한국가금학회지 (Korean Journal of Poultry Science)

  • 제40권3호
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  • Pages.163-169
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  • 2013
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  • 1225-6625(pISSN)
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  • 2287-5387(eISSN)
한국가금학회 (The Korean Society of Poultry Science)
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상업용 동결보호제를 이용한 한국재래닭(오계) 원시생식세포의 동결 보존

Cryopreservation of Primordial Germ Cells(PGCs) from Korean Native Chicken(Ogye) Embryos using Commercial Cryoprotectants

  • 김현 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 김동훈 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 한재용 (서울대학교 동물자원과학과) ;
  • 도윤정 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 김재환 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 김영신 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 성환후 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 고응규 (농촌진흥청 국립축산과학원 가축유전자원시험장) ;
  • 김성우 (농촌진흥청 국립축산과학원 가축유전자원시험장)
  • Kim, Hyun (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Kim, Dong Hun (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Han, Jae Yong (WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University) ;
  • Do, Yoon Jung (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Kim, Jae Hwan (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Kim, Young Sin (Animal Genetic Resources Station, National Institute of Animal Science, RDA) ;
  • Seong, Hwan Hoo (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)
  • 투고 : 2013.07.03
  • 심사 : 2013.08.13
  • 발행 : 2013.09.30
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초록

가금의 정액 동결 보존은 보고되고 있지만, 큰 난황의 구조 등과 같은 이유로 암컷의 유전물질의 보존은 불가능한 실정이다. 닭에 있어 닭원시생식세포(PGCs)의 동결 보존은 암컷과 수컷 양쪽의 유전물질을 보존할 수 있는 대체 방법이다. 본 연구에서 원시생식선으로부터 분리방법은 5.5일(stage 28 : 5.5일령(Hamburger and Hamilton, 1951)) 동안 발생한 수정란의 초기배자를 실체 현미경하에서 예리한 핀셋을 이용하여 원시생식선 부분만을 분리한 후, MACS 방법으로 정제하였다. 두 개의 상업적으로 사용되는 동결보호제(A와 B)와 10% EG + 15% FBS를 동결보호제로 하는 대조군을 각각 닭 PGCs의 동결 및 융해에 이용하였다. 동결 및 융해 후의 닭 PGCs의 회복율은 A(35.5%), B(60.5%) 그리고 대조군에서는 52.8%를 각각 확인하였다. 52.8%의 닭 PGCs의 회복율을 보인 대조군과 동결보호제 B 처리군 간에는 유의적인 차이를 보이지 않았다. 그러나 두 처리군에 비해 동결보호제 A는 35.5% 유의적으로 낮았다. 하지만, 동결 및 융해 후의 닭 PGCs 생존율은 각각 A(77.9%), B(77.4%) 그리고 대조군(81.6%)으로 보였다. 두 처리구 간에 유의적인 차이는 없었다. 본 연구는 배자 발생 초기의 원시생식선으로부터 채취한 닭 PGCs는 상업적으로 이용되고 있는 동결보호제(A와 B)를 사용해서 동결할 수 있다는 것을 확인했고, 생존율에 나쁜 영향을 주지 않고 액체질소에 성공적으로 보관할 수도 있음을 시사하고 있다.

Cryopreservation of poultry semen has been reported, but preservation of female genetic material has not been possible because of the unique anatomical and physiological characteristics of the avian egg. Thus an alternative strategy for conservation of oviparous species of animals must be developed. Recent technological developments for producing germline chimeras by the transfer of primordial germ cells (PGCs) into recipient embryos has enabled the conservation and retrieval of chicken genetic resources in their complete form. In the present study, fertilized eggs were incubated for about 5.5 days to obtain embryos at stage 28. The whole embryo was collected from the germinal gonad using a fine glass micro pipette under a microscope. The PGCs were then purified using MACS method. Two commercially available cryoprotectants (A and B) were used to preserve the PGCs, and EG were used as a control. The average recovery rate of PGCs after thawing was 35.5% and 60.5% with the A and B treatments, respectively. There was no significant difference between B treatments and control, which showed an average recovery rate of 52.8%. However, the recovery rate obtained using A cryoprotectant (35.5%) was significantly lower than using treatment control and B. The average viability of the PGCs after thawing were 77.9% and 77.4% for cryoprotectants A and B, respectively, and the control were was 81.6%. There was no statistically significant difference between the two treatments and control. It was concluded that all of the available cryoprotectants examined in this study could be used for preservation of PGCs from embryos. Further experiments to produce germline chimera from PGCs preserved using this techniques are strongly recommended.

