• Journal of Embryo Transfer
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• v.16 no.3
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• pp.239-243
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• 2001

Selection of oocyte cryopreservation method is a prerequisite factor for developing an effective bank system. Compared with slow freezing method, the vitrification has various advantages such as avoiding intracellular ice crustal formation. In our previous, we attempted to employ a vitrification method using ethylene glycol and an electron microscope grid for cryopreservation of mouse oocytes. However, A high incidence of spindle and chromosome abnormalities was detected in thawed oocytes after vitrification. We examined whether the addition of a cystoskeleton stabilizer Taxol $^{TM}$, to the vitrification solution could promote the post-thawed survival and subsequent development of stored oocytes. More oocytes developed to the 4-cell (44.7% vs. 69.7%), 8-cell (31.8% vs. 64.2%), morula (24.7% vs. 54.3%), and blastocyst (20.3% vs. 49.2%) stages after the addition of Taxol$^{TM}$ to the cryoprotectant than after no addition. 21 and 26 mouse pups were born after transfer of blastocyst derived from oocytes vitrified without and with Taxol. The addition of Taxol to vitrification solution greatly promoted post-thaw preimplantation development of ICR morose oocytes.tes.

• Journal of Embryo Transfer
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• v.10 no.3
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• pp.193-202
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• 1995

In order to improve the cryopreservatory techniques of livestock embryos, the quick freezing method which is directly plunged in liquid nitrogen via prefreezing procedure without freezing machine was carried out for mouse embryos treated with permeable and nonpermeable cryoprotectants. The viability of frozen-thawed embryos were evaluated by FDA vital dye test. The results obtained was summaried as follows: 1. A total of 720 embryos were recovered from frozen embryos for viability test. Evalution of the fluorescein diacetate(FDA) vital dye test with mice embryos were resulted of 2.3 total mean score - evaluted in orderly higher mean grade of P3 453 (63%), P2 133(18%), P1 51(7%) and P0 83(12%). 2. An all-round evalution of these combination, the highest viability was showed in 3M ethylene glycol + 0. 25M trehalose treated with the copper prefreezing. 3. Effects of permeable and nonpermeable cryoprotectants combination were evaluated by means FDA score. 3M ethylene glycol + 0.25M trehalose showed the highest survival rates of 2.8 mean FDA score. 4. Effects of permeable cryoprotectants were evaluated by mean FDA score but the results were not significantly different each other. 5. In evalution of the nonpermeable cryoprotectants, 0. 25M trehalose obtalned higher mean FDA score than of 0.25M sucrose and it was significantly different(P<0.05). 6. There was no significantly difference between copper and stainless-steel in prefreezing procedures.

• Proceedings of the Korean Institute of Information and Commucation Sciences Conference
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• 2013.10a
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• pp.943-945
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• 2013

The study of Schwann cell myelination has been facilitated by the availability to isolate and establish pure population of primary Schwann cells. Dorsal root ganglia (DRG) of mouse embryo as source of Schwann cells were used in this study. This method includes three steps: first step of dissociation of the embryonic DRG, second step of expansion of Schwann cell precursors, followed by mechanical separation of the Schwann cell-neuronal network from the underlying fibroblasts, and third step of purification of Schwann cells from the associated neurons and subsequent expansion of the purified Schwann cells. We made a highly purified population of Schwann cells and Schwann cell-neuron networks in a short period using this procedure.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.127-137
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• 1994

This study was carried out to investigate the inhibiting or promoting effect of fetal bovine serum fractionated by the molecular weight and to examine the effect of reconstruction of serum fractions on the development of 1- and 2-cell mouse embryos fertilized in vitro (IVEE) . The serum was separated by ultrafiltration or gel filtration methods and added in m-KRB medium for culture of IVFE. The developemental ability(cavitation and hatching) of embryos following culture of day 4 and 6 was compared among fractions. Small molecular weight fraction( <3 kDa) significantly inhibited the development of 1-and 2-cell IVFE to the blastocyst stages, compared with other fractions. One-cell IVFE were more sensitively damaged than 2-cell embryos by that fraction and arrested mainly at 2~4 cell stages. Moreover, small amount(<3%,v /v) of the inhibiting fraction acted even with protein rich fraction(100~30 kDa) and arrested the embryonic development. On the other hand, 100~30 kDa fraction promoted the embryonic development and no inhibiting effect was observed at the level of 50%(v /v) in culture medium In the experiment of gel filtraton, =30 kDa fraction showed the highest promoting effect on the embryonic development, but <4 kDa fraction inhibited significantly the development. These results suggest that serum contains not only small molecular weight inhibitory component(s) but also promoting one rather than albumin on embryonic development. And serum can be more effectively used in the IVF program after removal of inhibitory component(s) by one of above separation methods.

• Development and Reproduction
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• v.23 no.3
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• pp.285-295
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• 2019

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. The actin-regulatory activity of JMY is based on a cluster of three actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domains that nucleate actin filaments directly and promote nucleation of the Arp2/3 complex. In addition to these activities, we examined the activity of JMY generation in early embryo of mice carrying mutations in the JMY gene by CRISPR/Cas9 mediated genome engineering. We demonstrated that JMY protein shuttled expression between the cytoplasm and the nucleus. Knockout of exon 2, CA (central domain and Arp2/3-binding acidic domain) and NLS-2 (nuclear localization signal domain) on the JMY gene by CRISPR/Cas9 system was effective and markedly impeded embryonic development. Additionally, it impaired transcription and zygotic genome activation (ZGA)-related genes. These results suggest that JMY acts as a transcription factor, which is essential for the early embryonic development in mice.

