• Journal of Embryo Transfer
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• v.25 no.4
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• pp.251-258
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• 2010

In the present study, effects of concentration and time of culture in presence of roscovitine on nuclear maturation and meiotic spindle configuration, chromosomal alignment were examined in porcine oocytes. In experiment 1, porcine cumulus oocyte complexes (COCs) were cultured at $39^{\circ}C$ in a 5% $CO_2$ atmosphere in North Carolina State University 23 (NCSU-23) supplemented with 25, 50, 75 or $100\;{\mu}M$ roscovitine for 22 h and then were cultured for additional 22 h after removal of roscovitine. Nuclear maturation and morphology of the meiotic spindle and chromosomal alignment were examined to determine the optimal concentration of roscovitine in oocyte maturation. In experiment 2, COCs were cultured in NCSU-23 supplemented with $50\;{\mu}M$ roscovitine for 17, 20, 27 or 42 h and then an additional 22 h without roscovitine was followed to determine the optimal time of culture. The optimal concentration of roscovitine to arrest and resume meiosis of porcine oocyte was $50\;{\mu}M$ by examining nuclear status (p<0.05) and normal spindle and chromosome configuration. The optimal time of culture in presence of roscovitine to arrest meiosis of porcine oocyte was 17 h (p<0.05), although MII rates and normal morphology of the meiotic spindle and chromosomal alignment were not significantly different among various times of culture. In conclusion, the optimal concentration and time of culture in presence of roscovitine to arrest porcine oocytes are $50\;{\mu}M$ and 17 h, respectively.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.283-289
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• 2004

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.

• Development and Reproduction
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• v.18 no.2
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• pp.117-125
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• 2014

Vitrification method is widely used in oocyte cryopreservation for IVF but the birth rates are lower than that of the fresh oocyte. One of the known main reasons is structural instability of meiotic spindle and chromosome systems of mature oocyte. To get the best way for keeping competence of matured oocytes, we studied the best conditions for vitrification focused on equilibration times. The mature oocytes were underwent vitrification with current popular method and analyzed the survival rates, microtubule stability and DNA integrity. The survival rates of recovered oocyte are almost same between groups and are more than 93%. The structural configuration of meiotic spindle was well kept in 10 min equilibration group and the stability rate was almost same with that of control. The chromosomal breakdown was observed in all experimental groups, but the chromosomal stability was higher in 10 min equilibration group than the other groups. The 10 min equilibration group showed best condition compared with the other groups. Based on these results, the equilibration time is one of the key factors in successful keeping for competence of mature oocyte. Although, more fine analysis about the effects of physical stress on oocyte during vitrification is needed to define the optimal condition, it is suggested that the optimal equilibration time to get competent oocyte in mouse is 10 min. Information acquired this study may provide insight into intracellular structural events occurring in human oocytes after vitrification and application for cryopreservation of human oocyte.