• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.9-9
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• 2002

This study was to evaluate the in vitro survival of vitrified-thawed bovine MII enucleated (MIIe) oocytes according to activation timing and minimun volume cooling (MVC) method and their in vitro development after somatic cell nuclear transfer (SONT). Bovine oocytes were recovered from slaughtered bovine ovary and matured in TCM-199 supplemented with 10% FBS. (omitted)

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.116-116
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• 2002

Further development of reconstructed embryos may be dependent upon the synchronization of donor nucleus and recipient cytoplasm at cell fusion, To control the synchronization of donor and recipient cells, the enucleated MII arrested oocytes are artificially stimulated prior to embryo reconstruction. Destruction of cyclin B results in the exit of cells from M-phase of cell cycle. This study was designed to investigate the effects of single or combined stimulation affected cyclin B1 mRNA and protein levels in mouse oocytes. The oocyte activation was induced by 7% ethanol or 10$\mu\textrm{g}$/$m\ell$ Ca-ionophore without (single) or with (combined) 10$\mu\textrm{g}$/$m\ell$ cycloheximide. Competitive quantitative PCR for cyclin Bl mRNA and western blot analysis for cyclin B1 protein was preformed in mouse oocytes. Cyclin B1 mRNA level was significantly reduced in single (P<0.05) and combined (P<0.05) stimulation groups. However, this level did not change in non-activated group and increased in intact group. Cyclin B1 protein level was also significantly reduced in both single (P<0.05) and combined (P<0.05) stimulation groups. In conclusion, single and combined stimulation induces the degradation of cyclin B1 mRNA and protein after activation in enucleated mouse oocytes.

• Korean Journal of Animal Reproduction
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• v.18 no.2
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• pp.121-126
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• 1994

This study was conducted to examine the condition of activation of the nuclear transplant bovine embryos. In vitro fertilized(IVF) and nuclear transplant embryos(NTs) were co-cultured with bovine oviduct epithelial tissue(BOET). NTs were treated with cycloheximide(CHXM) for 0 to 6 h after electrofusion to investigate the activation conditin of recipient ooplast. Then, the infljence of the CHXM treatment timing on the cleavage and development of NTs were investigated in relation to the nuclear transplant time. The cleavage rates of NTs were increased with the increasing time of the CHXM treatment from 0 to 6 h (54.7 to 91.3%, P<0.01). Similar trend was shown in the development into the morula or blastocyst stage, but very limitted. Activation of enucleated oocytes prior to fusion enhanced development of NTs compared with that post fustion. This result suggests that the frequency of activation of NTs can be greatly enhanced by treating with CHXM for 6 h. The result also suggests that if blastomeres of unknown cell cycle stage are used, activation of enucleated oocytes prior to fusion enhances development of NTs.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.133-139
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• 1997

Large scale production of cloned embryos requires the technology of multiple generational nuclear transfer(NT) by using NT embryos itself as the subsequent donor nuclei. In this work we investigated comparatively the effects of enucleated oocytes treated with ionomycin and 6-DMAP on the electrofusion rate and in vitro developmental potential in the first and second NT embryos. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 15 hours after hCG injection. The enucleated oocytes were pre-activated by 5 min incubation in 5$\mu$M ionomycin and 2 hours incubation in 2 mM 6-DMAP at 19~20 hours post-hCG before microinjection. In the first and second generation NT, the unsynchronized 16-cell stage embryos were used as nuclear donor. The separated donor blastomeres were injected into the enucleated activated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of single pulse for 60 $\mu$sec at 1.25kV/cm in $Ca^2$+, $Mg^2$+ - free 0.28 M mannitol solution. In the non-preactivation group, the electrofusion and electrical stimulation was given 3 pulses for 60 $\mu$sec at 1.25 kV/cm in 100$\mu$M $Ca^2$+, $Mg^2$+ 0.28 M mannitol solution. The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in TCM-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The results obtained were summarized as follows: 1. In the first generational NT embryos, the electrofusion rate of preactivated and non-activated oocytes(80.4 and 87.8%) was not significantly different, but in the second generational NT embryos, the electrofusion rate was significantly(P<0.05) higher in the non-activated oocytes(85.7%) than in the preactivated oocytes(70.1%). 2) In the first and second generational NT embryos, the developmental potential to biastocyst stage was significantly(P<0.05) higher in the preactivated oocytes(39.3 and35.7%) than in the non-preactivated oocytes(16.0 and 13.3%). No significant difference in the developmental potential was shown between the first and second generational NT embryos derived from the preactivated oocytes. In conclusion, it may be efficient to use the oocytes preactivated with ionomycin and 6-DMAP for the multiple production of cloned embryos by recycling nuclear transfer.

