• Proceedings of the KSAR Conference
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• 2000.10a
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• pp.5-6
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• 2000

Nitric oxide (NO) is a simple combined molecule of oxygen and nitrogen, and has a wide variety of action on the physiological and pathophysiological function of the body. It is a key transducer of the vasodilator message from the endothelium to vascular cells. However, its different roles have been elucidated by numerous researches, which was undertaken in the 80's and 90's. Three types of NO synthase were involved in synthesizing NO and they are identified in different tissues and cells including macrophage, endothelial cells and even tumor cells. In the late 90's, we undertook a number of researches for elucidating the effect of NO on embryo development, since developmentally arrested bovine embryos contained large amount of NO metabolites in their cytoplasm. Subsequently, we found that the addition of a spontaneous NO donor to culture medium markedly inhibited embryo development and that its inhibitory role was independent of embryonic genome activation. Research was focused to find a way to prevent the inhibitory action of NO on embryo development and demonstrated that the addition of hemoglobin, a NO scavenger, to embryo culture medium greatly stimulated in vitro-development of bovine and mouse embryos. Based on these research outcomes, we developed a NO action-free culture system for embryos and other tissues. The efficacy of such system has subsequently been confirmed by achieving the high rates of preimplantation development and blastocyst formation in the NO action-free culture of mouse and bovine embryo. In this article, we briefly introduced the nature of NO and our research outcomes on the role of NO in embryo development.

• Journal of Embryo Transfer
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• v.11 no.1
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• pp.35-40
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• 1996

This study was carried out to select the best cryoprotectant and to establish optimal concentration of the cryoprotectant in ultrarapid freezing of mouse 4-cell embryos and morulae, respectively. We investigated survival of ultrarapid frozen embryos according to various cryoprotectants such as glycerol, ethylene glycol, propylene glycol and dimethyl sulfoxide (DMSO). Suvival of the embryos frozen at different concentrations (3.0, 4.0 and 5.0 M) of indivisual cryoprotectant was also tested. Preimplantation developmental rate (96.3%, 83/86) of 4-cell mouse embryos treated with 4.0 M ethylene glycol after ultrarapid freezing and thawing was higher than those of other cryoprotectants (glycerol, propylene glycol and DMSO). In the ultrarapid freezing of mouse morulae, the highest developmental rate (98.8%, 89 /90) of the embryos to blastocysts was shown in the group of 5.0 M glycerol. Thus, these results demonstrate that 4.0 M ethylene glycol and 5.0 M glycerol are optimal for ultrarapid freezing of 4-cell mouse embryos and morulae, respectively.

• Clinical and Experimental Reproductive Medicine
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• v.27 no.3
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• pp.253-259
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• 2000

Objective: To verify the effect of Matrigel, a ECM complex from Engelbreth-Holm-Swarm (EHS) mouse sarcoma on the preimplantation development and apoptosis of mouse fertilized eggs. Method: Late pronucleus stage eggs were cultured through the blastocyst stage in the presence of Matrigel (0.5%, v/v). Characteristics of apoptosis and cell number assesed by Hoecst staining and TUNEL labeling at the blastocyst stage, respectively. Results: Morphological development, number of cells per embryo was significantly increased but rate and number of TUNEL positive nuclei of the embryo were decreased in the presence of Matrigel. Conclusion: This result suggested that at low concentration of Matrigel improves both viability and morphological development in the preimplantation mouse embryos.

• Journal of Embryo Transfer
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• v.27 no.3
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• pp.189-192
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• 2012

Although embryonic stem cells (ESCs) or ES-like cells are reported from many mammalian species other than the mouse, the culture system for murine ESCs may not be suitable to the other species. Previously many other research groups have modified either human or mouse ESC culture systems for bovine ESC culture. In this study, we compared three different culture mediums consisting of DMEM, ${\alpha}$-MEM or KnockOut$^{TM}$-DMEM (KO), which are modified from human or mouse ESC culture system, for the generation of bovine ESCs. In this study, some pre-requisite events which are important for establishment and long-term propagation of ESCs such as inner cell mass (ICM) attachment on feeder cells, primary colony formation and sustainability after passaging. Once the ICM clumps attached on feeder cells, this was designated as passage 0. In regards to the rate of ICM attachment, ${\alpha}$-MEM was superior to the other systems. For primary colony formation, there was no difference between DMEM and ${\alpha}$-MEM whereas KO showed lower formation rate than the other groups. For passaging, the colonies were split into 2~4 pieces and passed every 5~6 days. From passage 1 to passage 3, DMEM system seemed to be appropriate for maintaining putative bovine ESCs. On the other hand, ${\alpha}$-MEM tended to be more suitable after passage 6. Although ${\alpha}$-MEM support to maintain a ES-like cell progenies to passage 15, all three culture systems which are modified from human or mouse ESC culture media failed to retain the propagation and long-term culture of putative bovine ESCs. Our findings imply that more optimized alternative culture system is required for establishing bovine ESC lines.

