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KMID : 1023620110350030319
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Reproductive and Developmental Biology
2011 Volume.35 No. 3 p.319 ~ p.327
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The Effect of Porcine Sperm Cytosolic Factor (SCF) on In Vitro Development of Porcine PA and NT Embryos
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Shim Joo-Hyun
Kim Dong-Hoon
Ko Yeoung-Gyu
Hwang Seong-Soo
Oh Keon-Bong
Yang Boh-Suk
Jin Dong-Il
Park Jin-Ki
Im Gi-Sun
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Abstract
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This study investigated whether the addition of porcine sperm cytosolic factor (SCF) at fusion/activation affects in vitro development of porcine parthenogenetic(PA) and nuclear transfer (NT) embryos. To determine the optimum concentration of SCF, control group of oocytes was activated with 0.3M mannitol (1.0 mM CaCl2 ? 2H2O), other three groups of oocytes were parthenogentically activated with the fusion medium (0.1mM CaCl2 ? 2H2O) supplemented with 100, 200 or 300 μg/ml SCF, respectively. Matured oocytes were activated with two electric pulses (DC) of 1.2 kv/cm for 30 μsec. The activated embryos were cultured in PZM-3 under 5% CO2 in air at 38.5℃ for 6 days. Oocytes activated in the presence of SCF showed a significantly higher blastocyst rate than control (p<0.05). Apoptosis rate was significantly lower in 100 μg/ml SCF group than other groups (p<0.05). Cdc2 kinase activity in control and SCF treatment group of oocytes was determined using MESACUP cdc2 kinase assay kit at 1, 5, 10, 15, 30, 45 and 60 min after activation. Cdc2 kinase activity was significantly decreased (p<0.05) in SCF group than MII oocytes or control within 5 min. For NT embryo production, reconstructed oocytes were fused in the fusion medium supplemented with 0.1 mM CaCl2 ? 2H2O (T1), 1.0 mM CaCl2 ? 2H2O (T2) and 0.1 mM CaCl2 ? 2H2O with 100 μg/ml SCF (T3). Fused embryos were cultured in PZM-3 under 5% CO2 in air at 38.5℃ for 6 days. Developmental rate to blastocyst stage was significantly higher in T3 than other groups (23.0% vs. 13.5 to 15.2%) (p<0.05). Apoptosis rate was significantly lower in T3 than T1 or T2 (p<0.05). The relative abundance of Bax-α/Bcl-xl was significantly lower in in vivo or SCF group than that of control (p<0.05). Moreover, the expression of p53 and caspase3 mRNA was significantly lower in in vivo or SCF group than that of control (p<0.05). These results indicate that the addition of SCF at fusion/activation might improve in vitro development of porcine NT embryos through regulating cdc2 kinase level and expression of apoptosis related genes.
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KEYWORD
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SCF, Porcine NT embryo, Developental ability, Cdc2 kinase activity
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