• Korean Journal of Animal Reproduction
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• v.26 no.4
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• pp.329-338
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• 2002

This study was carried out to investigate the developmental rates of embryo reconstructed with different cell type and to estimate correlation of transcriptional level of octamer-binding transcription factor 4 (Oct4) and fibroblast growth factor 4 (FCF4) gene on peri-implantation stage embryos. Donor cells were transferred into perivitelline space of enucleated oocytes. The karyoplast-cytoplast couplets were accom- plished by cell to cell fusion and activated with ionomycin and 6-dimethylaminopurine. Reconstructed embryos were co-cultured with bovine oviduct epithelial cells in CR 1 aa medium. There is no difference in blastocyst formation rate following nuclear transfer UT) with fetal fibroblast cell (16/50; 32.0%), cumulus cell (16/49; 32.6%) and ear cell (17/52; 32.6%). The expression level of Oct4 and FCF4 in peri-implantation bovine embryo derived from in vitro fertilization (IVF) and NT were determined by reverse-transcription polymerase chain reaction (RT-PCR) technique. In peri-implantation of IVF result in a transient increased of FCF4 paralleled by an increased expression of Oct4. However, Oct4 gene was highly expressed in hatching blastocysts derived from NT compared to IVF. Also, FGF4 expression level in hatching blastocysts and outgrowth stage derived from NT was lower than that of IVF. In conclusion, it is suggested that the different transcription patterns observed in nuclear transfer embryos may lead to a lower rate of embryo development, implantation and pregnancy.

• Development and Reproduction
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• v.3 no.1
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• pp.75-85
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• 1999

To investigate the origin and action mechanism of cytoplasmic factors as regulators of morphogenesis, the embryonic development, RNA synthesis and protein phosphorylation were examined in reconstituted embryos. A half of 1-cell mouse embryo with both pronuclei was electrofused with the enucleated cytoplasm of 1- or 2-cell embryos which were cultured for 24 hrs from post 20 hrs hCG in CZB with or without cycloheximide (CHX, an inhibitor of protein synthesis; P+P-CHX group), genistein (Gen, an inhibitor of tyrosine protein kinase; P+2-Gen group) and 6-dimethylaminopurine (6-DMAP, an inhibitor of serine-threonine protein kinase; P＋2-DMAP group), and co-cultured with Vero cells for 5 days. And their development, cell numbers at compaction, [5, 6-$^3$H]-uridine incorporation into RNA and the pattern of protein phosphorylation after labeling of [$^{32}$ P] orthophosphate were compared with that of the reconstituted embryos such as P+2 or P+P (control group). Embryonic development and the time of RNA synthesis in P+P-CHX were similar to those in P+P. But the time and the cell stages of embryonic compaction in P+P-CHX were similar to those in P+2. The compaction was initiated at 4-cell in P+2 and P+2-Gen, but at 8-cell in P+P and P+2-DMAP. On a two-dimensional gel electrophoresis, phosphorylation of 80KD and 110KD proteins were inhibited after 3 hrs of reconstruction in the embryo of P+2-DMAP when compared with that of P+2 and P+2-Gen. These results suggest that protein synthesis between 1- and 2-cell stage affects the timing of embryonic genome activation, and that cytoplasmic factors derived from oocyte or their modification regulates the time schedule of embryonic compaction in mouse. Also, serine-threonine protein kinase has an important role on the regulation of compaction.

• Reproductive and Developmental Biology
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• v.33 no.1
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• pp.7-11
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• 2009

This study was performed to confirm the microtubule assemblies and methylation patterns of porcine IVF and parthenogenetic embryos. Cumulus-oocyte complexes were collected and matured in vitro for 42 hr. Oocytes were fertilized by prepared fresh sperm or activated parthenogenetically by exposure to electric stimulation and 6-dimethylaminopurine. Porcine IVF and parthenogenetic embryos were cultured in vitro for 6 days. Embryos were stained by immunofluorescence staining method to observe the dynamic of nucleus and microtubules in the first mitotic phase and the methylation patterns in different developmental stages. After then, samples were confirmed and analyzed through a laser-scanning confocal microscope. IVF embryos had a centrosome originated from sperms, which was shown a $\gamma$-tubulin spot. However, $\gamma$-tubulin spot was not observed in parthenogenetic embryos. A lower methylation level was observed in IVF embryos compared to parthenogenetic ones at the morula and blastocyst stages. In conclusion, it is considered that microtubule assemblies and genetic regulation mechanism differ between parthenogenetic and IVF embryos.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.107-115
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• 2001

