• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.75-75
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• 2002

The present study was conducted for the production of transgenic cloned cows by somatic cell nuclear transfer (SCNT) that secrete human prourokinase into milk. To establish an efficient production system for bovine transgenic SCNT embryos, the offset was examined of various conditions of donor cells including cell type, size, and passage number on the developmental competence of transgenic SCNT embryos. An expression plasmid far human prourokinase (pbeta-ProU) was constructed by inserting a bovine beta-casein promoter, a green fluorescent protein (GFP) marker gene, and a human prourokinase target gene into a pcDNA3 plasmid. Three types of bovine somatic cells including two adult cells (cumulus cells and ear fibroblasts) and fetal fibroblasts were prepared and transfected using a lipid-meidated method. In Experiment 1, developmental competence and rates of GFP expression in bovine transgenic SCNT embryos reconstructed with cumulus cells were significantly higher than those from fetal and ear fibroblasts. In Experiment 2, the effect of cellular senescence in early (2 to 4) and late (8 to 12) passages was investigated. No significant differences in the development of transgenic SCNT embryos were observed. In Experient 3, different sizes of GFP-expressing transfected cumulus cells [large (>30 ${\mu}{\textrm}{m}$) or small cell (<30 ${\mu}{\textrm}{m}$)] were used for SCNT. A significant improvement in embryo development and GFP expression was observed when small cumulus cells were used for SCNT. Taken together, these results demonstrate that (1) adult somatic cells could serve as donor cells in transgenic SCNT embryo production and cumulus cells with small size at early passage were the optimal cell type, and (2) transgenic SCNT embryos derived from adult somatic cells have embryonic development potential.

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.639-645
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• 1997

The objectives of the present study were improvements in the efficiency of developmental rates to morula and blastocyst stages to produce a large number of genetically identical nuclear transplanted embryos. The oocytes collected from slaughterhouse ovaries were matured 24h in TCM199+10% FBS and exposed to $39^{\circ}C$ or room temperature to allow cytoplasmic maturation and gain activation competence. Donor embryos were treated for 12h with $10{\mu}g/ml$ nocodazole or $0.05{\mu}g/ml$ demicolcine to synchronize the cell cycle stage at 26h after the onset of culture. The blastomeres and recipient oocytes were fused by electrofusion. The cloned embryos were then cultured in various conditions to allow further development. In the treatment of oocyte activation and cell cycle regulation of donor nuclei, the room temperature exposure and nocodazole treatment group had significant effect on the developmental rates to morula/blastocyst(21.7% vs 12.1~16.7%), but had no significant effect on the fusion rates between donor blastomeres and recipient oocytes. The developmental rates of bovine nuclear transplanted embryos appeared to be higher significantly in mTALP medium under 5% $O_2$ condition and in TCM199 with bovine oviduct epithelial cell under 20% $O_2$ condition(22.2%) than other groups. In embryo transfer of nuclear transplanted embryos, there were no significant differences in calving rates between the use of excellent and good grade donor embryos.

• Archives of Pharmacal Research
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• v.17 no.4
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• pp.209-212
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• 1994

Mophological normal of unfertilized oocytes, which was collected 12-14 hours after human Chorionic Gonadotropin(jCG) injection, was not influenced by chronically adiministration of cocaine for 2 weeks in mice. Proportion of normal unfertilized oocytes in non-cocaine treated group (control), `0 mg/kg and 20 mg/kg cocaine treated group based on body weight with subcutaneous(s.c.) daily injection of cocaine for 2 weeks were 92.9%, 85.6% and 90.9%, respectively. There is no significant difference between control and cocaine treated groups. Two to 8 cell stage embryos collected 24-48 hours post hCG in control group were 66.7%, whereas, 10 mg/kg and 20 mg/kg groups treated with cocaine was 12.5% and 27.3% respectively. Although control and treated groups are significantly different (p<0.05) the developmental score of 2 to 8 cell stage embryos collected at 24-48 hours post HCG, there is no difference between 10 mg/kg and 20 mg/kg treated with cocaine groups. These results indicated that the normal embryos of the roups of cocaine administration were significantly amested when compared with that of control group. The proportion of 2 to 8 cell stage embryo reaching the blastocyst stage, which were cultured 48-52 hours with 5% $Co_2$ in air at $37^{\circ}C$, were 93.9% in control group and, 70.4% and 71.9% in each 10 mg/kg and to blastocyst in vitro culture was significantly limited embryos obtained from cocanized mice compared with those of control mice. These results suggest that episode of cocaine intoxication can cause impaiment of early embrygenesis in the mouse.