• Journal of Embryo Transfer
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• v.9 no.2
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• pp.153-160
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• 1994

This study evaluated the influence of cell stage of donor nucleus on nuclear injection, electrofusion and in vitro development in the rabbit to improve the efficiency of nuclear transplantation in the rabbit. The embryos of 8-, 16- and 32-cell stage were collected from the mated does by flushing viducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FGS) at 44, 54 and 60 hours after hCG injection. The blastorneres separated from these embryos were used as donor nucleus. The ovulated oocytes collected at 14 hours after hCG injection were used as recipient cytoplasm following removing the nucleus and the first polar body. The separated blastomeres were injected into the enucleated oocytes by micromanipulation and were electrofused in 0.28 M mannitol solution at 1.5 kV /cm, 60 $\mu$sec for three times. The fused oocytes were cocultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FGS for 72~120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. The cultured nuclear transplant embryos were stained with Hoechst 33342 solution and the number of cells were counted by fluorescence microscopy. The successful injection rate of 8-, 16- and 32-cell-stageblastomeres into enucleated oocytes was 86.7, 91.0 and 93.9%, respectively. The electrofusion rate of 8-, 16- and 32-cell-stage blastomeres with enucleated oocytes was 93.3,89.3 and 79.0%, respectively. Development of blastomeres to blastocyst was similar with 8-,16- and 32-cell-stage donor nuclei(26.2, 25.8 and 26.6%, respectively, P<0.05). The mean number of cell cycle per day during in vitro culture in nuclear transplant embryos which received 8-, 16- and 32-cell- stage nuclei was 1.87, 1.81 and 1.43, respectively.

• Asian-Australasian Journal of Animal Sciences
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• v.8 no.6
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• pp.641-645
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• 1995

This study was conducted to develop a method for production of nuclear transplant bovine embryos using in vitro-matured (IVM) oocytes and to examine the effect of different conditions of electrofusion on fusion rate and developmental capacity of donor nucleus transplanted to enucleated oocytes. Eight- to sixteen-cell embryos derived from oocytes matured and fertilized in vitro used as donor blastomeres and IVM oocytes were used as recipient oocytes. Oocytes were enucleated immediately after 23-24 h IVM and then reconstituted with a donor blastomere in two different micromanipulation media. Fusion rate and subsequent development of the reconstituted oocytes was compared under the different electric stimuli and recipient oocyte ages. Success rate of enucleation was significantly higher in TCM-199 medium containing FCS than in DPBS. The high fusion rate(75-94%) and development (6.4-14.8%) to morulae and blastocyst (M + B) were obtained from 0.6-0.75 kV/cm DC voltage, although total cleavage was not different among the electric pulses. Most optimal condition of electric stimulation for fusion and development was 1 DC voltage of 0.75 kV/cm, in which 80.5% of oocytes were fused, 80.0% and 31.7% of which was cleaved and developed to M + B, respectively. No M + B was obtained from 1.2 kV/cm DC voltage regardless of pulse frequency. Recipint oocyte age at electrofusion greatly affected the cleavage and subsequent development to M + B, showing high rate at 40-41 h oocyte maturation. These results suggest that a suitable condition of electrofusion for donor nuclei derived from IVF may be 1-2 DC pulses of 0.7 kV/cm for $70{\mu}sec$ and that processing of a transplanted nucleus in IVM oocytes may be affected by maturation age of recipient oocytes.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.18-18
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• 2001

