Journal of Embryo Transfer (한국수정란이식학회지)

The Korean Society of Embryo Transfer (한국수정란이식학회)
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Effect of Incubation Time after Cooling on the Meiotic Spindle and Chromosomes of Mouse Oocytes

냉각 후 배양시간이 생쥐 난자의 방추체와 염색체에 미치는 영향

  • Published : 2004.12.01
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Abstract

This study was conducted to determine the effects of incubation time after cooling on mouse meiotic spindle and chromosome alignment and the optimal incubation time for their restoration. Oocytes at the metaphase II were obtained from superovulated mice. Control oocytes were held at 37$^{\circ}C$ during the experiment. Oocytes were rapidly cooled to $0^{\circ}C$, held for 30 minutes, warmed and incubated at 37$^{\circ}C$ for 5, 15, 30, 60 and 120 minutes, respectively. The morphological features of spindle and chromosomes in oocytes were evaluated by immunofluorescent staining. Meiotic spindle of control oocytes exhibited a normal-looking bipolar configuration(barrel-shaped) and highly fluorescent microtubles. The chromosomes were clustered in a discrete bundles at metaphase plate. Disassembly of meiotic spindle and chromosome dispersion were occurred immediately after chilling of oocyte. Fluorescence intensity index(FIS), normal chromosomes aligned and normal spindle configuration were compared according to incubation time at 37$^{\circ}C$. Restoration of a barrel-shaped spindle and normal chromosome alignment was occurring after 5 minutes incubation at 37$^{\circ}C$, improved as a incubation time increased, and decreased gradually after 120 minutes incubation(P<0.05). The optimal incubation time for restoration of meiotic spindle and chromosomes in cooled oocytes was 60 minutes.

동결 과정 중 필수적인 단계중 하나인 냉각(cooling)과 냉각 후 배양시간이 생쥐 난자의 방추체의 형태와 염색체의 배열에 미치는 영향을 알아봄으로서 냉각 후 손상되었던 난자의 방추체와 염색체가 정상적으로 회복하는데 필요한 최적의 배양시간을 알아보기 위해 본 실험을 실시하였다. 생후 4-6주령의 암컷 B6C3Fl 생쥐를 과배란 처리하여 metaphase II상태의 난자를 회수하여 다음과 같이 처리하였다. 대조군은 난자를 냉각처리하지 않았으며 실험군은 난자를 $0^{\circ}C$에서 30분간 냉각한 후 37$^{\circ}C$에서 가온하여 즉시 일부 난자는 면역형광 염색을 실시하고 나머지 난자는 5% $CO_2$ 37$^{\circ}C$가 유지된 배양기내에서 Ml6 배지에 각각 5분, 15분, 30분, 60분, 120분간 배양한 후 면역 형광염색을 실시하였다. 난자의 방추체와 염색체를 평가하기 위한 면역형광염색은 Zenes 등의 방법(2001)에 준하여 실시하였다. 냉각처리하지 않은 생쥐 난자를 면역형광 염색하여 방추체와 염색체를 관찰한 결과 생쥐 metaphase II 상태의 난자는 대칭성의 원통모양의 방추체 형태를 보였으며 염색체는 metaphase plate위에 분리된 다발모양으로 밀집되어 보였다. 냉각 직후 미세관의 소실에 의한 방추체 형태의 이상과 형광성의 소실이 나타났으며 염색체는 다발모양의 밀집된 형상에서 벗어나 비정상적인 배열상을 보였다. 냉각 처리된 난자를 37$^{\circ}C$에서 가온하고 배양하였을 때 미세관의 재중합이 일어나 미세관의 형광성을 회복하기 시작하였고 방추체는 정상적인 배열상으로 회복되었다. 생쥐 난자를 냉각처리한 후 배양시간에 따른 방추체 미세관의 형광성(FIS), 염색체의 배열, 방추체의 형태를 비교하였다. 배양 5분에서 60분까지 FIS, 정상 염색체 배열을 보인 난자의 비율, 정상 방추체의 형태를 보인 난자의 비율이 점진적으로 증가하였으나 120분 배양에서는 감소하였다(P<0.05). 위의 세 가지 평가를 기준으로 하여 냉각 후 난자의 회복율을 관찰하였을 때 배양 60분에서 최상의 회복율을 나타냈다.

