Chung Park

Association of castration-dependent early induction of c-myc expression with a cell proliferation of the ventral prostate gland in rat

The protooncogene c-myc is known to be associated with both cell proliferation and apoptosis. The possible cellular affects of castration on the ventral prostate gland of rat as well as the relationship to a castration induced c-myc expression were examined. Levels of c-myc mRNA in the ventral prostate gland peaked at 6 h (early induction) and 48 h (late induction) after castration, respectively. Castration-induced DNA fragmentation was not observed at an early induction of c-myc mRNA. DNA fragmentation appeared to be testosterone-dependent. On the other hand, cellular DNA synthesis measured by [3H]thymidine uptake in the ventral prostate gland was increased to maximum at 6 h after castration. These results suggest that an early induction of c-myc mRNA in ventral prostate gland after castration is closely associated with cell proliferation of the gland.

Modification of octamer binding transcriptional factor is related to H2B histone gene repression during dimethyl sulfoxide-dependent differentiation of HL-60 cells

Transcriptional regulation of H2B histone gene during dimethyl sulfoxide (DMSO)-dependent differentiation of HL-60 cells has been investigated using DNase I footprinting and DNA mobility shift assay. The level of histone H2B mRNA showed a slight decline at 2 days and hardly detectable at 4 days after DMSO treatment. H2B histone mRNA was repressed in proportion to the concentration of DMSO. In DNase I footprinting analysis, one nuclear factor (octamer binding transcription factor, OTF) bound at -42 bp (octamer motif, ATTTGCAT) in undifferentiated HL-60 cells. The binding pattern of OTF was unchanged during DMSO-dependent differentiation. One protein complex (OTF) was detected by DNA mobility shift assay in undifferentiated HL-60 cells. The mobility of OTF was partially retarded during DMSO-dependent differentiation and the retardant OTF was not newly synthesized protein. These results suggest that the posttranslational modification of OTF may be responsible for the repression of H2B histone gene during DMSO-dependent differentiation of HL-60 cells.

ATF is important to late S phase-dependent regulation of DNA topoisomerase IIα gene expression in HeLa cells

DNA topoisomerase IIalpha (Topo IIalpha) is regulated in late S phase-dependent manner. To identify late S phase-dependent cis-acting elements of Topo IIalpha gene, we have investigated the synchronized HeLa cells with chloramphenicol acetyltransferase and DNase I footprinting assays. The level of Topo IIalpha mRNA increased after release from aphidicolin block and reached a maximum in 8h (late S phase) in HeLa cells, and Topo II unknotting activity was also in parallel with the level of Topo IIalpha mRNA. The late S phase-regulatory element was found to be located in the region containing ATF-binding element between -290 and -90bp and the region was required for a maximal stimulation during late S phase. DNase I footprinting assay showed that ATF-binding element and novel cis-acting element (Topo IIalpha-specific sequence) were the principal protein-binding sites and the proteins interacting with these elements were induced during late S phase. One DNA-protein complex was formed by DNA mobility shift assay when ATF-binding site was incubated with nuclear extract prepared from late S phase cells, but no protein bound in non-S phase cells. Taken together, these results suggest that ATF may be essential transacting factor for maximal expression of Topo IIalpha gene during late S phase in HeLa cells.

32 EFFECTS OF FUSION/ACTIVATION METHODS ON DEVELOPMENT OF EMBRYOS PRODUCED BY NUCLEAR TRANSFER OF PORCINE FETAL FIBROBLASTS

Pigs have become increasingly important in the field of biomedical research, and interest has grown in the use of transgenic cloned pigs as potential xenograft donors. The present study were carried out to investigate the effects of intensity of DC pulse, number of DC pulses, and equilibration before fusion/activation on developmental ability of porcine embryos derived from nuclear transfer. Porcine cumulus-oocyte complexes (COCs) were cultured in modified TCM-199 (mTCM-199) medium for 44 h at 38.5°C, 5% CO2 in air. After in vitro maturation (IVM), metaphase II oocytes were selected for enucleation. Porcine fetal fibroblasts were obtained from a porcine fetus on Day 35 of gestation as donor cells. Oocytes were enucleated by removing, with a micropipette, the first polar body along with adjacent cytoplasm containing the metaphase plate; then a donor cell was injected in contact with the cytoplasm of each oocyte. In experiment 1, several different fusion/activation intensities (two DC pulses of 0.4, 0.8, 1.2, 1.6, and 2.0 kV cm-1 for 30 µs) were carried out to investigate the effect on the development of nuclear transfer embryos. In experiment 2, the reconstructed oocytes were fused and activated with 1, 2, or 3 DC pulses of 1.2 kV cm-1 for 30 µs. In experiment 3, reconstructed oocytes were equilibrated in mTCM-199 medium at 38.5°C, 5% CO2 for 0, 1, 2, 3, 4, 5, and 6 h. After equilibration, the reconstructed oocytes were fused and activated with one DC pulse of 1.2 kV cm-1 for 30 µs in fusion medium. The reconstructed embryos were transferred into PZM-3 medium containing 0.3% BSA for further culture. The rates of embryo cleavage and development of blastocyst stage were evaluated at 48 h and 6-7 days, respectively. The cell numbers of blastocysts were counted by using Hoechst 33342 epifluorescence staining. Data were analyzed by ANOVA and Duncan

