Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
권호 표지

Study on the Vitrification of Porcine GV and M II Oocytes after Removal of Cytoplasmic Lipid Droplets

세포질 내 지방구 제거가 돼지 난포란의 유리화 동결에 미치는 영향

  • Published : 2003.04.01
Download PDF
⟨ Previous Next ⟩

Abstract

This study was conducted to investigate that the immature and mature oocytes of porcine can be cryopreserved by vitrification. Oocytes were centrifuged to polarize the cytoplasmic lipid droplets. The lipids were removed from cytoplasm by micromanipulation. Delipated oocytes were centrifuged after being preincubated with cytochalasin B(CB) fer 10 min, and lipid droplets were removed. Centrifuged oocytes were treated with CB and centrifuged to polorize lipid droplets but not delipated and control oocytes is not-treatment. Oocytes of three types were vitrified in electron microscope(EM) grids. The results of survival, maturation and cleavage rates were as follows. 1 The survival rates of immature oocytes were 15.1%, 0% and 0% in the Delipated, Centrifuged and Control after vitrification, respectively, and its rate of Delipated wassignificantly higher than Centrifuged and Control(P<.01). 2. The survival rates of mature oocytes were 12.21%, 0% and 0% in the Delipated, Centrifuged and Control after vitrification, respectively, and its rate of Delipated was significantly higher than Centrifuged and Control(P<.01). 3 The maturation rates of immature oocytes were 37.5% and 68.9% for metaphase II in the Delipated after vitrification and Non-vitrification, respectively, and its rate of Non-vitrification was significantly higher than Delipated after vitrification(P<.01). 4. The cleavage rates of immature oocytes were 12.5%, 0%, 0% and 56.1% in the Delipated, Centrifuged, Control after vitrification and Non-vitrification, respectively. It's rate of Delipated was higher than Centrifuged and Control, but there were no significant difference, and its rate of Non-vitrification was significantly higher than Delipated, Centrifuged and Control(P<.05). 5 The cleavage rates of mature oocytes were 25.0%, 0%, 0% and 67.9% in the Delipated, Centrifuged, Control after vitrification and Non-vitrification, respectively. It's rate of Delipated was higher than Centrifuged and Control, but there were no significant difference, and its rate of Non-vitrification was significantly higher than Delipated, Centrifuged and Control(P<.05).

본 연구는 미성숙, 성숙 단계의 돼지 난포란이 유리화 동결에 의한 동결보존이 가능한지를 조사 하고자 실시하였다. 난포란은 세포질 내 지방구를 분극시키기 위해 원심분리를 실시하였고, 미세조작기를 이용하여 지방구를 제거하였다. 돼지 난포란을 CB 처리하여 원심분리 후 지방구를 제거한 지방제거구(Delipated), CB 처리 후 원심분리만 하여 지방구를 분극시킨 원심분리구(Centrifuged), 아무처리도 하지 않은 대조구(Control)를 EM grid를 이용한 유리화 동결, 융해 한 후 생존율과 체 외 발생율을 조사하였다. 그 결과를 요약하면 다음과 같다. 1. 미성숙 시기에 지방제거, 원심분리, 무처리 후 유리화 동결을 실시한 돼지 난포란의 생존율은 각각 15.1%, 0%, 0%로 지 방제 거구에서만 유의적으로 높은 생존율을 나타내었다(p<.01). 2. 성숙 시기에 지방제거, 원심분리, 무처리 후 유리화 동결을 실시한 돼지 난포란의 생존율은 각각 12.2%, 0%, 0%로 지방제거구에서만 유의적으로 높은 생존율을 나타내었다(P<.01). 3. 미성숙 시기에 지방제거 후 유리화 동결을 실시하여 생존한 난자치 핵 성숙율은 37.5%로 동결하지 않은 난포란의 성숙율 68.9%보다 유의적으로 낮은 것이었다(P<.01). 4. 미성숙 시기에 지방제거, 원심분리, 무처리 후 유리화 동결을 실시하여 생존한 난자의수정 후 배 발달율은 12.5%, 0%, 0%로 지방 제거구에서만 배 발달을 나타었으나, 세 처리간의 유의차는 없었고, 이것은 동결하지 않은 난포란의 배 발달율 56.1%보다는 유의 적으로 낮은 것이었다(P<.05). 5. 성숙 시기에 지방제거, 원심분리, 무처리 후 유리화 동결을 실시하여 생존한 난자의 수정 후 배 발달율은 25.0%, 0%, 0%로 지방제거구에서만 배 발달을 나타내었으나 세 처리간의 유의차는 없었고, 이것은 동결하지 않은 난포란의 배 발달율 67.9%보다는 유의적으로 낮은 것이었다(P<.05).