• Korean Journal of Animal Reproduction
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• v.18 no.1
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• pp.39-46
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• 1994

This experiment was carried out to develop an in vitro culture system for rabbit embryos. The zygotes or 2-cell embryos were collected from the oviducts of the superovulated and mated does with D-PBS/10% FCS at 24 hours after hCG injection. The in vitro developmental rate of blastocyst formation and the number of nuclei in the embryos were examined under the following treatments; 1) TCM-199 with 10% FCS, 2) EBSS with 10% FCS, 3) rabbit vitreous humor(VH), 4) TCM-199 with 10% FCS＋BOEC, 5) TCM-199 with 10% FCS＋ROEC, 6) EBSS with 10% FCS＋BOEC and 7) EBSS with 10% FCS＋ROEC. For a comparative study of in vivo and in vitro development, the fresh blastocysts, which were developed in vivo for 96 hours after hCG injection, were collected from the uterus and their numbers of nuclei were counted. 1. The zygotes or 2-cell embryos developed to the blastocyst stage in TCM-199, EBSS and VH at the rates of 93, 92 and 89%, respectively. 2. The higher developmental rates 95~98% of blastocyst formation was achieved when the embryos were co-cultured with a monolayer of bovine or rabbit oviductal epithelial cells in TCM-199 or EBSS. No significant difference in developmental rates was shown between bovine and rabbit oviductal epithelial cells. 3. In a comparative study of in vivo and in vitro development, the total numbers of nuclei were significantly less in the in vitro cultured embryos(104~224) than the in vivo developed embryos(1, 0090 at 96 hours after hCG injectin. 4. The mean cell cycle numbers in the embryos cultured for 72 hours in TCM-199 with 10% FCS, EBSS with 10% FCS, TCM-199 with 10% FCS＋BOEC, TCM-199 with 10% FCS＋ROEC, EBSS with 10% FCS＋BOEC and in vivo was 7.38, 6.63, 7.76, 7.69, 7.01 and 9.92, respectively. From these results, it can be suggested the optimal culture system for in vitro culture of rabbit embryos is a co-culture system with bovine or rabbit oviductal epithelial cells in TCM-199 with 10% FCS. Considering the significant reduction in total numbers of nuclei in the in vitro cultured embryos, the advanced research on development of in vitro culture system for rabbit embryos is expected.

• Journal of Embryo Transfer
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• v.29 no.3
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• pp.235-240
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• 2014

Ethylene glycol (EG) has been successfully used as a cryoprotectant for vitrification of mammalian embryos (including human embryos) due to its low formula weight and high permeation into cells compared with other cryoprotectants, including propylene glycol (PROH). Cryopreservation is able to store the surplus pre-embryos for freezing and furthermore thawing and transfer in a subsequent cycle. 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). Female ICR mice (6~8 weeks old) were induced to superovulate by sequential intraperitoneal injection of 5 IU PMSG and 5 IU hCG 48 h apart. Mouse embryos were collected according to its developmental stage after the injection of hCG. Embryos were cryopreserved not only during cryoprotectant step (1~4 step) but also in a variety of media (HTF, IVF medium, D-PBS) and cell stage. The results were as follows : There is no clear advantage in these freezing media of rapid method, but 4 cell and 8 cell of slow method (2, 3 and 4 step) have advantage in D-PBS. The development of embryos according to cell stage become greater in 8 cell stage. In the treatment steps of cryopreservation, the development of embryo to blastocyst was similar among rapid method, but the development of 4 cell and 8 cell embryos to blastocyst according to slow method was better than rapid method.

• Clinical and Experimental Reproductive Medicine
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• v.41 no.1
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• pp.1-8
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• 2014

Objective: Estrogen related receptor ${\beta}$ (Esrrb) is a member of the orphan nuclear receptors and may regulate the expression of pluripotencyrelated genes, such as Oct4 and Nanog. Therefore, in the present study, we have developed a method for delivering exogenous ESRRB recombinant protein into embryos by using cell-penetrating peptide (CPP) conjugation and have analyzed their effect on embryonic development. Methods: Mouse oocytes and embryos were obtained from superovulated mice. The expression of Oct4 mRNA and the cell number of inner cell mass (ICM) in the in vitro-derived and in vivo-derived blastocysts were first analyzed by real time-reverse transcription-polymerase chain reaction and differential staining. Then 8-cell embryos were cultured in KSOM media with or without $2{\mu}g/mL$ CPP-ESRRB protein for 24 to 48 hours, followed by checking their integration into embryos during in vitro culture by Western blot and immunocytochemistry. Results: Expression of Oct4 and the cell number of ICM were lower in the in vitro-derived blastocysts than in the in vivo-derived ones (p<0.05). In the blastocysts derived from the CPP-ESRRB-treated group, expression of Oct4 was greater than in the non-treated groups (p<0.05). Although no difference in embryonic development was observed between the treated and non-treated groups, the cell number of ICM was greater in the CPP-ESRRB-treated group. Conclusion: Treatment of CPP-ESRRB during cultivation could increase embryos' expression of Oct4 and the formation rate of the ICM in the blastocyst. Additionally, an exogenous delivery system of CPP-conjugated protein would be a useful tool for improving embryo culture systems.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.35-41
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• 2003