289 EFFECT OF FERTILIZATION TIME OF PIG OOCYTES MATURED IN-VITRO BY BOAR SPERM STORED AT 4°C

The use of boar sperm stored at 4°C may be a useful tool for in vitro production of pig embryos. Therefore, this study was undertaken to investigate the effect of fertilization time of pig oocytes matured in-vitro by boar sperm. The sperm-rich fraction (30–60 mL) was slowly cooled to room temperature (20–23°C) by 2 h after collection. Semen was transferred into 15-mL tubes, centrifuged at room temperature for 10 min at 800g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5 mL of the LEN (11.0 g lactose hydrate, 20 mL egg yolk, 0.05 g N-acetyl-D-glucosamine and 100 mL distilled water) diluent to provide 1.0 × 109 sperm mL−1 at room temperature. The resuspended semen was cooled in a refrigerator to 4°C. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 μg mL−1 insulin, 2 μg mL−1 vitamin B12, 25 mM HEPES, 10 μg mL−1 bovine apotransferrin, 150 μM cysteamine, 10 IU mL−1 PMSG, 10 IU mL−1 hCG, 10 ng mL−1 EGF, 0.4% BSA, 75 μg mL−1 sodium penicillin G, 50 μg mL−1 streptomycin sulfate and 10% pFF. After about 22 h of maturation, oocytes were cultured without cysteamine and hormones for 22 h at 38.5°C, 5% CO2 in air. Oocytes were inseminated with boar sperm stored at 4°C for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9 h in 500 μL TBM fertilization media with 1 × 106 mL−1 sperm concentration. Thereafter, oocytes were transferred into 500 μL NCSU-23 culture medium containing 0.4% BSA for further culture of 6, 48 and 144 h, fixed and stained for the evaluation of fertilization parameters and developmental ability. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times of 6 and 9 h than in those of 1 and 3 h. The percentage of polyspermic oocytes was highest in fertilization time of 9 h compared with other incubation times. The rates of cleaved oocytes were higher in the fertilization times of 6 and 9 h (85.0 and 84.6%) compared with those of 1 and 3 h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time of 6 h (33.6%) than in that of 1, 3 and 9 h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9 ± 3.3, 27.6 ± 2.7, 26.3 ± 2.2 and 24.4 ± 1.8 in the fertilization times of 6, 9, 3 and 1 h, respectively. In conclusion, we found out that boar sperm stored at 4°C could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend the coincubation time of 6 h in 500 μL TBM fertilization medium with 1 × 106 mL−1 sperm concentration for in vitro fertilization of pig oocytes matured in vitro.

303 PARTHENOGENETIC DEVELOPMENT OF PIG OOCYTES BY CHEMICAL ACTIVATION

Efficient activation is essential for the success of animal cloning by nuclear transfer. The aim of this study was to investigate the effects of chemical activation agents on parthenogenetic development of pig oocytes matured in vitro. The medium used for oocyte maturation was TCM-199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 μg mL-1 insulin, 2 μg mL-1 vitamin B12, 25 mM HEPES, 10 μg mL-1 bovine apotransferrin, 150 μM cysteamine, 10 IU mL-1 PMSG, 10 IU mL-1 hCG, 10 ng mL-1 EGF, 0.4% BSA, 75 μg mL-1 sodium penicillin G, 50 μg mL-1 streptomycin sulfate and 10% pFF. After about 22 h of maturation, oocytes were cultured without cysteamine and hormones for 22 h at 38.5°C, 5% CO2 in air. Cumulus-free oocytes showing first polar body were selected for activation. Oocytes were activated as follows. First, all oocytes were activated with 25 mM HEPES buffered NCSU-23 medium containing 8% ethanol for 10 min. After that, in treatment 1, oocytes were incubated in the NCSU-23 medium supplemented with 7.5 μg mL-1 cytochalasin B for 3 h. In treatment 2, oocytes were incubated in the NCSU-23 medium supplemented with 10 μg mL-1 cycloheximide for 3 h. In treatment 3, oocytes were incubated in the NCSU-23 medium supplemented with 7.5 μg mL-1 cytochalasin B for 1.5 h, and then were incubated in the NCSU-23 medium supplemented with 10 μg mL-1 cycloheximide for 1.5 h. In treatment 4, oocytes were incubated in the NCSU-23 medium supplemented with 7.5 μg mL-1 cytochalasin B plus 10 μg mL-1 cycloheximide for 3 h. Following activation, oocytes were transferred into 500 μL NCSU-23 culture medium containing 0.4% BSA for further culture for 20 and 144 h. Activated oocytes were fixed and stained for evaluation of activation rate, cleaved oocytes, blastocyst formation rate and cell numbers per blastocyst. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rate of oocyte activation was higher in treatment 4 (62.1%) than in treatment 1, 2 and 3 (52.0, 49.6 and 58.0%, respectively). The percentage of cleaved oocytes was lower in treatment 1 and 2 (56.9 and 55.2%) than in treatment 3 and 4 (68.8 and 68.5%). The rate of blastocyst formation from the cleaved oocytes was higher in treatment 3 and 4 (19.8 and 22.0%) than in treatment 1 and 2 (12.1 and 11.7%). Mean cells per blastocyst were lowest in treatment 2 (21.2 ± 0.9) compared to treatment 1, 3 and 4 (27.3 ± 2.2, 30.4 ± 3.8 and 30.9 ± 3.4, respectively). In conclusion, cytochalasin B combined with cycloheximide was more efficient for parthenogenetic development of pig oocytes matured in vitro.