Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Effect of Ethylene Glycol (EG) on the Viability of Mammalian Embryo during Cryopreservation

포유류 초기 배아의 동결 시 생존율에 미치는 Ethylene Glycol(EG)의 영향

  • 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)
  • 김현 (일본동경대학교 수의과학대학 수의생리학교실) ;
  • 조영무 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 고응규 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 김성우 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 성환후 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 야마노우치 케이타로 (일본동경대학교 수의과학대학 수의생리학교실)
  • Received : 2014.09.16
  • Accepted : 2014.09.26
  • Published : 2014.09.30
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Abstract

Ethylene glycol (EG) has been successfully used as a cryoprotectant for vitrification of mammalian embryos (including human embryos) due to its low formula weight and high permeation into cells compared with other cryoprotectants, including propylene glycol (PROH). Cryopreservation is able to store the surplus pre-embryos for freezing and furthermore thawing and transfer in a subsequent cycle. 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). Female ICR mice (6~8 weeks old) were induced to superovulate by sequential intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48 h apart. Mouse embryos were collected according to its developmental stage after the injection of hCG. Embryos were cryopreserved not only during cryoprotectant step (1~4 step) but also in a variety of media (HTF, IVF medium, D-PBS) and cell stage. The results were as follows : There is no clear advantage in these freezing media of rapid method, but 4 cell and 8 cell of slow method (2, 3 and 4 step) have advantage in D-PBS. The development of embryos according to cell stage become greater in 8 cell stage. In the treatment steps of cryopreservation, the development of embryo to blastocyst was similar among rapid method, but the development of 4 cell and 8 cell embryos to blastocyst according to slow method was better than rapid method.

생쥐 2세포기, 4세포기, 8세포기를 각 발생 단계에서 채취하여 동결 보호제를 첨가한 서로 다른 배양액에서 배양하고, 배아의 동결 보존 및 융해 시 급속 처리와 저속 처리 단계로 비교하여 이들 조건이 배아의 생존과 발현에 미치는 영향을 조사하여 다음의 결과를 얻었다. 동결 보호제로 처리하여 배양액을 달리한 경우, 급속 단계에서는 모든 배양액에서 비슷한 발생율을 보였고, 저속단계의 4세포기와 8세포기는 D-PBS에서 높은 발생율을 보였다(P<0.05, P<0.01). 배아의 발생 시기에 따른 동결 보존 후, 발생율은 2, 4, 8세포기로 넘어갈수록 발생율의 증가를 보여 8세포기에서 발생율이 가장 높았다(P<0.01). 동결 보호제의 처리단계에 따른 발생율은 2세포기의 급속 단계에서는 유사하였으나, 4세포기와 8세포기는 저속단계에서 높은 발생율을 보였으며(P<0.05), 특히 8세포기에서 가장 높았다(P<0.01).

Keywords

References

  1. Bernard A and Fuller BJ. 1996. Cryopreservation of human oocytes: a review of current problems and perspectives. Hum. Reprod. Update. 2:193-207. https://doi.org/10.1093/humupd/2.3.193
  2. Bryant G. 1995. DSC measurement of cell suspensions during successive freezing runs: implications for the mechanisms of intracellular ice formation. Cryobiology, 32:114-128. https://doi.org/10.1006/cryo.1995.1011
  3. 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
  4. Gilmore JA, McGann LE, Liu J, Gao DY, Peter AT and Kleinhans FW. 1995. Effect of cryoprotectant solutes on water permeability of human spermatozoa. Biol. Reprod. 53: 985-995. https://doi.org/10.1095/biolreprod53.5.985
  5. Hotamisligil S, Toner M and Powers RD. 1996. Chnges in membrane integrity, cytoskeletal structure, and developmental potential of murine oocytes after vitrification in ethylene glycol. Biol. Reprod. 55:161-168. https://doi.org/10.1095/biolreprod55.1.161
  6. Kim MK, Lee SJ, Uhm EU, Yoon SH, Park SP, Chung KS and Lim JH. 1996. Cryopreservation of mouse IVF zygotes by vitrification. J. Animal. Reprod. 20:119-126.
  7. Mandelbaum J, Junca AM, Plachot M, Alnot MO, Tibi C, Cohen J and Salat-Baroux J. 1988. Solutions provided by the freezing of embryos and questions posed by the freezing of human oocytes. Rev. Fr. Gynecol. Obstet. 83:619-622.
  8. Martino AN, Songsasen 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
  9. Rall WF and Fahy GM. 1985. Ice-free cryopreservation of mouse embryos at$-196^{\circ}C$ by vitrification. Nature 313:573- 575. https://doi.org/10.1038/313573a0
  10. Rall WF. 1987. Factors affecting the survival of mouse embryos cryopreserved by vitrification. Cryobiology 24:387-402. https://doi.org/10.1016/0011-2240(87)90042-3
  11. Rayos AA, Takahashi YM, Hishinuma A and Kanagawa H. 1994. Quick freezing of unfertilized mouse oocytes using ethylene glycol with sucrose or trehalose. J. Reprod. Fertil. 24:100-123.
  12. 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
  13. Zhu, SE, Kasai M, Otoge H, Sakurai T and Machida T. 1993. Cryopreservation of expanded mouse blastocysts by vitrification in ethylene glycol-based solutions. J. Reprod. Fert. 98:139-145. https://doi.org/10.1530/jrf.0.0980139