Despite of importance of integrated events of nucleus and microtubule remodeling in nuclear transferred embryos with somatic cells, little information is available on this subject. In this study we configured chromatin and microtubule organization following somatic cell nuclear transfer in pre- and non-activated bovine oocytes in order to clearify nuclear remodeling process and to demonstrate centrosome inheritance during nuclear transfer. The cumulus-oocyte complexes were collected from slaughterhouse and were matured in vitro for 20 h in TCM 199 supplemented hormone. Matured bovine oocytes were enucleated by aspirating the frist polar body and metaphase chromatin using a beveled pipette. Bovine fibroblast cells were fused into enucleated oocyte by electrical stimulation. Reconstructed oocytes were activated with ionomycine and 6-dimethylaminopurin, and then cultured in CRlaa medium. The organization of nuclear and microtubules were observed using laser-scanning confocal microscopy. At 1 hour after fusion, microtubule aster was seen near the transferred nucleus in most oocytes regardless activation condition. While most of fibroblast nuclei remodeled to premature chromosome condensation (PCC) and to the two masses of chromosome in non-activated oocytes, a few number of fibloblasts went to PCC and multiple pronuclear like structures in activated oocytes. Microtubular spindle was seen around condensed chromosome. Gamma-tubulin was detected in the vicinity of condensed chromosome, suggesting this is a transient spindle. The spindle seperated nucleus into two masses of chromatin which developed to the pronuclear like structures. Two pronuclear like structures were than apposed by microtubular aster and formed one syngamy like nuclear structure at 15 h following nuclear transfer. At 17 to 18 h after fusion, two centrosomes were seen near the nucleus, which nucleates micrtubules for two cell cleavage. While 31% of reconstructed oocytes in non-activated condition developed to morulae and blastocysts, a few reconstructed oocytes in pre-activated condition developed to the blastocyst. These results suggested introduction of foreign centrosome during nuclear transfer, which appeared to give an important role for somatic cell nuclear reprogramming.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.219-226
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• 1998

Enucleation of oocytes is an important limiting step for embryo cloning. We propose an enucleation technique based on the removal of chromatin after oocyte activation by aspirating the second polar body containing complemented chromatin. In a preliminary experiment to determine an optimal age of oocytes enucleation in rabbits, oocytes were enucleated at 15~20 hours post hCG. Recently ovulated oocytes were enucleated at a higher rate than aged oocytes. Microsurgical removal of the complemented chromatin in the second polar body was significantly more effective in enucleating than aspiration of a larger cytoplasm volume surrounding the first polar body of metaphase-arrested oocytes(96.8% versus 70.4%; P〈0.05). Moreover, compared with a nuclear transplantation protocol based on enucleation of metaphase-arrested oocytes and preactivated oocytes followed by treatment with 5 $\mu$M ionomycin for 5 min and 2 mM DMAP for 1 hr, there was no significant difference in the rate of blastocyst development. The ease with which modified technique can be performed is likely to render this technique widely useful for research and practice on mammalian cloning.

• 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.15 no.3
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• pp.247-253
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• 2000

This study was undertaken to optimize enucleation and reconstitution methods for the production of cloned mice by somatic cell nuclear transfer Outbred ICR mouse oocytes at the metapahse- II stage were retrieved from female mice superovulated by PMSG and hCG. In Experiment 1, oocytes were enucleated in medium supplemented with cytochalasin B (CCB) of 3 levels (0, 7.5 or 15 $\mu\textrm{g}$/mL), and higher rate of encleation was obtained at 7.5 and 15 $\mu\textrm{g}$/mL than at $\mu\textrm{g}$/mL. In Experiment 2, oocytes enucleated in 7.5 $\mu\textrm{g}$/mL CCB-containing medium were reconstituted with different types of somatic cell by following methods; 1) cumulus cells by direct cell injection, 2) cumulus cells by electric fusion (1.25 kV/cm, 2 pulses for each 70 $mutextrm{s}$) or 3) STO cells by the electrofusion. Electrofusion of STO cells with enucleated oocytes yielded the greatest (P<0.05) rate of reconstitution without lysis (76%) than any other combinations. Although significant decrease in the rate of somatic cell introduction was found, the electrofusion of cumulus cells yielded better rate of reconstitution than direct injection (0 vs. 18%). In Experiment 3, the duration of electric stimulation for the fusion was changed to either 50 $mutextrm{s}$ or 90 $mutextrm{s}$, but no significant improvement of reconstitution efficacy was obtained. In conclusion, this study showed that ICR mouse oocytes could be used for the production of reconstituted oocytes and a fusion method of 1.25 KV/cm with 2 pulses using 570 cell was the optimal.