Korean Journal of Poultry Science (한국가금학회지)

The Korean Society of Poultry Science (한국가금학회)
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The Evaluation of Various Conditions in the Cryopreservation of Primordial Germ Cells on Korean Native Chicken (Ogye)

한국재래닭(오계)의 원시 생식 세포의 냉동 보존에 있어서 여러 조건의 평가

  • Kim, Hyun (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Cho, Young Moo (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Han, Jae Yong (WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University) ;
  • Choi, Sung Bok (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Byun, Mi Jeong (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Kim, Young Sin (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Ko, Yeoung-Gyu (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Seong, Hwan-Hoo (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Kim, Sung Woo (Animal Genetic Resources Station, National Institute of Animal Science, RDA)
  • 김현 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 조영무 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 한재용 (서울대학교 동물자원과학과) ;
  • 최성복 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 변미정 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 김영신 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 고응규 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 성환후 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 김성우 (농촌진흥청 국립축산과학원 가축유전자원시험장)
  • Received : 2014.09.24
  • Accepted : 2014.11.11
  • Published : 2014.12.31
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Abstract

Cryopreserving cells which are maintaining their viability are the very complex process. This study has been carried out in order to find the effects of cryopreservation steps and freezing media on the rates of viability of cryopreserved chicken primordial germ cells (PGCs). PGCs obtained from the germinal gonade of 5.5~6 day (stage 28) chick embryos of Korean Ogye (KO) and Commercial breeds (C), using the MACS method were suspended in a freezing medium containing a freezing and protecting agents (e.g. dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG)). Gonads were harvested from stage 28 chick embryos and pooled in groups of 5, 10, 15, 20E embryos, contributing gonads to the cell suspension. The gonadal cells, including PGCs, were then frozen in 1 of the following cryoprotectant treatments : 2.5%, 5%, 10%, 15% and 0% cryoprotectant (DMSO, EG, PG) as a control. Effects of exposure to slow freezing and vitrification, with different concentrations of the cryoprotectant solution, were examined. After vitrification and slow freezing, survival rates of the frozen-thawed PGCs from the 10% EG plus FBS treatment were 85.63%, and 66.14% (p<0.05), respectively. The viability of PGCs after freeze-thawing was significantly higher for 10% EG plus FBS treatment than for 10% PG + FBS treatment (p<0.05) (85.63% vs 66.81%) by vitrification. This study established a method for preserving chicken PGCs that enables systematic storage and labeling of cryopreserved PGCs in liquid ($LN_2$) at a germplasm repository and ease of entry into a data base. In the future, the importance for this new technology is that poultry lines can be conserved while work is being conducted on improving the production of germline chimeras.

동결 닭 원시 생식 세포의 생식계열 키메라를 이용한 생체에의 복원을 실용화 하기 위해서는, 닭 원시 생식 세포의 동결 보존 기술의 향상에 의해 동결 및 융해 후의 많은 생존세포를 확보하는 것이 반드시 필요하다. 닭 원시 생식 세포는 배양 5.5일령의 닭 원시 생식선으로부터 채취하고, MACS 방법에 의해서 순수 닭 원시 생식 세포를 분리했다. 15% 각각의 EG를 동결 보호제로 사용한 처리군이 각 군의 농도에 상관없이 유의적(p<0.05)으로 PG 처리군보다 동결 및 융해 후의 세포의 생존율이 높음을 확인하였다. 특히, 동결 보호제로 10% EG를 이용한 유리화 처리군에서 85.63%로 동일한 농도의 PG 처리군(66.81%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 한편, 10% EG를 이용한 완만 동결 처리군에서 66.14%로 동일한 농도의 PG 처리군(50.11%)보다 유의적(p<0.05)으로 가장 높은 생존율을 보였다. 이상의 결과들로부터 유리화 동결에 있어서 가장 높은 생존율을 보인 10% EG이 최적의 동결 보호제로서 사용 가능함을 확인하였고, 이는 한국재래닭(오계)의 원시 생식 세포의 동결 보존의 실용화가 보다 더 향상될 수 있는 또 하나의 방법이 될 수 있음을 시사한다.