Keywords

References

  1. Abeydeera LR and Day BN. 1997. In vitro penetration of pig oocytes in a modified trisbuffered medium: effect of BSA, caffeine and calcium. Theriogenology, 48:537-544 https://doi.org/10.1016/S0093-691X(97)00270-7
  2. Aigner S, Van der Elst J, Siebzehnrubl E, Wildt L and Lang N. 1992. The influence of slow and ultra-rapid freezing on the organization of the meiotic spindle of the mouse oocyte. Human Reprod., 7:857-864 https://doi.org/10.1093/oxfordjournals.humrep.a137750
  3. Al-Hasani S, Diedrich K, Van der Ven H, Reinecke A and Krebs D. 1987. Cryopreservation of human oocytes. Human Reprod., 2:695-700 https://doi.org/10.1093/oxfordjournals.humrep.a136616
  4. Baka SG, Toth TL, Veeck LL, Jones HWJ, Muasher SJ and Lanzendorf SE. 1995. Evaluation of the spindle apparatus of in vitro matured human oocytes following cryopreservation. Human Reprod., 10:1816-1820 https://doi.org/10.1093/oxfordjournals.humrep.a136182
  5. Chung HM, Hong SW, Lim JM, Lee SH, Cha WT, Ko JJ, Han SY, Choi DH and Cha KY. 2000. In vitro blastocyst formation of human oocytes retrieved from unstimulated and stimulated cycles following vitrification at various maturational stages. Fertil. Steril., 73: 545-551 https://doi.org/10.1016/S0015-0282(99)00546-4
  6. Edidin M and Petit VA. 1977. The effect of temperature on the lateral diffusion of plasma membrane protein. In: Elliott K, Whelan J(eds.) The Freezing of Mammalian Embryos. Amsterdam: Elsevier, Excerpta Medica. 155-174
  7. Fuku E, Kojima T, Shioya Y, Marcus GJ and Downey BR. 1992. In vitro fertilization and development of frozen-thawed bovine oocytes. Cryobiology, 29:485-492 https://doi.org/10.1016/0011-2240(92)90051-3
  8. Gook DA, Osborn SM, Bourne H and Johnson WIH. 1994. Fertilization of human oocytes following cryopreservation: normal karyotypes and absence of stray chromosomes. Human Reprod., 9:684-691 https://doi.org/10.1093/oxfordjournals.humrep.a138572
  9. Gook DA, Osborn SM and Johnson WIH. 1993. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle. Humam Reprod., 9:1101-1109
  10. Hamlett DK, Franken DR, Cronje HS and Luus H. 1989. Murine oocyte cryopreservation: Comparison between fertilization success rates of fresh frozen metaphase I and II oocytes. Arch. Androl., 23:27 https://doi.org/10.3109/01485018908986785
  11. Hochi S, Akira K, Ken K and Akira H. 1997. In vitro fertilizing ability of bovine oocyte frozen-thawed at immature, maturing and mature stage. J. Mmalian Ova Res., 14:61-65 https://doi.org/10.1274/jmor.14.61
  12. Hong SW, Chung HM, Lim JM, Ko JJ, Yoon TK, Yee B and Cha KY. 1999. Improved human oocyte development after vitrification: a comparison of thawing methods. Fertil. Steril., 72:142-146 https://doi.org/10.1016/S0015-0282(99)00199-5
  13. Hotamisligil S, Toner M and Power R. 1996. Change in membranne integrity, cytoskeletal structure and developmental potential of murine oocytes after vitrification in ethylene glycol. BioI. Reprod., 55:161-168 https://doi.org/10.1095/biolreprod55.1.161
  14. Leibo SP. 1977. Fundamental biology of mouse ova and embryos. In: Elliott K., Whelan J. (eds.) The Freezing of Mammalian Embryos. Amsterdam: Elsevier, Excerpta Medica. 69-92
