• 한국가축번식학회지
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• 제24권3호
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• pp.311-317
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• 2000

본 연구는 체외에서 한우 난포란의 성숙과 수정후 배의 체외발생에 있어서 온도충격의 영향이 처리온도 및 처리시간에 미치는 효과를 검토하였다. 1. 체외에서 생산된 4~8 세포기 배의 배반포로의 배발달율에 있어서 온도충격의 적정온도는 41$^{\circ}C$, 처리시간은 30초였다. 2. 체외성숙 후의 온도충격이 체외성숙 전의 온도충격보다 수정란의 분할율을 유의하게 상승시켰다(p<0.05). 3. 체외성숙 후와 배양 5일째 배에 각각 온도처리를 하였을 경우 비처리군보다 배반포의 배발달율이 향상되었다.

• 농업과학연구
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• 제22권1호
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• pp.96-102
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• 1995

본 연구는 돼지 체외수정란의 발육에 있어서 적절한 에너지 급원을 구명하기 위하여 수행하였는 바, BMOC-II 배양액을 기본배양액으로 하여 여기에 해당과정을 통하여 에너지를 생산하는 glucose와 이 과정에 중요하게 작용하는 phosphate를 실험설계에 따라 첨가한 배 양액에 체외에서 발생된 돼지의 수정란을 배양하여 수정란의 발육에 미치는 영향을 조사하였다. 1. 배양액에 Glucose와 phosphate가 모두 첨가되지 않은 배양액에서 배양된 돼지의 수정란은 15.2%가 4-세포란 이상으로 발육하였으나, 상실배로는 전혀 발육하지 못하였다. 2. 배양액에 첨가된 glucose의 농도는 phosphate의 첨가유무에 관계없이 농도가 높은 처리에서 4-세포기 이상으로 발달한 비율은 감소하였으나, 상실배에 도달한 비율은 증가하는 경향을 나타냈고, phosphate와 함께 첨가되었을 때보다 단독으로 첨가하였을 때가 첨가농도에 관계없이 돼지 체외수정란의 발육능을 증가시켰다.

• Reproductive and Developmental Biology
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• 제29권3호
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• pp.145-147
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• 2005

Autocrine or paracrine mediators released by the early embryo are implicated in the support of embryonic development. Their mechanisms and optimal embryo density in the medium, however, are uncertain. This study was conducted to establish the optimal embryo density and culture medium volume in mouse parthenogenetic embryo culture. In experiment 1, culture of parthenogenetirally activated oocytes at a concentration of $2{\~}4$ embryos/${\mu}L$ significantly improved development to the blastoryst stage ($72{\%}{\leq}$) compared with culture at the lower ($0.2{\~}1$e mbryos/${\mu}L,\;0\~37.5\%$) and the higher ($5{\~}6$ embryos/${\mu}L,\;30\~53\%$) concentration for 120 h when the oocytes were cultured in a 5 ${\mu}L$ drop under mineral oil In experiment 2, the embryos cultured at a concentration of $2{\~}4$ embryos/${\mu}L$ in a 10 ${\mu}L$ drop ($81.1{\%}$) showed significantly higher blastocyst rates than those in a 5 ${\mu}L$ drop ($68.5{\%}$). This study optimizes in vitro culture condition by modifying embryo density and the volume of culture medium It may give appropriate level of autocrine and/or paracrine factors to enhance viability and subsequent normal development of mouse parthenogenetic embryos in vitro.

• Asian-Australasian Journal of Animal Sciences
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• 제8권4호
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• pp.317-320
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• 1995

The effect of co-culture with cumulus cells and granulosa cells during maturation and development on in vitro developmental potency of follicular oocytes was examined. TCM-199 supplemented with 15% FCS and hormones was used as maturation medium. Sperm from frozen semen was capacitated in modified mTALP medium containing 0.3% BSA, $10{\mu}g/ml$ heparin and 5 mM/ml caffeine. The fertilized embryos were co-cultured on monolayer of cumulus cells or granulosa cells in TCM-199. The embryo co-cultured with cumulus cells showed higher percentage of embryo developed to morula and blastocyst (73.3%) than the embryo co-cultured without cumulus cells (30.8%). The percentage of oocytes developed to morula and blastocyst among cleaved oocytes was significantly (p < 0.05) higher in the oocytes co-cultured with cumulus cells during development (62.4%) than in the oocytes co-cultured with granulosa cells during maturation and with cumulus cells during development (52.3%), and in the oocytes co-cultured with granulose cells during development (52.8%). The results of this study indicate that co-culture of in vitro fertilized embryos with cumulus cells in the development medium increased the rate of embryos developed to morula and blastocyst among cleaved oocytes.

