• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.263-268
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• 2000

The development of single blastomeres isolated from 2-, 4- and 8-cell mouse embryos and the ability of such blastomeres to survive slow freezing were studied. Of 223, 60 and 188 single blastomeres isolated from 2-, 4- and 8-cell mouse embryos, respectively, 111 blastomeres (49.8%) from 2-cell embryos, 12 blastomeres (20.0%) from 4-cell embryos and blastomeres (16.5) from 8-cell embryos developed into blastocysts after culture for 96 hrs. The recovery rate was 54.2% (65/120), 46.4% (13/28) and 24.3% (17/70) of blastomeres derived from 2-, 4- and 8-cell embryos following freezing and thawing and the survival of frozen-thawed blastomeres was 27.1% (16/59), 36.4% (4/11) and 17.6% (3/17), and respectively. The apparently six normal fetuses were obtained from frozen-thawed blastomere from 2-cell embryos after transferring into the recipients. These results indicate that mouse btastomeres isolated from preimplatation stage embryos can survive storage in liquid nitrogen following slow freezing.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.1
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• pp.25-33
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• 2006

Objective: The aim of this study was to investigate whether embryonic stem (ES) cells can be established from isolated blastomeres of mouse embryos. Methods: Blastomeres were separated from mouse (C57Bl/6J) 2- or 4-cell embryos. Isolated blastomeres or whole 4-cell embryos were co-cultured with mitosis-arrested STO feeder cells in DMEM supplemented with recombinant murine leukemia inhibitory factor and ES-qualified fetal bovine serum. After the tentative ES cell lines were maintained from isolated blastomeres or whole embryos, some of them were frozen and the others were sub-cultured continually. Characteristics of tentative ES cell lines as were evaluated for specific genes expressions with immunocytochemistry and RT-PCR. Results: One ES cell line (3.0%) was established from isolated blastomere of 2-cell embryo and one cell line (4.0%) from isolated two blastomeres of 4-cell embryo. And five cell lines (16.7%) were established from whole 4-cell embryos. Both cell lines from isolated blastomere and whole embryo expressed mouse ES cell specific markers such as SSEA-1, Oct-4 and alkaline phosphatase. Marker genes of three germ layers were expressed from embryoid bodies of both cell lines. Conclusion: This study suggests that mouse ES cells could be established from isolated blastomeres, although the efficiency is lower than whole embryos. This animal model could be applied to establishment of autologous human ES cells from biopsied blastomeres of preimplantation embryos in human IVF-ET program.

• 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.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.113-113
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• 2002

The stimulatory effect of EGF and FSH on oocyte maturation have been reported in various mammalian species. And some reports presented FSH enhanced the effect of EGF on oocyte maturation. But, the interaction between EGF and FSH on nuclear maturation of mammalian oocytes is not fully understood. We observed the effect of EGF and FSH on nuclear maturation during in vitro maturation of mouse oocytes. Also, we examined the interaction between EGF and FSH on nuclear maturation of mouse oocytes using the EGFR inhibitor or FSH inhibitor. Germinal vesicle (GV) stage oocytes were obtained from 3-4weeks PMSG primed BCFI hybrid mice and cultured in TCM-199 medium with 0.4%PVP supplemented with/without EGF (1ng/ml), FSH (1ug/ml), EGFR specific tyrosine kinase inhibitors: Tyrphostin AG 1478 (500nM), MAP kinase kinase inhibitor : U0126 (20uM) or PD 98059 (100uM) for 14-l5hr. Rapid staining method were used for the assessment of nuclear maturation. Nuclear maturation rates of EGF indjor FSH-treated group were significantly higher than those of control group. Treatment of EGFR inhibitor significantly block the nuclear maturation of GV oocyte in EGF-treated group, but it did not block those of GV oocyte in FSH-treated or FSH and EGF-treated group. Treatment of FSH inhibitor(U0126, PD98059) significantly block the nuclear maturation of EGF-treated group, FSH-treated and FSH and EGF-treated group. These results show that EGF has a stimulatory effect as well as different action pathway with FSH on in-vitro maturation of mouse oocyte in vitro. Therefore, further studies will be needed to find the signaling pathway of EGF associated with nuclear maturation.