Development of effective activation protocols is of great importance for improving the success of cloning and subsequent transgenic. Three methods for oocyte activation, including 5$\mu$M ionomycin (5 min) alone, ionomycin + 1.9 mM 6-dimetylaminopurine (DMAP, 3 hrs) and ionomycin + 10 $\mu\textrm{g}$/ml cycloheximide (CHX, 3 hrs) were compared for their effects of pronuclei (PN) formation, development, developmental velocity and ploidy of parthenotes to IVF control in bovine. In group of ionomycin + DMAP, the oocytes having more 3PN were significantly (P<0.05) higher than in groups of ionomycin alone and of ionomycin + CHX (45.5% vs. 0 and 0%, respectively). Activation with the ionomycin alone, ionomycin + DMAP and ionomycin + CHX resulted in cleavage rates of 30, 85.5 and 57.9%, respectively. The blastocysts rate of parthenotes activated by ionomycin + DMAP treatment was significantly higher (12.3%. p<0.05) than those of other treated groups. Chromosome analysis shows that ionomycin + DMAP treatment greatly enhances the incidence of chromosomal abnormality of the parthenotes. From the results, we may conclude that DMAP treatment to the oocytes accelerates developmental velocity resulting in both the higher incidence of chromosome abnormality and of PN formation, and strongly suggest that CHX combined with ionomycin is better than DMAP for the purpose of successful nuclear transplantation. Developmental velocity of parthenotes activated by ionomycin + DMAP treatment was significantly (P<0.05) faster than others.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.281-288
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• 2000

This experiment was carried out to investigate the optimal activation condition for parthenogenetic development. In order to activate oocytes at 22 h post onset of maturation, the oocytes were subjected to 5 $\mu$M ionomycin(I) for 5 min ,10 $\mu$M calcium ionophore(Ca) for 5 min, 2 mM 6-dimethylamino-purine(DMAP) for 3 h and 10 $\mu\textrm{g}$/$m\ell$ cycloheximide(CH) for 6 h alone or in combination. The activated oocytes were cultured in modified CR$_1$aa at 5% $CO_2$, 5% $O_2$, 90% $N_2$. l. The cleavage rates after 48 h culture of oocytes treated with I, Ca, DMAP and CH were 12.7%, 14.1%, 28.9% and 22.9%, respectively. There was no blastocyst formation. 2. The cleavage rates after 48 h culture of oocytes treated with I ＋ DMAP, I ＋ CH, Ca ＋ DMAP and Ca ＋ CH were 96.9%, 82.1%, 93.1% and 34.7%, respectively. Developmental rates to blastocysts were 10.4%, 5.3%, 17.6% and 7.1 %, respectively. When oocytes were treated with Ior Ca followed by DMAP, the blastocyst formation rate was significantly higher than other groups(P <0.05). 3. According to single activation treatment, pronucleus formation rates were 5.4%, 3.6%, 28.3% and 28.8%, respectively, Whereas, all oocytes treated with the combined activation agents formed 100% pronucleus.

• Korean Journal of Microbiology
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• v.25 no.3
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• pp.221-228
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• 1987

The extracellular adenine deaminase from Nocardioides sp. J-275L was purified by the following techniques: ammonium sulfate fractionation, DEAE-Cellulose, DEAE-Sephadex A-50 column chromatography, and Sephacryl S-200 superfine gel filtration. The enzyme was partially purified about 3889.5-fold with about 5.2% yield by these procedures. The molecular weight of the enzyme was 39,000 by a calibrated Sephacryl S-200 superfine column chromatography. The enzyme was stable at pH 7.5 and up to $40^{\circ}C$. Glycerol was effective on the stabilization of the enzyme during storage. The optimum pH and temperature of the enzyme were around pH 7.5 and $40^{\circ}C$, respectively. The apparent Michaelis constant Km of the enzyme for adenine was $7.4\times 10^{-5}$M. The purine analogues, 6-chloropurine, 2,6-diaminopurine, 6-bromopurine, 4-aminopyrazolo [3.4-d]pyrimidine, and 8-azaadenine were substrates for the enzyme. 6-Dimethylaminopurine was a competitive inhibitor of the enzyme. The enzyme was inhibited by 1mM of $Cu^{2+}, Fe^{3+}, Pb^{2+}, Hg^{2+}$, and $Ag^{+}$, and 1mM of $\alpha$,$\alpha$'-dipyridyl, pentachlorophenol, and pCMB.

• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.759-764
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• 2001

This study was carried out to investigate the effect of activation timing, cell cycle and passage on the development of embryos produced by cumulus cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at $38.5^{\circ}C$ in a humidified atmosphere of 5% $CO_2$ in air. The 1~6 passages of serum deprived or actively dividing cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One pulse of 180 volts for $15{\mu}s$ was applied to induce the fusion between karyoplast and cytoplast. The activation was done before or after the fusion. To activate, oocytes were treated with $10{\mu}M$ calcium ionophore for 5 min immediately followed by 2 mM 6-dimethylaminopurine for 3 h. The nuclear transfer embryos were cultured in $500{\mu}l$ of modified CRlaa supplemented with 3 mg/ml BSA in four well dish covered with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/ml BSA and 5% FBS for 4 days. The incubation environment was 5% $CO_2$, 5% $O_2$, 90% $N_2$ at $38.5^{\circ}C$. There was no blastocyst formation when the nuclear transfer embryos were activated before the fusion, whereas, 29.9% of blastocyst formation was shown when the nuclear transfer embryos were activated after the fusion. When serum deprived and actively dividing cumulus cells were used as nuclear donor cells, the developmental rates to blastocyst were 38.5% and 40.6%, respectively. There was no significant difference between serum deprived and actively dividing cells in the developmental rates. The developmental rates to blastocyst according to 1~6 passages were 37.5~44.4%. However, there were no significant differences among passages. These results indicate that 1~6 passage cumulus cell irrespective of cell cycle could support development of nuclear transfer embryos activated after the fusion.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.124-124
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• 2003

The successful development of embryos cloned by nuclear transfer (NT)have been dependent on a wide range of known factors including cell cycle of donor and recipient ooplast, oocyte quality, NT procedure and oocyte activation. The present study compared the development of cloned porcine embryos following different activation treatments. Cumulus-oocyte complexes (COCs) were aspirated from 26 mm follicles of slaughterhouse ovaries and cultured for 22 h in NCSU #23 medium supplemented with 10% porcine follicular fluid, 0.57 mM cysteine, 0.5 g/mL LH, 0.5 g/mL FSH and 10 ng/mL EGF. The COCs were further cultured for an additional 22 h in the same medium at $39{\cird}C$ in an atmosphere of 5% $CO_2$ in air, without hormonal supplements. Primary cultures of fibroblasts isolated from a female fetus on day 40 of gestation were established in DMEM + 15% FCS. For nuclear donation, cells at the 5th-6th passage were cultured in DMEM +0.5% FCS for 5 days in order to arrest the cells in G0/Gl. After enucleation, oocytes were reconstructed by transfer of donor cells and fusion with three DC pulses (1.4 KV/cm, 30 sec) in 0.28 M mannitol containing 0.01 mM $CaCl_2$ and 0.01 mM $MgCl_2$. Eggs were then divided into three treatment groups, control (without further treatment, Group 1), eggs cultured in 10 g/ml cycloheximide (CHX) for 5 h (Group 2), and eggs cultured in 1.9 mM 6-dimethylaminopurine (6-DMAP) for 5 h (Group 3). The eggs were then cultured in sets of 30 in 60 I drops of NCSU#23 supplemented with 4mg/ml BSA (essentially fatty acid free) until day 7 at $39{\circ}C$ in a humidified atmosphere of 5% $CO_2$. On day 4 the culture were fed by adding 20 I NCSU #23 supplemented with 10% FBS. Development rates into blastocysts were significantly higher (P<0.05) in Group 3 embryos compared to Group 1 controls ($27.6 \mu 2.7% vs. 20.1 \mu 4.1%$, respectively), but rates did not differ in Group 2 compared to control ($23.8 \mu 5.7%$). Total cell number in Group 3 blastocysts was however significantly higher (P<0.05) than in Groups 1 and 2 ($44.6 \mu 2.4 vs. 19.9 \mu 1.9 and 21.9 \mu 2.1$, respectively). These results suggest that 6-DMAP is more efficient than cycloheximide in the activation of electrically fused NT oocytes during in vitro production of cloned porcine embryos.