Keywords

References

  1. Arav A, Yavin S, Zeron Y, Natan D, Dekel I, Gacitua H 2002 New trends in gamete's cryopreservation. Mol Cell Endocrinol 187:77-81. https://doi.org/10.1016/S0303-7207(01)00700-6
  2. Blackburn, HD 2006 The national animal germplasm program: Challenges and opportunities for poultry genetic resources. Poult Sci 85:210-215. https://doi.org/10.1093/ps/85.2.210
  3. Bautistaa JA, DelaPena EC, Katagiri S, Takahashi Y, Kanagawa H 1998 In vitro viability of mouse oocytes vitrified in an ethylene glycol-based solution. Jpn J Vet Res 46:13-18.
  4. Bautistab JA, Kanagawa H 1988 Current status of vitrification of embryos and oocytes in domestic animals: Ethylene glycol as an emerging cryoprotectant of choice. Jpn J Vet Res 45:183-191.
  5. Boutron P 1984 A more accurate determination of the quantity of ice crystallized at low cooling rates in the glycerol and 1,2-propandiol aqueous solutions: Comparison with equilibrium. Cryobiology 21:183-191. https://doi.org/10.1016/0011-2240(84)90210-4
  6. Chi HJ, Koo JJ, Kim MY, Joo JY, Chang SS and Chung KS 2002 Cryopreservation of human embryos using ethylene glycol in controlled slow freezing. Hum Reprod 17:2146-2151. https://doi.org/10.1093/humrep/17.8.2146
  7. Fulton JE and Delany ME 2003 Poultry genetic resources-operation rescue needed. Science 300:1667-1668. https://doi.org/10.1126/science.1085407
  8. Freshney RI 2005 Culture of Animal Cells: A Manual of Basic Technique. 5th Edn Wiley-Liss Inc Hoboken.
  9. Friedler S, Giudice LC, Lamb EJ 1988 Cryopreservation of embryos and ova. Fertil Steril 49:743-764.
  10. Gardner DK, M Lane 2000 Embryo culture systems. In "Handbook of In Vitro Fertilization", 2nd Ed. CRC Press Boca Raton FL 205-264.
  11. Hamburger V, Hamilton HL 1951 A series of normal stage in the development of the chick embryo. Journal of Morphology 8:49-92.
  12. Han JY, Park TS, Hong YH, Jeong DK, Kim JN, Kim KD, Lim JM 2002 Production of germline chimeras by transfer of chicken gonadal primordial germ cells maintained in vitro for an extended period. Theriogenology 58:1531-1539. https://doi.org/10.1016/S0093-691X(02)01061-0
  13. Hey JM, MarFarlane DR 1996 Crystallization of ice in aqueous solutions of glycerol and dimethly sulphoxide. Cryobiology 33:205-216. https://doi.org/10.1006/cryo.1996.0021
  14. IUCN Red List 2012 URL http://www.iucnredlist.org/documents/Summarystatistics
  15. Kasai M, Hamaguchi Y, Zhu SE, Miyake T, Sakurai T, 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
  16. Kasai M, Ito M, 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
  17. Kim JN, Kim MA, Park TS, Kim DK, Park HJ, Ono T, Lim JM, Han JY 2004 Enriched gonadal migration of donorderived gonadal primordial germ cells by immunomagnetic cell sorting in birds. Mol Reprod Dev 68:81-87. https://doi.org/10.1002/mrd.20051
  18. Kim, MK, Lee SJ, Uhm EY, Yoon SH, Park SP, Chung KS, Lim JH 1996 Cryopreservation of mouse IVF zygotes by vitrification. J Animal Reprod 20:119-126.
  19. Kino K, Pain B, Leibo SP, Clark ME, Etches RJ 1997 Production of chicken chimeras from injection of frozenthawed blastodermal cells. Poultry Sci 76:753-760. https://doi.org/10.1093/ps/76.5.753
  20. Kotobuki N, Hirose M, Machida H, Katou Y, Muraki K, Takakura Y, Ohgushi H 2005 Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells. Tissue Eng 11:663-673. https://doi.org/10.1089/ten.2005.11.663
  21. Kroll G 2001 The "Silent springs" of rachel carson; Mass media and the origins of modern environmentalism. Public Underst Sci 4:403-20.
  22. Kuleshova LLa, MacFarlane DR, Trounson AO, Shaw JM 1999 Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes. Cryobiology 38:119-30. https://doi.org/10.1006/cryo.1999.2153
  23. Kuleshova LLb, Shaw JM, Trounson AO 2001 Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation. Cryobiology 43:21-31. https://doi.org/10.1006/cryo.2001.2335