• Asian-Australasian Journal of Animal Sciences
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• v.23 no.9
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• pp.1152-1158
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• 2010

In differentiating human embryonic stem (d-hES) cells there are a number of types of cells which may secrete various nutrients and helpful materials for pre-implantation embryonic development. This study examined whether the d-hES could function as a feeder cell in vitro to support mouse embryonic development. By RT-PCR analysis, the d-hES cells revealed high expression of three germ-layered differentiation markers while having markedly reduced expression of stem cell markers. Also, in d-hES cells, LIF expression in embryo implantation-related material was confirmed at a similar level to undifferentiated ES cells. When mouse 2PN embryos were cultured in control M16 medium, co-culture control CR1aa medium or co-cultured with d-hES cells, their blastocyst development rate at embryonic day 4 (83.9%) were significantly better in the d-hES cell group than in the CR1aa group (66.0%), while not better than in the M16 group (90.7%)(p<0.05). However, at embryonic days 5 and 6, embryo hatching and hatched-out rates of the dhES cell group (53.6 and 48.2%, respectively) were superior to those of the M16 group (40.7 and 40.7%, respectively). At embryonic day 4, blastocysts of the d-hES cell group were transferred into pseudo-pregnant recipients, and pregnancy rate (75.0%) was very high compared to the other groups (M16, 57.1%; CR1aa, 37.5%). In addition, embryo implantation (55.9%) and live fetus rate (38.2%) of the d-hES cell group were also better than those of the other groups (M16, 36.7 and 18.3%, respectively; CR1aa, 23.2 and 8.7%, respectively). These results demonstrated that d-hES cells can be used as a feeder cell for enhancing in vitro and in vivo developmental potential of mouse pre-implantation embryos.

• Korean Journal of Veterinary Research
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• v.35 no.1
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• pp.95-105
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• 1995

The human homolog of position specific element of mouse Hoxa-7 was studied using transgene. It contains a 1.1 kb human DNA (HCR)- a homolog to the intergenic region between Hoxa-7 and -9, which directs the position specific expression of Hoxa-7-, tk promoter, LacZ (${\beta}$-galactosidase) gene as a reporter, and polyadenylation signal of SV40 large T antigen. It was injected into the mice embryos, and the resulting transgenic embryos were analysed through PCR as well as genomic Southern blotting with placenta DNA. Out of 20 embryos analysed, two were transgenic. Among them, one transgenic embryo expressed transgene when stained with X-gal. The expression pattern was in analogy to that of the mouse Hoxa-7, showing spatially restricted expression pattern, Since the expression of ${\beta}$-galactosidase is regulated by the upstream human HCR sequence, it implies that the HCR is the plausible position specific regulatory element of human.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.112-112
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• 2003

• Korean Journal of Animal Reproduction
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• v.11 no.2
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• pp.122-126
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• 1987

This study was carried out to investigate the effects of the glycerol equilibration methods and embryo grades before freezing on the survival rate after thawing in mouse embryos. The results obtained from this study were as follows: 1. The number of embryos of grade I(Excellent), II(Good) and III(Fair) before freezing in this study was 97(27.4%), 160(45.2%) and 97(27.4%), respectively. 2. The average survival rate of frozen-thawed embryos in 3 and 5 steps glycerol equilibration was 66.7% and 64.1%, and the rate of transfererable embryos after culture was 68.9% and 69.0%, respectively. 3. Out of embryo grade I and II before freezing, the transferable rate after thawing was 75.2% and 48.1%, respectively, and grade I embryos before freezing was higher transferable rate than that of grade II.

• 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.

• Journal of Embryo Transfer
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• v.23 no.1
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• pp.1-4
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• 2008

This study was performed to investigate the effect of laser-assisted hole in the zona pellucida (ZP) of frozen-thawed ICR mouse embryos on the process of hatching that is critical for expanded blastocysts to implant into endometrium, Vitrification medium, composed of ethylene glycol and sucrose supplemented with 7.5% (w/v) PVP, was used to freeze $2{\sim}4$ cell stage embryos recovered from oviducts of superovulated and mated female mice before storing them in $LN_2$. Right after thawing them, a laser beam was shot to make a hole in ZP followed by culturing in KSOM for $96{\sim}120\;hr$ and examining development to blastocyst and hatching every 12 hr. Laser-treated embryos showed significantly higher hatching rate compared to control (92.9% vs. 22.1%, p<0.05). From around Day 4, blastocysts developed from laser-treated embryos started hatching while the blastocysts of control group failed to hatch showing a lot of shrinkage. This study shows that a laser-assisted hole in ZP improves the hatching rate of blastocysts developed from frozen-thawed, in vitro cultured ICR mouse embryos.