Three experiments were performed to develop an optimal culture system of in-vitro derived porcine embryos. In experiment 1, embryos were cultured in two different basic culture media (NCSU-23 and PZH) supplemented with BSA (4mg/ml), FBS (10%), PVA (3mg/ml). Although no difference on the percentage of embryos developed to blastocysts was found (P>0.05), more 2-cell embryos (59.5% vs. 44.7 to 52.0%) were obtained when embryos were cultured in NCSU-23 added with BSA. In experiment 2, identical treatment was conducted using PZM medium. More (P<0.05) 2-cell embryos (54.4% vs. 40.7 to 44.5%) and blastocysts (12.5% vs. 5.8 to 9.0) were produced when embryos were cultured in PZM added with BSA (4mg/ml). In experiment 3, embryos were cultured in media as follows; 1) NCSU-23 without BSA for 192 hours; 2) NCSU-23 with BSA for 192 hours; 3) NCSU-23 with BSA for 96 h and then subsequently injected with FBS into culture drops. Although no difference on the percentage of embryos developed to blastocysts was found (P>0.05), more 2-cell embryos (63.8 to 69.2% vs. 52.0%) were obtained when embryos were cultured both in NCSU-23 added with BSA throughout culture duration and in FBS supplemented medium. In conclusion, no difference was found when porcine preimplantation embryos were cultured in NCSU-23 added with various macromolecules. However, when PZM medium was used, PZM medium supplemented with BSA showed more potential to support embryo development. Finally, FBS supplemented into culture drop 96h post-insemination did not show any benefits on embryo development.

• Clinical and Experimental Reproductive Medicine
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• v.20 no.2
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• pp.161-164
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• 1993

To forsee appropriate developmental cell stage in human embryos for cryopreservation, we observed blastocyst development in culture medium after cryopreservatlon and thawing of one cell, two cell, four cell stage of mice embryos. According to our results, development of the blastocyst of cryopreserved two cell mice embryos was significantly higher than that of cryopreserved one cell mice zygotes or four cell mice embryos.

• Journal of Animal Science and Technology
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• v.58 no.3
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• pp.13.1-13.7
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• 2016

Background: In the present study, various freezing containers were tested for mouse embryos of respective developmental stages; embryos were vitrified and then their survival rate and developmental rate were monitored. Mouse two cell, 8 cell, and blastula stage embryos underwent vitrification freezing-thawing and then their recovery rate, survival rate, development rate, and hatching rate were investigated. Methods: EM-grid, OPS, and cryo-loop were utilized for vitrification freezing-thawing of mouse embryos. Results: It was found that recovery rate and survival rate were higher in the group of cryo-loop compared to those of EM-grid (p < 0.05). Embryonic development rate, two cell embryos to blastocyst, as well as hatching rate were higher in the control group compared to the EM-grid group and OPS group (p < 0.05), yet no difference was noted between the control group and cryo-loop group. Development rate and hatching rate of eight cell morulae and blastocysts were all lower in the treatment groups than the control group whilst hatching rate of blastocysts was higher in the control group compared to the groups of EM-grid and OPS (p < 0.05); although the cryo-loop group was shown to be slightly higher than other groups, it was not statistically significant. Conclusions: In the study, we investigate effects of freezing containers on vitrified embryos of respective developmental stages; it was demonstrated that higher developmental rate was shown in more progressed (or developed) embryos with more blastomeres. There was however, no difference in embryonic development rate was shown amongst containers. Taken together, further additional studies are warranted with regards to 1) manipulation techniques of embryos for various vitrification freezing containers and 2) preventive measures against contamination via liquid nitrogen.

• Korean Journal of Veterinary Research
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• v.35 no.3
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• pp.635-641
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• 1995

The purpose of this study was to investigate the effects of developmental stage and equilibration time on survival of rabbit embryos following freezing by vitrification. Adult New Zealand White female rabbits were superovulated with PMSG and hCG. The 8-cell stage embryos were collected from 40 to 45 hours after hCG injection by flushing oviducts with Dulbecco's phosphated buffered saline and in vitro cultured in TCM-199 containing 10% fetal calf serum(FCS). Each embryos developed in vitro to 16-cell, compact morula and blastocyst was cryopreserved and cultured following thawing to examine their developmental potential to expanded blastocyst stage in vitro. The frozen-thawed-cultured embryos were stained with Hoechst 33342, and their nuclei were counted using a fluorescence microscope. On the toxicity test of EFS solution as cryopreservation, the survival rates of 8-cell stage embryos was decreased in reverse to increasing of exposure time over 5 minutes. The post-thaw survival rates of embryos on equilibration times was significantly(P<0.05) higher for 2 min. than for 5 or 10 minutes. From morula to blastocyst of rabbit embryos was more suitable than 8-cell stage for cryopreservation by vitrification. The higher post-thaw survival rate of embryos can be achieved by keeping the cryoprotectant at $4^{\circ}C$ than at $20^{\circ}C$. The mean number of nuclei per embryo following freezing by vitrification and in vitro culture to expanded blastocyst at compacted morula and blastcyst was not significantly differ from fresh blastocyst.