• Korean Journal of Animal Reproduction
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• v.22 no.2
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• pp.171-176
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• 1998

This study was carried out to confirm whether the in vivo developmental potential of mouse hatched blastocysts (HBs) can be obtained by vitrification method using the cryoprotectant EFS35. The HBs ($\theta$ 130$\mu\textrm{m}$) were cultured in vitro until day 5 from zygotes produced in vivo and were equilibrated in 10% ethylene glycol(EG) for 5 min, and then were exposed or vitrified in EFS35 (35% EG, 18% Ficoll and 0.3M sucrose). After 30 min thawing, re-expanding HBs were transferred into one or both uterine horns of pseudopregnant recipients on day 3 (4~6 embryos /horn). Pregnancy rates of recipients and implantation were assessed by autopsy on day 15 of gestation. The results obtained in these experiments were summarized as follows : After thaw-ing, in vitro survival of HBs was not significantly different between exposed (65.5%) and vitrified(54.5%) group. Also, when the in vivo development potential was examined, total implantation was not different between control (58.5% ) and vitrified (41.0%) group, although the live fetus formation of vitrified group (24.0%) was significantly lower than that of control (58.3%) group (p< 0.05). These results suggested that vitrification freezing method of mouse HBs using EFS35 can be used to make wide the utility of embryo transfer of the more embryos produced in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.29 no.4
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• pp.323-335
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• 2002

Object: The present study was accomplished to obtain a gene expression profile of the luminal epithelium during embryo apposition in comparison of implantation (1M) and interimplantation (INTER) sites. Material and Method: The mouse uterine luminal epithelium from IM and INTER sites were sampled on day 4.5 (Day of vaginal plug = day 0.5) by Laser Captured Microdissection (LCM). RNA was extracted from LCM captured epithelium, amplified, labeled and hybridized to microarrays. Results from microarray hybridization were analyzed by Significance Analysis of Microarrays (SAM) method. Differential expression of some genes was confirmed by LCM followed by RT-PCR. Results: Comparison of IM and INTER sites by SAM identified 73 genes most highly ranked at IM, while 13 genes at the INTER sites, within the estimated false discovery rate (FDR) of 0.163. Among 73 genes at IM, 20 were EST/unknown function, and the remain 53 were categorized to the structural, cell cycle, gene/protein expression, immune reaction, invasion, metabolism, oxidative stress, and signal transduction. Of the 24 structural genes, 14 were related especially to extracellular matrix and tissue remodeling. Meanwhile, among 13 genes up-regulated at INTER, 8 genes were EST/unknown function, and the rest 5 were related to metabolism, signal transduction, and gene/protein expression. Among these 58 (53+5) genes with known functions, 13 genes (22.4%) were related with $Ca^{2+}$ for their function. Conclusions: Results of the present study suggest that 1) active tissue remodeling is occurring at the IM sites during embryo apposition, 2) the INTER sites are relatively quiescent than IM sites, and 3) the $Ca^{2+}$ may be a crucial for apposition. Search for human homologue of those genes expressed in the mouse luminal epithelium during apposition will help to understand the implantation process and/or implantation failure in humans.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.315-323
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• 1994

Mammalian oviductal epithelial cells have been known to improve in vitro fertilization and embryonic development. Recently, co-cultured human embryos with the epithelial cells in human genital tract has been reported to improve the pregnancy rate. The purpose of the study was to investigate the effects of the epithelial cells of human genital tract on the development of mouse early embryos and human fertilized oocytes. The epithelial cells of human genital tract were collected from the fallopian tubes which were obtained during hysterectomy in fertile women and from the endometrium during endometrium biopsy. Collected human ampullary cells(HACs) and endometrial cells(HECs) were cultured for 10 days to establish primary monolayer. Second passaged HACs and HECs were obtained by trypsinization were cryopreserved in PBS with 1.5 M DMSO for later use. To investigate the effect when co-cultured with HACs and HECs, we tried to apply strict quality control on mouse embryo, from two cell to blastocyst prior to human trial. The results of quality control were as follows; In Group I (Ham's F10 with 10% FCS), Group IT (co-cultured with HACs) and Group ill (co-cultured with HECs), developmental rates to blastocyst were 63.3%(253/400), 76.0%(304/ 400),74.0%(296/400), respectively. Hatching rates were 36.8%(147/400), 41.80/0(167/400), 38.0%(152/400), respectively(p<0.05). To perform the human IVF, cryopreserved HACs were thawed at 37$^{\circ}C$ waterbath, seeded on the well dish and cultured for 48 hI'S. The pronuclear stage embryos were transferred to the seeded well dish. After 24 hRS, co-cultured embryos were examined and transferred to patient's uterus. The results of human IVF when co-cultured with HACs were that fertilization and developmental rates were 61.8% (256/414), 95.3% (244/256) as compared with 57.2% (279/488) and 94.6%(264/279) in Ham's F10 supplemented with 10% FCS(control). However, 62.9% (161/256) of co-cultured human embryos showed good embryos(no or slight fragmentation) as compared with 53.8 % (150/279) in control(p < 0.05). Pregnancy rate was 40.0% (12/30) when co-cultured with HACs whereas 30.6%(11/36) in control. In conclusions, co-culture system using HACs and HECs improved the developmental and hatching rates of mouse embryo. Also, in human IVF system when co-cultured with HACs, it improved both the quality of human embryos and the pregnancy rate.

