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.

This study was conducted to examine the effect of oocyte donor age and micromanipulation medium on the development of mouse cloned embryos receiving cumulus cells. Mouse oocytes were obtained from 6 to 11 week-old mice BDF1 female mice(experiment 1) and cumulus cells were used as donor cells. Micromanipulation procedures for nuclear transfer(NT) were performed in FHM, M2 or Hepes-buffered TCM199(TCM199) medium(experiment 2). After nuclear transfer, the reconstructed oocytes were activated by 10 mM SrCl2 in Ca-free CZB medium in the presence of 5 μg/ml cytochalasin B for 5 h and cultured in KSOM medium for 4 days. In experiment 1, the survival rate of oocytes after injection of cumulus cells were significantly(p<0.05) lower in oocytes from 6～7 week-old mice(53.3%) than in oocytes from 8～9(80.9%) and 10～11 week-old mice(77.1%). In experiment 2, the survival rate of oocytes after cell injection were significantly(p<0.05) higher in FHM and M2 medium(71.7% and 76.9%) than in TCM199 medium (51.2%). The activation rates of cloned embryos were not different among the micromanipulation media. However, the embryos developed to blastocyst stage were significantly(p<0.05) higher in FHM medium(13.9%) than in M2 and TCM199 medium(0.0% and 0.0%). In conclusion, the present study suggest that oocytes from above 8 week-old mice are superior to oocytes from 6～7 week-old mice as a source of recipient cytoplasm and FHM is superior to M2 and TCM199 as a micromanipulation medium for mouse somatic cell cloning.

The overexpression of Phosphoprotein Enriched in Astrocytes (PEA15) gene is commonly found in human diabetic patients. The overexpression of this gene in skeletal muscle and fat tissues have been reported to cause insulin resistance, thereby impairing insulin stimulated glucose uptake. We introduced a gene of mouse PEA15 (mPEA15) and enhanced green fluorescent protein (EGFP) into fertilized one cell pig zygotes using microinjection, and produced a piglet that showed overexpression of mPEA15 in the muscle tissues and expression of EGFP in the ear tissues and hooves. RT-PCR RFLP, southern blot and FISH analysis showed that the tissues carried the transgene. Real-time RT-PCR and western blots demonstrated that PEA15 gene was overexpressed in the various tissues and muscle tissues, respectively. These facts suggest that expression vector system is normally expressed in the trnasgenic (TG) pigs. To use as animal diseases model for type 2 diabetes, further study is necessary to confirm whether diabetes occur in these TG pigs, especially insulin resistance.