• Korean Journal of Animal Reproduction
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• v.25 no.1
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• pp.51-61
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• 2001

This study was conducted to determine developmental ability of reconstructed embryos by nuclear transfer using somatic cell of Korean bull with high genetic value. Fibroblast cells obtained from ear biopsy of the bull were cultured in Dulbecco's Modified Eagle's medium (DMEM) at 37$^{\circ}C$ in air containing 5% $CO_2$. The cummulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 culture medium and the oocytes were then enucleated in modified phosphate buffered saline with cytochalasin B. Matured bovine oocytes were enucleated by aspirating the first polar body and metaphase chromatin using a beveled pipette in modified phosphate buffered saline. The ear fibroblast cells were fused into enucleated oocyte by electrical stimulation. The reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurine, and then cultured in CR1aa medium for 7.5 days. Out of 524 bovine eggs reconstructed by nuclear transfer 65.6%(277/422) embryos were cleaved, and 30.7% (85/277) cleaved embryos were developed to the morula to blastocysts. There was no difference of developmental ability in vitro of reconstructed embryos regardless of donor cell passages. In order to determine fate of foreign mitochondria of donor nucleus, the Mito Tracker stained cells were fused into enucleated oocytes. The donor mitochondria were detected early stage of embryos, but disappeared rapidly. The developmental ability of reconstructed embryos was not impaired by Mito Tracker treatments. The results indicate that viable reconstructed embryos can be producted by nuclear transfer using somatic cell of Korean bulls.bulls.

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.133-139
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• 2013

The objective of this study was to determine the effect of post-activation treatment with cytoskeletal regulators in combination with or without 6-dimethylaminopurine (DMAP) on embryonic development of pig oocytes after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT). PA and SCNT oocytes were produced by using in vitro-matured pig oocytes and treated for 4 h after electric activation with $0.5{\mu}M$ latrunculin A (LA), $10.4{\mu}M$ cytochalasins B (CB), and $4.9{\mu}M$ cytochalasins D (CD) together with none or 2 mM DMAP. Post-activation treatment of PA oocytes with LA, CB, and CD did not alter embryo cleavage (85.8~88.6%), blastocyst formation (30.7~ 32.4%), and mean cell number of blastocysts (33.5~33.8 cells/blastocyst). When PA oocytes were treated with LA, CB, and CD in combination with DMAP, blastocyst formation was significantly (P<0.05) improved by CB+DMAP (42.5%) compared to LA+DMAP (28.0%) and CD+DMAP (25.1%), but no significant differences were found in embryo cleavage (77.5~78.0%) and mean blastocyst cell number (33.6~35.0 cells) among the three groups. In SCNT, blastocyst formation was significantly (P<0.05) increased by post-activation treatment with LA+DMAP (32.9%) and CD+DMAP (35.0%) compared to CB+DMAP (22.0%) while embryo cleavage (85.5~85.7%) and blastocyst cell number (41.1~43.8 cells) were not influenced. All three treatments (LA, CB, and CD with DMAP) effectively inhibited pseudo-polar body extrusion in SCNT oocytes. The proportions of oocytes showing single pronucleus formation were 89.6%, 83.9%, and 93.3%, respectively with the increased tendency (P<0.1) by LA+DMAP and CD+ DMAP compared to CB+DMAP. Our results demonstrate that post-activation treatment with LA or CD in combination with DMAP improves pre-implantation development of SCNT embryos and the stimulating effect of cytoskeletal modifiers on embryonic development is differentially shown depending on the origin (PA or SCNT) of embryos in pigs.