• Reproductive and Developmental Biology
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• v.34 no.2
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• pp.73-79
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• 2010

We investigated the microtubule dynamics, including the inheritance of donor centrosomes and the mitotic spindle assembly occurring during the first mitosis of somatic cell nuclear transfer (SCNT) embryos in pigs. SCNT embryos were fixed 15 min and 1 h after fusion in order to assess the inheritance pattern of the donor centrosome. The distribution and dynamic of the centrosome and microtubule during the first mitotic phase of SCNT embryos were also evaluated. The frequency of embryos evidencing $\gamma$-tubulin spots (centrosome) was 93.2% in the SCNT embryos 15 min after fusion. In the majority of the SCNT embryos (61.5%), however, no centrosome was observed 1 h after fusion. The frequency of the embryos with no or abnormal mitotic spindles 20 h after fusion was 19.6%. The $\gamma$-tubulin spots were detected near the nuclei of somatic cells regardless of cell cycle phase, whereas $\gamma$-tubulin spots in the SCNT embryos were observed only during the inter-anaphase transition. These results showed that the donor centrosome is inherited into the SCNT embryos, but failed to assemble the normal mitotic spindles during first mitotic phase in some SCNT embryos.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.35-42
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• 1998

This experiment was carried out to improve in vitro development of rabbit one-cell embryos to the blastocyst stage. One-cell rabbit embryos were collected at 19\ulcorner20hr after superovulation induction and incubated at 39\ulcorner in 5% CO2 for 72hr. In order to find optimum conditions in medium that affects the rabbit embryo's development in vitro, RDH medium which mixed with RPMI1640, DMEM and Ham's F10 was compared with the previously reported mediums (Ham's F10 and RD) for embryo development and cell numbers. Three additives (BSA, taurine and glucose) were tested for the development of rabbit one-cell embryos in vitro. When the embryos were cultured in RDH medium, their development was markedly promoted as compared with Ham's F-10 or RD alone. Glucose exhibited no significant effects on embryo development and cell numbers. BSA a, pp.ared to promote transition from morula to blastocyst stage and taurine increased cell numbers of cultured embryos markedly regardless of medium. BSA and taurine together in RDH medium showed the additive effects on embryos development and cell number.

• Current Research on Agriculture and Life Sciences
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• v.7
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• pp.83-97
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• 1989

This study was carried out to obtain basic information necessary for aggregation and in-vitro culture of mouse embryos by treating phytohemagglutinin-p (PHA-P). The 4-, 8-cell and morula embryos were obtained from female mice of albino BALE/C, CBA and C57BL strains, those were injected 5 i.u pregenant mare serum gonadotrophin and 5 i.u human chorionic gonadotrophin to superovulation. The zona pellucidia was removed by placing the embryos in Acidic Tyrode solution containing 1.0% protease or/and 5 ug/ml PHA-P. The pairs of zona free embryos were subjected to aggregation by glassneedle in BMOC-3 containing 5 ug/ml PHA-P. The aggregation embryos were cultured in Brinster's mouse ova culture-3(BMOC-3) medium under the gas phase of 5% $CO_2$ in air $37^{\circ}C$ for 13 to 50 hours. The results obtained in this study are summarised as follows : 1. When 4-, 8-cell and morula embryos were zona-freed in acidic Tyrode solution containing 1.0% protease or/and 5 ug/ml PHA-P, and cultured in vitro to blastocysts, the 4- and 8-cell embryos showed slightly less development rates than the morula one did, and solution of 5 ug/ml PHA-P brought some higher development rate than negative control. 2. As 2, 5 or 10 ug/ml PHA-P was added to the solution to aggregate 4-, 8-cell or morula embryos, 2 ug/ml solution represented slightly lower aggregation rate than the higher levels solutions, and 4- and 8-cell embryos showed higher rates than morula one did (P<.05). 3. In respect to the development rates of aggregated embryos to morula no significant difference was found among PHA-P levels and between 4-and 8-cell embryos. With respect to those of aggregated embryos to blastocysts the different levels of PHA-P showed similar results, however, the 4- and 8-cell embryos represented higher rates than the morula one did (P<.05). 4. The mean time necessary for development of aggregated 4-, 8-cell and morula embryos to blastocysts were 38.5-40, 26-27 and 19-20hrs. Respectively in solution for aggregation. 5. The aggregation rates of embryos were 34-94%, when treated protease or/and PHA-P. Supplementation of 5 ug/ml PHA-P to the solution for aggregation showed a trend demonstrating higher aggregation rate compared to negative control, although no significance was found. However, 4- and 8-cell embryos represented significantly higher aggregation rates than the morula one did (P<.05). 6. The development rates of 4- and 8-cell embryos to morula were 52.7-84.7 and 73.8-87.2%, respectively, showing no significant difference between two cell stages. However, the aggregation rates of embryos treated with solution containing PHA-P were higher than negative control (P<.05). 7. The development rates of 4- and 8-cell and morula embryos to blastocysts were 41.7-77.7 78.7-83.0 and 0-19.2%, respectively. The rates of 4-cell embryos treated with PHA-P were significant higher than the negative control (P<.05). The 8-cell and morula embryos also showed more rates when treated PHA-P.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.77-86
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• 1998