Keywords

References

  1. Almeida PA and Bolton VN. 1995. The effect of temperature fluctuations on the cytoskeletal organization and chromosomal constitution of the human oocyte. Zygote, 3:357-365 https://doi.org/10.1017/S0967199400002793
  2. Aman RR and Parks JE. 1994. Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocytes. Biol. Reprod., 50:103-110 https://doi.org/10.1095/biolreprod50.1.103
  3. Azambuja RM, Kraemer DC and Westhusin ME. 1998. Effects of low temperatures on in-vitro matured bovine oocytes. Theriogenology, 49: 1155-1164 https://doi.org/10.1016/S0093-691X(98)00063-6
  4. Chen SU, Lien YL, Cheng YY, Chen HF, Ho HN and Yang YS. 2001. Vitrification of mouse oocytes using closed pulled straws(CPS) achieves a high survival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws (OPS) and grids. Hum. Reprod., 16:2350-2356 https://doi.org/10.1093/humrep/16.11.2350
  5. Chen SU, Lien YR, Lien KH, Chao HN, Ho YS, Yang YS and Lee TY. 2003. Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing: clinical implications in oocyte freezing-a review article. Mol. Cell. Endocrinol., 202:101-107 https://doi.org/10.1016/S0303-7207(03)00070-4
  6. Chen CK, Wang CW, Tsai WJ, Hsieh LL, Wang HS and Soong YK. 2004. Evaluation of meiotic spindles in thawed oocytes after vitrification using polarized light microscopy. Fertil. Steril., 82:666-672 https://doi.org/10.1016/j.fertnstert.2003.12.053
  7. Emiliani S, Van den Bergh M, Vannin AS, Biramane J and Englert Y. 1999. The outcome of cryopreserved human embryos after intracytoplasmic sperm injection and traditional IVF. J. Assist. Reprod. Genet., 16:405-409 https://doi.org/10.1023/A:1020561307110
  8. Eroglu A, Toth TL and Toner M. 1998. Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes. Fertil. Steril., 69:944-957 https://doi.org/10.1016/S0015-0282(98)00030-2
  9. Gook DA, Osborn SM and Johnston WIH. 1993. Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration and the meiotic spindle. Hum. Reprod., 8:1101-1109 https://doi.org/10.1093/oxfordjournals.humrep.a138201
  10. Magistrini M and Szollosi D. 1980. Effects of cold and isoprophyl-N-phenylcarbamate on the second meiotic spindle of mouse oocytes. Eur. J. Cell. Biol., 22:699-707
  11. Mandelbaum J, Belaisch-Allart J, Junca AM, Antoine JM, Plachot M, Alvarez S, Alnot MO and Salat-Baroux J. 1998. Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes. Hum. Reprod. Suppl., 3:161-174
  12. Martino A, Pollard JW and Leibo SP. 1996. Effect of chilling bovine oocytes on their developmental competence. Biol. Reprod., 45:503-512
  13. Mazur P. 1990. Equilibration, quasi-equilibration, and non-equilibrium freezing of mammalian embryos. Cell. Biophys., 17:53-92 https://doi.org/10.1007/BF02989804
  14. Parks JE and Ruffing NA. 1992. Factors affecting low temperature survival of mammalian oocytes. Theriogenology, 37:59-73 https://doi.org/10.1016/0093-691X(92)90247-O
  15. Parks SE, Chung HM, Cha KY, Hwang WS, Lee ES and Lim JM. 2001. Cryopreservation of ICR mouse oocytes: imporved post-thawed preimplantation development after vitrification using Taxol, a cytoskeleton stabilizer. Fertil. Steril., 75:1171-1184
