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KMID : 0894520000040020161
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Development & Reproduction
2000 Volume.4 No. 2 p.161 ~ p.173
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Effect of Different Infusion Frequency of Liquid Nitrogen on Actin Filament , Mitochondria , Apoptosis and Development in Mouse 2-Cell Embryo after Freezing and Thawing
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손인표
안학준/계명찬/최규완/민철기/강희규/이호준/권혁찬
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Abstract
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The aim of this study was to assess the effect of the frequency of the LN2 infusion on the ultrastructure, metabolism and development of the embryo after freezing and thawing by computerized cell freezer. Two-cell embryos of ICR mouse were randomly allocated into fresh (control), high-frequency freezing (group 1) and low-frequency freezing (group 2). For fresh and frozen-thawed intact 2-cell embryos, total ceil number in the blastocyst was counted by fluorescent microscope after Hoechst 33258 staining. Relative amount of H2O2 was measured by DCHFDA. Intracellular location and membrane potential of the mitochondria were evaluated by staining with rhodamine 123 and JC-1. The structure of actin filament was also evaluated by confocal microscope. DNA fragmentation was assessed by TUNEL method after development into blastocyst. The survival rate of intact embryo was higher in group 1 than group 2 (50.7％ vs. 34.6％ respectively, p<0.05). The blastocyst developmental rate was significantly low in group 2 (86.7％, 76.7％ vs. 44.0％ for control, group 1 and group 2 respectively, p<0.05). Total cell number in the blastocyst was also significantly lower in group 2 than control (79.5±12.9, 71.6±8.0, and 62.5±4.7 for control, group 1 and group 2 respectively, p<0.05). The relative amount of H2O2 was higher in group 2 than other groups (15.3±3.0, 16.6±1.6 vs. 23.4±1.8, p<0.05). After JC-1 staining, relative intensity of mitochondria with high membrane potential was significantly lower in group 2 than control and group 1 (17.2±3.8, 17.4±1.3 vs. 13.2±2.0, p<0.05). In group 2, partial deletion and aggregation of the actin filament was found. DNA fragmentation rate was also hieher for group 2 versus other groups (30.8％, 36.0％ vs. 65.6％, p<0.05). The frequency of the LN2 infusion is an important factor for the development of frozen-thawed mouse embryo. High-frequency infusion may prevent damages of cytoskeleton and mitochondria in the embryo probably by preventing the temperature fluctuation during dehydration phase. We speculate that the application of high-frequency infusion method in human embryo may be promising.
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KEYWORD
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