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free T6 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at l20hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUT1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture ($93.8{\pm}3.1$, n=35) is significantly higher than that of control and glucose-free group ($76.6{\pm}3.8$, n=35 and $68.2{\pm}4.3$, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

• Journal of Embryo Transfer
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• v.10 no.1
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• pp.73-82
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• 1995

large scale production of cloned embryos requires the technology of multiple generation nuclear transplantation(NT) using NT embryos as the subsequent donor nuclei. The purposes of this study were producing the second generation cloned rabbit embryos, and also to determine the electrofusion rate and in vitro developmental potential comparatively in the cloned embryos of the first and second NT generation. 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 In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gi /S transition of 32-cell stage. The first generation NT embryos which were developed to 8-cell were synchronized in Gi /S transition phase of the following 16-cell stage and used as donor nuclei for second generation Synchronization of the cell cycle of blastomeres was induced, first, using an inhibitor of microtuble polymerization, colcemid for 10 hours to arrest blastomeres in M phase, and secondly, using a DNA synthesis inhibitor, aphidicolin for 1.5 to 2 hours to arrest them in Gi /S transition boundary. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 14 hours after hCG injection. The separated donor blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.25 kV /cm in 0.28 M rnannitol solution The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. Following in vitro culture of the first and second generation cloned embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The results obtained were summarized as follows: 1. The electrofusion rate was found to be similar as 79.4 and 91.5% in the first and second generation NT rabbit embryos, respectively. 2. The in vitro developmental potential to blastocyst stage of the second generation NT embryos (23.3%) was found significantly(p<0.05) lower, compared with that of the first generation NT embryos (56.8%). 3. The mean blastomeres counts of embryos developed to blastosyst stage following in vitro culture for 120 hours and also their daily cell cycles during the culture period were decreased significantly (p<0.05) to 104.3 cells and 1.33 cylces in the second NT generation, compoared with 210.4 cells and 1.54 cycles in the first NT generation, respectively.

• Current Research on Agriculture and Life Sciences
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• v.9
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• pp.29-36
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• 1991

The study was carried out to investigate the viability and the offspring production rate of single blastomeres and biopsied 4-cell mouse embryos and, also to examine the efficiency of biopsy. The results obtained are summerised as follows: 1. The separated blastomeres from 4-cell embryos and intact 4-cell embryos which were cultured in Medium 2 were developed to trophoblastic vesicle and blastocyst embryo by 82.6% and 89.5%. respectively. 2. The biopsied embryos from 4-cell embryos and intact 4-cell embryos which were cultured in Medium 2 were developed to blastocyst embryo by 83.3% and 90.4%, respectively. 3. The biospied blastomeres and the separated blastomeres from 4-cell embryos which were cultured in Medium 2 were developed to trophoblastic vesicle by 80.8% and 83.3%, respectively. 4. The biopsied embryos from 4-cell embryos and intact 4-cell embryos were transferred to recipients, and the offspring rate was 36% and 48.6%, respectively.