• Clinical and Experimental Reproductive Medicine
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• v.44 no.4
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• pp.193-200
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• 2017

Objective: This study was conducted to investigate the efficacy of laser-assisted hatching (LAH) and various vitrification times for embryonic development and blastocyst cell numbers. Methods: First, 2-cell and 8-cell embryos were collected by flushing out the oviducts. In the control groups, they were vitrified for 8 or 10 minutes without LAH. The LAH groups underwent quarter laser zona thinning-assisted hatching before vitrification (4, 6, and 8 minutes or 4, 7, and 10 minutes, respectively). After incubation, double-immunofluorescence staining was performed. Results: The hatched blastocyst rate 72 hours after the 2-cell embryos were thawed was significantly higher in the 2LAH-ES8 group (33.3%) than in the other groups (p< 0.05). In the control-8 group ($22.1{\pm}4.6$), the cell number of the inner cell mass was higher than in the LAH groups (p< 0.05). The number of trophectoderm cells was higher in the 2LAH-ES6 group ($92.8{\pm}8.9$) than in the others (p< 0.05). The hatched blastocyst rate 48 hours after the 8-cell embryos were thawed was higher in the 8LAH-ES4 group (45.5%) than in the other groups, but not significantly. The inner cell mass cell number was highest in the 8LAH-ES7 group ($19.5{\pm}5.1$, p< 0.05). The number of trophectoderm cells was higher in the 8LAH-ES10 group ($73.2{\pm}12.1$) than in the other groups, but without statistical significance. Conclusion: When LAH was performed, 2-cell embryos with large blastomeres had a lower hatched blastocyst rate when the exposure to vitrification solution was shorter. Conversely, 8-cell embryos with small blastomere had a higher hatched blastocyst rate when the exposure to vitrification solution was shorter.

• Journal of Embryo Transfer
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• v.30 no.4
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• pp.299-303
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• 2015

KO mice provide an excellent tool to determine roles of specific genes in biomedical filed. Traditionally, knockout mice were generated by homologous recombination in embryonic stem cells. Recently, engineered nucleases, such as zinc finger nuclease, transcription activator-like effector nuclease and clustered regularly interspaced short palindromic repeats (CRISPR), were used to produce knockout mice. This new technology is useful because of high efficiency and ability to generate biallelic mutation in founder mice. Until now, most of knockout mice produced using engineered nucleases were C57BL/6 strain. In the present study we used CRISPR-Cas9 system to generate knockout mice in FVB strain. We designed and synthesized single guide RNA (sgRNA) of CRISPR system for targeting gene, Abtb2. Mouse zygote were obtained from superovulated FVB female mice at 8-10 weeks of age. The sgRNA was injected into pronuclear of the mouse zygote with recombinant Cas9 protein. The microinjected zygotes were cultured for an additional day and only cleaved embryos were selected. The selected embryos were surgically transferred to oviduct of surrogate mother and offsprings were obtained. Genomic DNA were isolated from the offsprings and the target sequence was amplified using PCR. In T7E1 assay, 46.7% among the offsprings were founded as mutants. The PCR products were purified and sequences were analyzed. Most of the mutations were founded as deletion of few sequences at the target site, however, not identical among the each offspring. In conclusion, we found that CRISPR system is very efficient to generate knockout mice in FVB strain.

• The Korean Journal of Zoology
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• v.39 no.3
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• pp.239-247
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• 1996

An embryonic stem (ES) cell-based in vitro model system was examined to determine whether a simple differentiation of embryoid bodies (EB) in the suspension medium is useful to dissect early erythropoiesis. Characteristics of the differentiating EBs were monitored for their differentiation potential to generate hematopoietic cell types by general morphology, benzidine staining and two-step colony assays, and expressivity of several erythroid marker genes by the RT-PCR analysis for total cellular RNA prepared from the differentiating EBs. Every ematopoietic lineage cells were generated from the differentiating EBs with reproducible frequencies, similar to the other sophisticated differentiation protocols. Furthermore, the globin gene switching in differentiating ES cells paralleled the sequence of events found in the mouse embryo, and such that their expression was activated by at least 12 hrs later than those of erythroid-specific transcription factors, GATA-1 and Tal-1 The erythropoietic differentiation program initiated reproducibly and efficiently in this simple differentiation system in a suspension culture, such that this system may be useful for dissection of the molecular events of early erythropoiesis.