  15. Leibo SP and Loskutoff NM. 1993. Cryobiology of in vitro-derived bovine embryos. Theriogenology, 39:81-94 https://doi.org/10.1016/0093-691X(93)90025-Z
  16. Lim JM, Fukui Y and Ono H. 1991. The post-thaw developmental capacity of frozen bovine oocytes following in vitro maturation and fertilization. Theriogenology, 35:1225-1235 https://doi.org/10.1016/0093-691X(91)90368-N
  17. Lim JM, Fukui Y and Ono H. 1992. Developmental competence of bovine oocytes frozen at various maturation stages followed by in vitro maturation and fertilization. Theriogenology, 37:351-362 https://doi.org/10.1016/0093-691X(92)90193-U
  18. Mahmoudzadeh AR, van Soom A, Ysebaert MT and De Kruif A. 1994. Comparison of twostep vitrification versus controlled freezing on survival of in vitro produced cattle embryos. Theriogenology, 42: 1389-1397 https://doi.org/10.1016/0093-691X(94)90259-L
  19. Mandelbaum J, Junca AM, Placho M and Alnot MD. 1987. Cryopreservation of human embryos and oocytes. Human Reprod., 3:117
  20. Martino A, Songsasen N and Leibo SP. 1996. Development into blastocysts of bovine oocytes cryopreserved by ultra-rapid cooling. BioI. Reprod., 54: 1059-1069 https://doi.org/10.1095/biolreprod54.5.1059
  21. Men H, Chen W, Shang E, Yang S and Zou R. 1997. Cryopreservation of mouse oocytes using slow cooling, ultra-rapid cooling and vitrification protocols. Theriogenology, 47: 1423-1431 https://doi.org/10.1016/S0093-691X(97)00133-7
  22. Mohr LR and Trounson AO. 1981. Structural changes associated with freezing of bovine embryos. BioI. Reprod., 25:1009-1025 https://doi.org/10.1095/biolreprod25.5.1009
  23. Nagashima H, Cameron RDA, Kuwayama M, Young M, Beebe L, Blackshaw AW and Nottle MB. 1999. Survival of porcine delipated oocytes and embryos after cryopreservation by freezing or vitrification. J. Reprod. Dev., 45(2):167-176 https://doi.org/10.1262/jrd.45.167
  24. Nagashima H, Kashiwazaki N, Ashman RJ, Grupen CG, Seamark RF and Nottle MB. 1994. Removal of cytoplasmic lipid enhances the tolerance of porcine embryos to chilling. BioI. Reprod., 51:618-622 https://doi.org/10.1095/biolreprod51.4.618
  25. Nagashima H, Kato Y, Yamakawa H, Matsumoto T and Ogawas S. 1989. Changes in freezing tolerance of pig blastocysts in peri-hatching stage Jpn. J. Anim. Reprod., 35:130-134 https://doi.org/10.1262/jrd1977.35.130
  26. Nagashima H, Kuwayama M, Grupen CG, Ashman RJ and Nottle MB. 1996. Vitrification of porcine early cleavage stage embryos and oocytes after removal of cytoplasmic lipid droplets. Theriogenology, 45(1):180 https://doi.org/10.1016/0093-691X(96)84653-X
  27. Nagashima H, Yamakawa H and Niemann H. 1992. Freezability of porcine blastocysts at different peri-hatching stages. Theriogenology, 37:839-850 https://doi.org/10.1016/0093-691X(92)90045-S
  28. Niimura S and Ishida K. 1980. Histochemical observation of lipid droplets in mammalian eggs during the early development. Jpn. J. Anim. Reprod., 26:46-49 https://doi.org/10.1262/jrd1977.26.46
  29. Norberg HS. 1973. Ultrastructural aspects of the preattached pig embryo : cleavage and early blastocyst stages. Z Anat. Entwicklung sgesch, 143:95-114 https://doi.org/10.1007/BF00519913
  30. Park SE, Lee KA, Son WY, Ko JJ, Lee SH and Cha KY. 1997. Chromosome and spindle configuration of human oocytes maturated in vitro after cryopreservation at the germinal vesicle stage. Fertil. Steril., 68(5):920-926 https://doi.org/10.1016/S0015-0282(97)00365-8