• Korean Journal of Animal Reproduction
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• v.14 no.1
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• pp.9-16
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• 1990

This study was carried out in order to investigate effects of cryoprotectant concentration and equilibration time on survival of ultrarapidly frozen 2-cell mouse embryos. Mouse 2-cell embryos, following dehydration by exposure to DMSO and sucrose, were directly immersed into liquid nitrogen and thawed in 37$^{\circ}C$ water. Viability was defined by development rate to the blastocyst stage after in vitro culture for 72 hours. The results are summarized as follows ; 1. When 0.25M of sucrose was added into the freezing medium at various concentrations of DMSO and dilution medium, higher development rate of embryo was obtained in 3.0M DMSO concentrations (82.6%). However, when sucrose concentraitons of 0.25 and 0.5 M were added to the freezing medium with 3.0 M DMSO and dilution medium, development rate of embryos were 81.7% and 24.1%, respectively. 2. In the equilibration time at room temperature, higher development rate was attained after short period of time (2.5min) in 3.0 M DMSO＋0.25 M sucrose (85.9%). 3. The development rate of embryos at in vitro 2-cell, in vitro 2-cell, solution control and untreated control was 84.6%, 90.9%, 89.9%,, and 89.7%, respectively.

• Korean Journal of Veterinary Research
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• v.33 no.1
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• pp.151-169
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• 1993

The present study was carried out to investigate the best condition for nuclear-cytoplasm fusion and in vitro culture of nuclear transplanted embryos and to investigate the production of nuclear transplanted offsprings. The nuclei from 2-, 4- and 8-cell mouse embryos were transferred into enucleated 2-cell embryos, and the reconstituted embryos were submitted to direct current(DC) pulses at output voltage of 1.0, 1.5 and 2.0 kV/cm for 100, 150 and $200{\mu}$ sec to induce cell fusion. 1. The culture of intact or zona cut 2-cell embryos in the medium supplemented with cytochalasin B($5{\mu}g/m{\ell}$) and colcemide($0.1{\mu}g/m{\ell}$)for 30 and 60 minutes did not affect the development to later stage. 2. The in vitro developmental rates of group A(a nucleus from one of the blastomeres was removed) and B(electrofusion of group A) were significantly lower than that of control group(p<0.01). 3. When nuclear transplanted embryos were submitted to electrofusion, the significantly higher fusion rates of 2-cell donor nuclei were achieved at the electric field strength of DC 1.5kV/cm for 100 and $150{\mu}$ sec, DC 2.0 kV/cm for $100{\sim}200{\mu}$ sec than DC 1.0 kV/cm for 100 and $150{\mu}$ sec(p<0.01). The significantly higher fusion rates of 4-cell donor nuclei were achieved at DC 2.0 kV/cm for 100 and $150{\mu}$ sec than DC 1.0kV/cm for $100{\sim}200{\mu}$ sec(p<0.01). These fusion rates in 8-cell donor nuclei were 88.7~99.3%. 4. The developmental potency to blastocyst in 2- and 4-cell donor nuclei was significantly higher in DC 1.0 and 2.0 kV/cm for $100{\sim}200{\mu}$ sec treated group and DC 2.0 kV/cm for 150 and $200{\mu}$ sec treated group (p<0.01). The developmental potency to blastocyst in 8-cell donor nuclei was significantly higher in DC 2.0 kV/cm for $100{\mu}$ sec treated group than in DC 1.0 kV/cm for $100{\mu}$ sec treated group and DC 2.0 kV/cm for 150 and $200{\mu}$ sec treated group(p<001). 5. The developmental potency to blastocyst after nuclear transplantation was significantly higher in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01). 6. The success rate of nuclear injection into enucleated 2-cell embryos was significantly higher in 2-cell donor nuclei than in 4- or 8-cell donor nuclei(p<0.01). 7. The culture time taken for the nuclear transplanted 2-cell embryos to blastocyst stage was significantly longer in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01). 8. There was no significant difference in the developmental potency of nuclear transplanted embryos within the concentration of EGF at 0 to 15 ng per $m{\ell}$ of BMOC-3 solution. 9. The production rates of offspring after transfer of nuclear transplanted embryos to recipient mouse were significantly higher in 2-cell donor nuclei than in 8-cell donor nuclei(p<0.01).