• Korean Journal of Animal Reproduction
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• v.16 no.1
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• pp.39-46
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• 1992

In viro fertilizatin is very important in both human clinical practice and animal breeding. However, the success rate of in vitro fertilization is not high. The purpose of this study ws to determine wheter in the vitro fertilization and culture of porcine oocyte using a hydrogel chamber were possible or not. Hydrogel chambers were made of polymerized 2-hydroxyethyl methacrylate. Matured follicular oocytes in Waymouth's medium and T L Hepes medium, tubal oocytes, and preincubated sperm in M199 medium were treansferred into the lumen of the hydrogel chambers. The chambers containing porcine oocytes and spermatozoa implanted into the mouse peritioneal cavity, and ova were examined after the recovery of the chambers at 84 hours after preservation start. The result was shown that fertilization and culture of porcine oocytes were successfully achieved inside of the hydrogel chamber.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 1998.07a
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• pp.43-44
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• 1998

Effect of somatic cell coculture on hatching of mouse blastocyst was examined. Mid-blastocysts were cocultured with granulosa cell primary culture or Sertoli cell line ($TM_{4}$) derived from mouse testis for 48 hr. Blastocysts cultured in medium (10% FBS) started to hatch more faster than cocultured embryos during 12 hr of coculture. After then blastocysts cocultured with somatic cell hatched faster than control. Degeneration of embryos was also greately reduced by coculture. This result suggested the potentiation of hatching as well as embryonic viability by coculture with somatic cell and Sertoli cell line can be used for embryo coculture.

• The Korean Journal of Zoology
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• v.38 no.2
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• pp.196-203
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• 1995

The c-myc proto-oncogene, one of the immediately earlY genes, is expressed in various mammalian cell types and heavily involved in the regulation of cell proliferation and differentiation. To determine endogeneous expression pattern of c-myc gene in preimpBantation mouse embwos, we employed a reverse transcription coupled to polvrnerase chain reaction (RT-PCR). Transcript of c-myc was detected at fertilized embryos as a maternal transcript. At the early two-cell stave, transcript of c-myc gene was hardly detected, bu, appeared at late two-cell embryos as a zygotic transcript. The level of c-myc expresion was increased at later stases and peaked at blastocvst stage. To examine the functional role of promoter region for c-myc gene transcription, we fused the 5'upstream region (1.8 kb) including econ 1 of c-myc genomic DNA with E. coli lacE gene fnamed as pcMYC-laczl. pcMYC-lacZ was microiniected into the pronscleus of mouse one-cell embryovs, and p·salactosidase activity was determined tv histochemical staining with X-gal at different stases. f-galactosidase activity was detected only at blastocyst, but not at the earlier stage embryos. This result indicates that c-myc gene is transcriptionallv active during mouse preimplantation development.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.1
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• pp.13-20
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• 1997

The objective of this study was to determine the effect of EGF on the preimplantation development of mouse IVF embryos and their ICM and TE cell number. And also, we examined the expression of EGF-R protein on embryonic development by indirect immunofluorescence. The results obtained in these experiments were summarized as follows; Group culture (5 embryos/ 25 ${\mu}l$) showed more improved development rate to blastocyst than singly culture. This inferior development of singly cultured 2-cell embryos improved by the addition of EGF. Especially, 2-cell embryos cultured singly in 10 ng/ml of EGF (62.4%) indicated significant difference in development to blastocyst compared with control group (47.9%). Also, cell number of ICM and TE by differential labelling showed the increased pattern in the EGF treatment group. The stimulating effect of EGF with the development level was significantly increased after 4-cell stage (p<0.05). ICM proportion also showed the increased pattern with the developmental level in the EGF treatment group. In addition, expression of EGF-R by indirect immunofluorescence detected after 4-cell stage. Therefore, EGF could stimulate preimplantation mouse embryo development by binding with expressed EGE-R after 4-cell stage and produce the more increased ICM and TE cell number of blastocyst.

• Biomedical Science Letters
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• v.23 no.4
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• pp.372-379
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• 2017

This study was investigated to test whether paternal DNA that was destined for degradation was properly licensed by testing for the presence of mini-chromosome maintenance protein (MCM) 7 and origin recognition complex (ORC) 2 in the paternal pronuclei. ORC2 is one of the first licensing protein to come on and MCM7 is one of the last licensing protein to come on. Zygotes were prepared by injection of control and treated sperm injection (ICSI). To control for DNA breakage, epididymal spermatozoa were treated with DNase I to fragment the DNA, then injected into oocytes. The presence of MCM7 and ORC2 in the pronuclei of mouse zygotes was tested by immunohistochemistry, just before the onset of DNA synthesis, at 5 h after fertilization, and after DNA synthesis began, at 9 h post fertilization. We found that in all cases, both MCM7 and ORC2 were present in both pronuclei at 5 h after sperm injection, just before DNA synthesis began. This indicates that no matter how extensive the DNA damage, recruitment of licensing proteins to the origins of replication was not inhibited. Sperm DNA fragmentation does not prevent licensing of DNA replication origins. Furthermore, the embryo recognizes DNA that is damaged by nucleases. Our data indicate that the one-cell embryo does harbor a mechanism to prevent the replication of severely damaged DNA from spermatozoa, even though the embryos do not undergo classical apoptosis.