  31. Parkening TA, Tsunoda Y and Chang MC. 1976. Effects of various low temperatures, cryoprotective agents and cooling rates on the survival, fertilizability and development of frozen-thawed mouse egg. J. Exp. Zool., 369-374
  32. Parks JE and Ruffing NA. 1992. Factors affecting low temperature survivals of mammalian oocytes. Theriogenology, 37:59-73 https://doi.org/10.1016/0093-691X(92)90247-O
  33. Pellicer A, Lightman A, Parmer TG, Behrman HR and De Cherney AH. 1988. Morphologic and functional studies of immature rat oocytecumulus complexes after cryopreservation. Fertil. Steril., 50:805-810 https://doi.org/10.1016/S0015-0282(16)60320-5
  34. Pickering SJ, Braude PR, Johnson MH, Cant A and Currie J. 1990. Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocytes. Fertil. Steril., 54:102-108 https://doi.org/10.1016/S0015-0282(16)53644-9
  35. Polge C. 1977. The freezing of mammalian embryos: perspectived and possibilities. In: Elliott K, Whelan J(eds.), The Freezing of Mammalian Embryos. Amsterdam: Elsevier, Excerpta Medica, 3-18
  36. Pollard JM and Leibo SP. 1994. Chilling sensitivity of mammalian embryos. Theriogenology, 41: 101-106 https://doi.org/10.1016/S0093-691X(05)80054-8
  37. Quinn P, Kerin J, Stone B and Wilson L. 1986. Successful cryopreservation of human oocytes. In: 42nd Ann Mtg Am Fertil Soc and 18th Ann Mtg Canadian Fertil Androl Soc Abstr, 72(Abst)
  38. Rall WF. 1992. Cryopreservation of oocytes and embryos; Methods and applications. Animal Reprod. Science, 28:237-245 https://doi.org/10.1016/0378-4320(92)90110-Y
  39. 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
  40. Rall WF, Wood MJ, Kirby C and Whittingham DG. 1987. Development of mouse embryos cryopreserved by vitrification. J. Reprod. Fert., 80:499-504 https://doi.org/10.1530/jrf.0.0800499
  41. Rubinsky B, Arav A and Devires AL. 1991. Cryopreservation of oocytes using directional cooling and antifreeze glycoproteins. CryoLetters, 12:93-106
  42. Sathanathan AH, Ng, SC, Trounson AO, Bongso A, Ratnam SS and Ho J. 1988. The effect of ultrarapid freezing on meiotic spindles of mouse oocytes and embryos. Gamete Res., 21:385-401 https://doi.org/10.1002/mrd.1120210407
  43. Schmidt M, Hyttle P, Greve T and Avery B. 1993. Ultrastructure of frozen thawed bovine in vitro matured oocytes. Theriogenology, 39:304 https://doi.org/10.1016/0093-691X(93)90159-3
  44. Schellander K, Peli J, Schmoll F and Brem G. 1994. Effects of cryopreservation and carbohydrates on freezing of matured and unmatured bovine oocytes. Theriogenology, 23:909-915
  45. Schroeder AC, Chanplin AK, Mobraaten LE and Eppig JJ. 1990. Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro. J. Reprod. Fert., 89:43-50 https://doi.org/10.1530/jrf.0.0890043
  46. Son WY, Ko JJ, Park SE, Yoon TK, Lee KA, Cha KY and Lee WS. 1996. Effects of 1,2 -propanediol and freezing-thawing on the in vitro developmental capacity of human immature oocytes. Fertil. Steril., 66:995-999 https://doi.org/10.1016/S0015-0282(16)58696-8
  47. Suzuki T and Nishikata Y. 1992. Fertilization and cleavage of frozen thawed bovine oocytes by one step dilution method in vitro. Theriogenology, 37:306 https://doi.org/10.1016/0093-691X(92)90375-2
