hazout A, Forman R, Rainhorn JD and Fryman R. 1986. High pregnancy rate after early human embryo freezing. Fertil. Steril. 46:268- 272. https://doi.org/10.1016/S0015-0282(16)49524-5
  29. Todorow SJ, Siebzehnrubl ER, Koch R, Wildt L and Lang N. 1989. Comparative results on survival of human and animal eggs using different cryoprotectants and freeze-thawing regimens. I. Mouse and hamster. Hum. Reprod. 4:805-811. https://doi.org/10.1093/oxfordjournals.humrep.a136990
  30. Trounson A and Mohr L. 1983. Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 305:707-709. https://doi.org/10.1038/305707a0
  31. Van-den-Abbeel E, Van-der-Elst J and Van-Steirteghem AC. 1994. The effect of temperature at which slow cooling is terminated and of thawing rate on the survival of one-cell mouse embryos frozen in dimethyl sulfoxides or 1,2-propanediol solutions. Cryobiology 31:423-433. https://doi.org/10.1006/cryo.1994.1052
  32. Whittingham DG and Leibo SP. 1972. Mazur. Survival of mouse embryos frozen to $-196^{\circ}C$and $-296^{\circ}C$. Science NY. 187: 411-414.
  33. Whittingham DG. 1980. Principles of embryo preservation. In: Ashwood Smith MJ, Farrant J. editor. Low Temperature Preservation in Medicine and Biology. Pitman Medical, Tunbridge Wells. 65-83.
  34. Wilmut L. 1972. The effect of cooling rate, warming rate cryoprotective agent and stage of development on survival of mouse embryos during freezing and thawing. Life Sci. 2: 1071-1079.
  35. Willadsen S, Polge C and Rowson LEA. 1978. The viability of deep-frozen cow embryos. Reprod. Fertil. 52:391-393. https://doi.org/10.1530/jrf.0.0520391
  36. Yoon TK, Lee DR, Cha SK, Chung HM, Lee WS and Cha KY. 2007. Survival rate of human oocytes and pregnancy outcome after vitrification using slush nitrogen in assisted reproductive technologies. Fertil. Steril. 88:952-956. https://doi.org/10.1016/j.fertnstert.2006.12.071
  37. Zeilmarker GH, Alberta AT and Van Gent I. 1984. Two pregnancies following transfer of intact frozen-thawed embryos. Fertil. Steril. 42:293-296. https://doi.org/10.1016/S0015-0282(16)48029-5