  16. 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 oocyte. Fertil. Steril., 54:102-108 https://doi.org/10.1016/S0015-0282(16)53644-9
  17. Pickering SJ and Johnson MH. 1987. The influence of cooling on the organization of the meiotic spindle of the mouse oocyte. Hum. Reprod., 2: 207-216 https://doi.org/10.1093/oxfordjournals.humrep.a136516
  18. Sathananthan AH, Kirby C, Trouson A, Philipatos D and Shaw J. 1992. The effects of cooling mouse oocytes. J. Assist. Reprod. Genet., 9: 139-148 https://doi.org/10.1007/BF01203754
  19. Songsasen N, Yu IJ, Ratterree MS, Vandevoort CA and Leibo. 2002. Effect of chilling on the organization of tublin and chromosomes in rhesus monkey oocytes. Fertil. Steril., 77:818-825 https://doi.org/10.1016/S0015-0282(01)03240-X
  20. Stachecki JJ, Munne S and Cohen J. 2004. Spindle organization after cryopreservation of mouse, human, and bovine oocytes. Reprod. Biomed. Online, 8:664-672 https://doi.org/10.1016/S1472-6483(10)61647-1
  21. Sun XF, Zhang WH, Chen XJ, Xiao GH, Mai WY and Wang WH. 2004. Spindle dynamics in living mouse oocytes during meiotic maturation, ageing, cooling and overheating: a study by polarized light microscopy. Zygote, 12:241-249 https://doi.org/10.1017/S0967199404002850
  22. Tarin JJ, Vendrell FJ, Ten J, Blanes R, van Blerkom J and Cano A. 1996. The oxidizing agent tertiary butyl hydroperoxide induces distrubances in spindle organization, c-meiosis, and aneuploidy in mouse oocytes. Mol. Hum. Reprod., 2:895-901 https://doi.org/10.1093/molehr/2.12.895
  23. Van Blerkom J and Davis PW. 1994. Cytogenetic, cellular, and developmental consequences of cryorpreservation of immature and mature mouse and human oocytes. Microsc. Res. Tech., 27: 165-193 https://doi.org/10.1002/jemt.1070270209
  24. Van der Elst J. 2003. Oocyte freezing: here to stay? Human. Reprod. Update., 9:463-470 https://doi.org/10.1093/humupd/dmg032
  25. Wang WH, Meng L, Hackett RJ, Odenbourg R and Keege DL. 2001. Limited recovery of meiotic spindles in living human oocytes after cooling- rewarming observed using polarized light microscopy. Hum. Reprod., 16:2374-2378 https://doi.org/10.1093/humrep/16.11.2374
  26. Woods EJ, Benson JD, Agca Y and Critser JK. 2004. Fundamental cryobiology of reproductive cells and tissues. Cryobiology, 48:146-156 https://doi.org/10.1016/j.cryobiol.2004.03.002
  27. Yin H, Baart E, Betzendahl I and Eichenlaub-Ritter U. 1998. Diazepam induces meiotic delay, aneuploidy and predivision of homologous and chromatids in mammalian oocytes. Mutagenesis, 13:567-580 https://doi.org/10.1093/mutage/13.6.567
  28. Zenzes MT, Bielecki R, Casper RF and Leibo SP. 2001. Effects of chilling to 0$^{\circ}C$ on the morphology of meiotic spindles in human metaphase II oocytes. Fertil. Steril., 75:769-777 https://doi.org/10.1016/S0015-0282(00)01800-8
  29. Zhou J, Shu HB and Joshi HC. 2002. Regulation of tublin synthesis and cell cycle progression in mammalian cells by gamma-tublin-mediated microtuble nucleation. J. Cell. Biochem., 84: 472-483 https://doi.org/10.1002/jcb.10033