  48. Toner M, Cravalho EG, Ebert KM and Overstrom EW. 1986. Cryobiophysical properties of porcine embryos. BioI. Reprod., 34(suppl. 1):98 (absract)
  49. Trounson AO, Willadsen SM, Rowson LEA and Newcomb R. 1976. The storage of cow eggs at room temperature. J. Reprod. Fert., 46:173-178 https://doi.org/10.1530/jrf.0.0460173
  50. Tsunoda Y, Parkening TA and Chang MC. 1976. In vitro fertilization of mouse and hamster egg after freezing and thawing. Experientia, 32: 223-224 https://doi.org/10.1007/BF01937777
  51. Ushijima H, Yamakawa H and Nagashima H. 1996. Cryopreservation of bovine IVM / IVF embryos at early cleavage stage following removal of cytoplasmic lipid droplets. Theriogenology, 45(1):159 https://doi.org/10.1016/0093-691X(96)84632-2
  52. Vajta G, Booth PJ, Holm P, Greve T and Callesen H. 1997a. Successful vitrification of early stage bovine in vitro produced embryos with the open pulled straw(OPS) method. CryoLetters, 18:191-195
  53. Vajta G, Holm P, Greve T and Callesen H. 1997b. Vitrification of porcine embryos using open pulled straw method. Acta. Vet. Scand., 38: 349-352
  54. Van Blerkom J. 1989. Maturation at high frequency of germinal vesicle-stage mouse oocyte after cryopreservation: alterations in cytoplasmic, nuclear, nucleolar and chromosomal structure and organization associated with vitrification. Human Reprod., 4:883-893 https://doi.org/10.1093/oxfordjournals.humrep.a137006
  55. Van Blerkom J and Davis PW. 1994. Cytogeneic, cellular and developmental consequences of cryopreservation of immature and mature mouse and human oocytes. Micro Res. and Tech., 27:165-193 https://doi.org/10.1002/jemt.1070270209
  56. Van der Elst J, Nerinckx S and Van Steirtghem A. 1992. In vitro maturation of mouse germinal vesicle-stage oocytes following cooling, exposure to cryoprotectants and ultrarapid freezing; limited effect on the morphology of the second meiotic spindle. Human. Reprod., 7: 1140-1446
  57. Van der Elst J, Van den Abbeel E, Jacobs R, Wisse E and van Steirtghem A. 1988. Effect of 1,2-propanediol and dimethylsulphoxide on the meiotic spindle of the mouse oocyte. Human Reprod., 3:960-967 https://doi.org/10.1093/oxfordjournals.humrep.a136826
  58. Vincent C, Pickering SJ, Johnson MN and Quick SJ. 1990. Dimethylsulfoxide affects the organization of microfilaments in the mouse oocytes. Mol. Reprod. Dev., 26:227-235 https://doi.org/10.1002/mrd.1080260306
  59. Whittngham DG. 1977. Fertilization in vitro and development to term of unfertilized mouse oocyte previously stored at - $196^{\circ}C$. J. Reprod. Fertil., 7:29(Abst.)
  60. Whittingham DG, Leibo SP and Mazur P. 1972. Survival of mouse embryos frozen to - $196^{\circ}C$ an - $269^{\circ}C$. Science, 178:411-414 https://doi.org/10.1126/science.178.4059.411
  61. Wood MJ, Whittingham DG and William FR. 1987. The low temperature preservation of mouse oocytes and embryos. In: Monk M (ed.), Mammalian Development. IRL Press, Oxford, England. 255-258
  62. Zhao J, Hattori M and Fujihara N. 1997. Ultrastructural comparison between immature and in vitro matured bovine oocyte cryopreserved in propanediol. J. Mamm. Ova Reserch, 14:84-94 https://doi.org/10.1274/jmor.14.84
  63. 최인경, 송해범. 1998. 각 성숙단계에서 동결, 융해한 돼지 난포란의 발달능력에 관한 연구. 한국가축번식학회지, 22(4):319-329