키워드

참고문헌

  1. 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
  2. Bernard A, 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
  3. 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
  4. Fulton JE, Delany ME 2003 Poultry genetic resources-operation rescue needed. Science 300:1667-1668. https://doi.org/10.1126/science.1085407
  5. FAO 2003 World Watch List for Domestic Animal Diversity 3rd edition (Scherf BD ed.) Food and Agriculture Organization of the United Nation. Rome.
  6. Fahy GM, MacFarlane DR, Angell CA, Meryman HT 1984 Vitrification as an approach to cryopreservation. Cryobiology 21:407-426. https://doi.org/10.1016/0011-2240(84)90079-8
  7. Freshney RI 2005 Culture of animal cells: A manual of basic technique. 5th Edn Wiley-Liss Inc Hoboken.
  8. Friedler S, Giudice LC, Lamb EJ 1988 Cryopreservation of embryos and ova. Fertil Steril 49:743-764. https://doi.org/10.1016/S0015-0282(16)59879-3
  9. Gardner DK, Lane M 2000 Embryo culture systems In "Handbook of in vitro Fertilization". 2nd ed CRC Press, Boca Raton, FL 205-264.
  10. Hamburger V, Hamilton HL 1951 A series of normal stage in the development of the chick embryo. Journal of Morphology 8:49-92.
  11. Hammerstedt RH, Graham JK 1992 Cryopreservation of poultry sperm: the enigma of glycerol. Cryobiology 29:26- 38. https://doi.org/10.1016/0011-2240(92)90004-L
  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. Hotamisligil S, Toner M, Powers RD 1996 Changes in membrane intergrity, 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
  15. Kim JT, Lee SH, Lee YJ, Jo DI 2004 Utility of bolus suture and silicone protector in auricular reconstruction of microtia. J Korean Soc Plast Reconstr Surg 31:9-16.
  16. Kino K, Pain B, Leibo SP, Cochran M, Clark ME, Etches RJ 1997 Production of chicken chimeras from injection of frozen-thawed blastodermal cells. Poultry Sci 76:753- 760. https://doi.org/10.1093/ps/76.5.753
  17. Kobayashi T, Takeuchi Y, Yoshizaki G, Takeuchi T 2003 Cryopreservation of trout primordial germ cells. Fish Physiol Biochem 28:479-480. https://doi.org/10.1023/B:FISH.0000030634.37486.fa
  18. Lake PE, Stewart JM 1978 Preservation of fowl semen in liquid nitrogen an improved method. Br Poult Sci 19:187-197. https://doi.org/10.1080/00071667808416462
  19. 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
  20. Martino A, Songsasen N, Leibo SP 1996 Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. Biol Reprod 54:1059-1069. https://doi.org/10.1095/biolreprod54.5.1059
  21. Naito M, Nirasawa K, Oishi T 1992 Preservation of quail blastoderm cells in liquid nitrogen. Br Poult Sci 33:449-453. https://doi.org/10.1080/00071669208417482
  22. Naito M, Tajima A, Yasuda Y, Kuwana T 1994a Production of germline chimeric chickens, with high transmission rate of donor-derived gametes, produced by transfer of primordial germ cells. Mol Reprod Dev 39:153-161. https://doi.org/10.1002/mrd.1080390206
  23. Naito M, Tajima A, Tagami T, Yasuda Y, Kuwana T 1994b Preservation of chick primordial germ cells in liquid nitrogen and subsequent production of viable offspring. J Reprod Fertil 102:321-325. https://doi.org/10.1530/jrf.0.1020321
  24. Naito M 2003 Cryopreservation of avian germline cells and subsequent production of viable offspring. J Poult Sci 40:1-12. https://doi.org/10.2141/jpsa.40.1
  25. Nakagata N 1989 High survival rate of unfertilized mouse oocytes after vitrification. J Reprod Fertil 87:479-483. https://doi.org/10.1530/jrf.0.0870479
  26. Park TS, Jeong DK, Kim JN, Song GH, Hong YH, Lim JM, Han JY 2003 Improved germline transmission in chicken chimeras produced by transplantation of gonadal primordial germ cells into recipient embryos. Biol Reprod 68:1657-1662. https://doi.org/10.1095/biolreprod.102.006825
  27. Rall WF, Wood MJ, Kirby C, Whittimgham DG 1987 Development of mouse embryos cryopreserved by vitrification. J Reprod Fertil 80:499-504. https://doi.org/10.1530/jrf.0.0800499
  28. 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
  29. Sommerfeld V, Niemann H 1999 Cryopreservation of bovine in vitro produced embryos using ethylene glycol in controlled freezing or vitrification. Cryobiology 38:95-105. https://doi.org/10.1006/cryo.1999.2159
  30. Sutton RL 1991 Critical cooling rate to avoid ice crystallization in solutions of cryoprotective agents. J Chem Soc Faraday Trans 87:101-105. https://doi.org/10.1039/ft9918700101
  31. Tagami T, Kagami H, Matsubara Y, Harumi T, Naito M, Takeda K, Hanada H, Nirasawa K 2007 Differentiation of female primordial germ cells in the male testes of chicken (Gallus gallus domesticus). Mol Reprod Dev 74:68-75. https://doi.org/10.1002/mrd.20499
  32. Tajima A, Naito M, Yasuda Y, Kuwana T 1998 Production of germ-line chimeras by transfer of cryopreserved gonadal primordial germ cells (gPGCs) 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
  33. Vajta G, Holm P, Kuwayama M 1998 Open pulled straw (OPS) vitrification : a new way to reduce cryoinjuries of bovine ova and embryos. Mol Reprod Dev 51:53-58. https://doi.org/10.1002/(SICI)1098-2795(199809)51:1<53::AID-MRD6>3.0.CO;2-V