• Korean Journal of Animal Reproduction
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• v.16 no.3
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• pp.217-224
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• 1992

These studies were carried out ot investigate on the transferred embryo development following ultrarapid frozen for 8-cell and morula of in vitro fertilization mouse embryos. The post-thaw embryo survival was evaluated and compared by cell stage of embryos and by equilibration time before ultrarapid freezing. The results obatined were summerized as follows: 1. The effects of equilibration time of 3 vs. 6 minutes before ultrarapid freezing and after thawing on the morphological survival and the viability of 8-cell and morulas embryos were not significant. 2. When the ultrarapid frozen-thawed 32 eight-cell and 33 morula embryos, and 30 fresh blastocysts were transferred to pseudopregnant recipient mice, the number of normal offsprings produced were 9(28.1%), 14(42.4%) and 18(60.0%), respectively. From the above resutls, it was concluded that the optimal conditions of pH osmolality of the media for mouse IVF and embryo culture, and the period of sperm preincubation might be 7.1, 310 mOsm and 120 min., respectively,a nd somewhat high conception rate might be resulted from transfer of frozen embryos of morula stage and fresh embryos of blastocyst stage.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.241-248
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• 2014

This study was carried out to evaluate the effects of embryonic stage, cryoprotectant, and freezing-thawing method on the rates of survival and development of the cryopreserved mouse early embryo and finally to establish the cryopreservation method of surplus embryos obtained during assisted reproductive technology (ART). Two to eight cell embryos were obtained from oviducts of mated $F_1$ hybrid female mice superovulated by PMSG and hCG. Two-step EG, DMSO and 4-step EG, DMSO were used as cryoprotectant and dehydration and rehydration method of embryos, and slow-cooling or rapid-cooling method was used as frozen program. The survival rates of embryos were measured after thawing and rehydration, and the developmental rates of embryos were compared and observed during culturing embryos for 24, 48, 72, 96 hrs. As for the survival and development rates of embryos according to embryonic stage, the survival rate of 2 cell stage in EG and DMSO was significantly higher than 4~8 cell (65.4% versus 61.2%, 81.1% versus 72.5%) (p<0.01, p<0.01), but the development rates of 4~8 cell embryos in EG and DMSO were significantly higher than 2 cell embryos for whole culture period (p<0.01) and the development rates of 4~8 cell embryos in EG were significantly higher than 2 cell embryos in DMSO (p<0.01). As for the survival and development rates of embryos according to cryoprotectant, the survival rate of 2 cell embryo in DMSO was significantly higher than that in EG(77.0% versus 64.4%) (p<0.01), whereas the development rate of embryos was not differ till 24 hrs. The development rate from morular to hatching blastocyst, however, was sinificantly higher in EG than in DMSO during 48 hr (p<0.01). The survival rate of 4~8 cell embryo was 62.5% in EG and 73.3% in DMSO. The development rates of embryo in EG were significantly higher for whole culture periods (p<0.01, 0.05). In respect to the effect of freezing and thawing program on the survival and development rates of embryos, method of slow cooling and rapid thawing was more effective than that of rapid cooling and rapid thawing. The survival rate of embryo in 2 cell stage was higher than in 4~8 cell stage, and EG appears more effective cryoprotectant than DMSO because EG showed better development rates of embryos in 2 and 4~8 cell stage. Moreover, slow cooling and rapid thawing method was considered as the best cryopreservation program.