  24. Lee DR, Yoon TK 2009 Effect of slush-nitrogen on the cryopreservation of oocytes and embryos using vitrification. Korean J Reprod Med 36:1-7.
  25. Lovelock JE, Bishop MWH 1959 Prevention of freezing damage to living cells by dimethyl sulphoxide. Nature 183:1394-1395. https://doi.org/10.1038/1831394a0
  26. Martino A, Songsasen N, 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
  27. Meryman HT 2007 Cryopreservation of living cells: Principles and practice. Transfusion 47: 935-945. https://doi.org/10.1111/j.1537-2995.2007.01212.x
  28. Miyake T, Kasai M, Zhu SE, Sakurai T, Machida T 1993 Vitrification of mouse oocytes and embryos at various stages of development in an ethylene-glycol based solution by a simple method. Theriogenology 40:121-134. https://doi.org/10.1016/0093-691X(93)90346-7
  29. Mozdziak PE, Angerman-Stewart J, Rushton B, Pardue SL, Petitte JN 2005 Isolation of chicken primordial germ cells using fluorescence-activated cell sorting. Poult Sci 84:594-600. https://doi.org/10.1093/ps/84.4.594
  30. Natio T, Tajima A, Tagami T, Yasuda Y, Kuwana T 1994 Preservation of chick primordial germ cells in liquid nitrogen and subsequent production of viable offspring. J Reprod 102:321-325. https://doi.org/10.1530/jrf.0.1020321
  31. Naito M 2003 Cryopreservation of avian germline cells and subsequent production of viable offspring. J Poultry Sci 40:1-12. https://doi.org/10.2141/jpsa.40.1
  32. Petitte JN 2006 Avian germplasm preservation: embryonic stem cells or primordoial germ cells. Poultry Sci 85:237-242. https://doi.org/10.1093/ps/85.2.237
  33. Pokorny P 2002 Obtaining chicken chimeras after injecting cryopreserved blastoderm cells into the subgerminal cavity of recipient embryos. Anim Sci Pap Rep 20:55-66.
  34. 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
  35. Rayos AA, Takahashi YM, Hishinuma, Kanagawa H 1994 Quick freezing of unfertilized mouse oocytes using ethylene glycol with sucrose or trehalose. J Reprod Fertil 24:100-123.
  36. Rezazadeh VM, Eftekhari-Yazdi P, Karimian L, Hassani F, Movaghar B 2009 Vitrification versus slow freezing gives excellent survival, post warming embryo morphology and pregnancy outcomes for human cleaved embryos. J Assist Reprod Genet 26:347-354. https://doi.org/10.1007/s10815-009-9318-6
  37. Tajima A, Naito M, Yasuda Y, Kuwana T 1998 Production of germ-line chimeras by transfer of cryopreserved gonadal primordial germ cells (gPGC) in chicken. J Exp Zool 280:265-267. https://doi.org/10.1002/(SICI)1097-010X(19980215)280:3<265::AID-JEZ8>3.0.CO;2-L
  38. Takase K, Sawai M, Yamamoto K, Yata J, Takasaki Y, Teraoka H, Tsukada K 1992 Reversible G1 arrest induced by dimethyl sulfoxide in human lymphoid cell lines: kinetics of arrest and expression of the cell cycle marker proliferating cell nuclear antigen in Raji cells. Cell Growth Differ 3:515-521.
  39. Whittingham DG, Leibo SP, Mazur P 1972 Survival of mouse embryos frozen to $-196^{\circ}C$ and $-269^{\circ}C$. Science NY 187: 411-414.
  40. Wilmut I, Schnieke AE, McWhir J, Kind AJ, Campbell KH 1997 Viable offspring derived from fetal and adult mammalian cells. Nature 385:810-813. https://doi.org/10.1038/385810a0
  41. Yasuda Y, Tajima A, Fujimoto T, Kuwana T 1992 A method to obtain germ-line chimaeras using isolated primordial germ cells. Journal of Reproduction and Fertility 96:521-528. https://doi.org/10.1530/jrf.0.0960521
  42. Yavin S, Aroyo A, Roth Z, Arav A 2009 Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush. Hum Reprod 24:797-804.
  43. Yoon TK, Lee DR, Cha SK, Chung HM, Lee WS, 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
  44. Zhao DF, Kuwana T 2003 Purification of avian circulating primordial germ cells by nycodenz density gradient centrifugation. Br Poult Sci 44:30-35.
  45. Zhu SE, Kasai M, Otoge H, Sakurai T, 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