• Korean Journal of Animal Reproduction
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• v.23 no.1
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• pp.85-92
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• 1999

This study was designed to determine effect of cryoprotectant kinds and cell stages on OPP vitrification method in mouse embryos. The freezing speed, cryoprotectants and cell stage could affect of embryo viability following various vitrification methods. The vitrification solution used were consisting of 40% (v/v) ethylene glycol, 18% (w/v) Ficoll, 0.3 M sucrose solution in holding medium (D-PBS supplemented with 5% FCS: HM) (EFS) or 16.5% ethylene glycol , 16.5% dimethyl sulfoxide, 0.5 M sucrose in HM (EDS). The embryos were collected from oviduct at 18 h after hCG injection and then washed and cultured in mHTF medium until use. In experiment 1, the blastocysts were vitrified by OPP straw to determine the optimal vitrification solution of EFS or EDS. The post-thaw survival rates at re-expanded stage rates were significantly different between EFS and EDS (95.0 vs 100%), but at hatching stage was not different between EFS and EDS (90.0 vs 95.0%). respectively. In experiment 2, zygotes, 2-, 4-cell, morula and blastocysts were vitrified by OPP method to determine the acceptable of early stage embryos. The development rates to expanded blastocyst in zygote (70.0%) were significantly lower rather than those in 2-, 4- 8-cell, compacted morula or blastocyst (89.7, 90.0, 92.8, 97.6 or 97.5%), respectively. However, the cell number of post-thaw developed to expanded blastocyst in blastocyst and control blastocyst stage (39.6$\pm$2.81, 35.7$\pm$2.98) were significanty higher than those in zygote, 2-, 4-, 8-cell, compacted morula (29.8$\pm$3.21, 31.3$\pm$3.83, 29.3$\pm$3.58, 28.9$\pm$3.21 or 30.8$\pm$2.93). In experiment 3, the zygotes were exposed in VSl for 1, 2, and 3 min to the optimal exposed time. The cleavage rates (91.6, 88.5, 88.9%) and develop mental rates to blastocyst (83.3, 74.3 and 69.4%) depends on the exposed time in VSl were not significantly different among 1, 2, or 3 min, respectively. The cell number also were not significantly different among exposed time in VS1. respectively. These results indicate that OPP method could be useful for vitrification either EFS or EDS vitrification solution. The post-thaw survival rates at zygote were significantly lower than those at 2-, 4-, 8-cell, morula or blastocyst, respectively. The zygote stage were more sensitive rather than late stage embryos. The exposing time in VS1 for 1 min was better than that for 2 or 3 min, even it was not significantly different. The OPP vitrification method could be useful of mouse embryos either with EFS or EDS vitrification solution.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.45-53
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• 2002

The main goal of this study was to produce transgenic porcine embryos by direct injection of sperm-mediated exogenous DNA. Spermatozoa (6$\times$10$^{6}$ sperms of final concentration) were mixed with pcDNA LAC Z (20 ng/$\mu$l) and subjected into electroporation (300~750 volts, 25 $\mu$F, 0.4 cm electrode). After sperm injection, the oocytes were activated electrically (1.7 KV/cm, 30$\mu$sec, single pulse) in 0.3 M mannitol solution or not. The sperm injected eggs were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air fur 144 h. The rates of cleavage and development into blastocyst stage in activation group were significantly higher than those of non-activation group (79.6% and 24.1% vs. 46.3% and 14.4%, respectively, p<0.05). Control oocytes and shame injection were developed to blastocysts low (2.5%). Sixty five (27.1%) out of 240 embryos observed in activation and non-activation groups were showed positive by X-gal staining. However, all embryos in both groups were expressed partial or mosaic pattern. These results suggested that electrical stimulation far oocytes activation after sperm injection enhances the incidence of both fertilization and development fellowing sperm injection in the pig. Our study also suggested that sperm-mediated transfer of exogenous DNA by ICSI would be used as a valuable tool for the production of transgenic porcine embryos.

• THE JOURNAL OF KOREAN ORIENTAL ONCOLOGY
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• v.6 no.1
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• pp.81-97
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• 2000

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.