냉각 후 배양시간이 생쥐 난자의 방추체와 염색체에 미치는 영향 = Effect of Incubation Time after Cooling on the Meiotic Spindle and Chromosomes of Mouse Oocytes

국문 초록 (Abstract)

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

다국어 초록 (Multilingual 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 during the experiment. Oocytes were rapidly cooled to , held for 30 minutes, warmed and incubated at 37 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. Restoration of a barrel-shaped spindle and normal chromosome alignment was occurring after 5 minutes incubation at 37, 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.

참고문헌 (Reference)

1 "and non-equilibrium freezing of mammalian embryos" 53-92, 1990

2 "and developmental consequences of cryorpreservation of immature and mature mouse and human oocytes" 165-193, 1994

3 "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." 16 : 2350-2356, 2001

4 "Transient cooling to room temperature can cause irreversible disruption of the meiotic spindle in the human oocyte" 102-108, 1990

5 "The oxidizing agent tertiary butyl hydroperoxide induces distrubances in spindle organization, c-meiosis, and aneuploidy in mouse oocytes." 2 : 895-901, 1996

6 "The outcome of cryopreserved human embryos after intracytoplasmic sperm injection and traditional IVF" 16 : 405-409, 1999

7 "The influence of cooling on the organization of the meiotic spindle of the mouse oocyte" 2 : 207-216, 1987

8 "The effects of cooling mouse oocytes" 139-148, 1992

9 "The effect of temperature fluctuations on the cytoskeletal organization and chromosomal constitution of the human oocyte" 3 : 357-365, 1995

10 "Spindle organization after cryopreservation of mouse, human, and bovine oocytes." 8 : 664-672, 2004

11 "Spindle dynamics in living mouse oocytes during meiotic maturation, ageing, cooling and overheating: a study by polarized light microscopy" 241-249, 2004

12 "Regulation of tublin synthesis and cell cycle progression in mammalian cells by gamma-tublin-mediated microtuble nucleation" 84 : 472-483, 2002

13 "Oocyte freezing: here to stay?" 9 : 463-470, 2003

14 "Limited recovery of meiotic spindles in living human oocytes after cooling- rewarming observed using polarized light microscopy" 16 : 2374-2378, 2001

15 "Fundamental cryobiology of reproductive cells and tissues" 48 : 146-156, 2004

16 "Factors affecting low temperature survival of mammalian oocytes" 1992

17 "Evaluation of meiotic spindles in thawed oocytes after vitrification using polarized light microscopy" 82 : 666-672, 2004

18 "Effects of low temperatures on in-vitro matured bovine oocytes" 49 : 1155-1164, 1998

19 "Effects of cryopreservation on meiotic spindles of oocytes and its dynamics after thawing clinical implications in oocyte freezing-a review article" 202 : 101-107, 2003

20 "Effects of cooling and rewarming on the meiotic spindle and chromosomes of in vitro-matured bovine oocytes" 50 : 103-110, 1994

21 "Effects of cold and isoprophyl-N-phenylcarbamate on the second meiotic spindle of mouse oocytes" 699-707, 1980

22 "Effects of chilling to 0 on the morphology of meiotic spindles in human metaphase II oocytes" 75 : 769-777, 2001

23 "Effect of chilling on the organization of tublin and chromosomes in rhesus monkey oocytes" 77 : 818-825, 2002

24 "Effect of chilling bovine oocytes on their developmental competence" 45 : 503-512, 1996

25 "Diazepam induces meiotic delay, aneuploidy and predivision of homologous and chromatids in mammalian oocytes" 13 : 567-580, 1998

26 "Cryopreservation of mouse and human oocytes using 1" 2 1101-1109, 1993

27 "Cryopreservation of ICR mouse oocytes imporved post-thawed preimplantation development after vitrification using Taxol" 75 : 1171-1184, 2001

28 "Cryopreservation in human assisted reproduction is now routine for embryos but remains a research procedure for oocytes" 3 : 161-174, 1998

29 "Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes" 69 : 944-957, 1998