• 한국수정란이식학회지
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• 제13권2호
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• pp.97-106
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• 1998

The pronuclear injection of metallothionein-human growth hormone (MT-hGH) gene into rabbit zygotes was performed to establish in vitro developmental system and to detect the presence of the injected gene by nested PCR. Mature female New Zealand White rabbits were superovulated by eGG and hCG treatments. The rabbits were mated and the zygotes were collected from the oviducts 18-22 h after hCG injection by flushing with D-PBS. Two to three picoliters of MT-hGH gene was microinjected into male pronuclei. The foreign gene-injected zygotes were cultured in TCM-199 or RD mediurn containing 10% FCS with a monolayer of rabbit oviductal epithelial cefls in a 5% $CO_2$ incubator. The presence of injected DNA in rabbit embryos or blastomeres at different developmental stages .vas detected by a nested PCR analysis. The results are summarized as follows ; 1.The developmental rate of the MT-hGH gene-injected zygotes to blastocyst was significantly higher in TCM-199 medium (68.1%) than in RD medium (42.9%). 2.The gene injection into pronuclei at 18 or 22 hours post hCG treatment during pronuclear stage did not much affect on the in vitro development of the rabbit embryos. 3.The rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased when they developed to blastocysts. The results indicate that the screening of transgene in rabbit embryos by nested PCR analysis could be a prornisible method for the preselection of transgenic embryos. Furthermore, the preselection of transgenic embryos would greatly reduce hoth the cost and effort of production of transgenic animals.

• Toxicological Research
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• 제14권3호
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• pp.357-363
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• 1998

The teratogenic potential of the anticonvulsant drug phenytoin (PHT) has been well documented both in the human and in the experimental animals. However there are few reports on the effects of PHT on embryonic development in rats in vitro. The present study was performed to evaluate the teratogenic effects of PHT using whole-embryo culture system in rats. Sprague-Dawley rat embryos were explanted on gestational day (GD) 9.5 and cultured for 48 hrs in the immediately centrifuged and heat-inactivated rat serum containing 0,25,50, or $100{\mu}g$ PHT/mL. At the end of culture period the embryos were scored for morphological development according to the procedure of Van Maele-Fabry, and their total protein contents were determined. At 100 ${\mu}$g/mL of culture medium. PHT caused significant reduction in developmental score and protein content of embryos and a high incidence morphological abnormalities (100%). Characteristic malformations included altered yolk and embryonic circulation, craniofacial hypoplasia, neural tube schisis, branchial arch defects, abnormal ratation, and limb bud hypoplasia, among others. There were no adverse effects on embryonic growth and development at concentrations of 25 and 50 ${\mu}$g /mL of culture medium. The results indicated that the dysmorphogenic effect of PHT on cultured embryos is due to a direct interference with embryonic development.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2001년도 춘계학술발표대회
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• pp.66-66
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• 2001

Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제21권11호
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• pp.1665-1672
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• 2008

Interspecies somatic cell nuclear transfer (iSCNT) is a useful method to preserve endangered species and to study the reprogramming event of a nuclear donor cell by the oocyte. Although several studies of iSCNT using murine cells and bovine oocytes have been reported, the development of murine-bovine iSCNT embryos beyond the 8-cell stage has not been successful. In this paper, we examined the developmental potential of embryos reconstructed with a murine embryonic fibroblast as the nuclear donor and a bovine oocyte as the cytoplasm recipient. The reconstructed embryos were cultured in CZB (murine medium) or CR1aa (bovine medium). In addition, for the development of a murine-bovine iSCNT blastocyst, the antioxidant ${\beta}$-mercaptoethanol (${\beta}ME$) was supplemented to CR1aa medium. Furthermore, to verify the mouse genome activation in murine-bovine iSCNT embryos, RT-PCR analysis of murine Xist was performed. The development of the murine-bovine iSCNT embryos cultured in CR1aa was significantly higher than that in CZB (p<0.05). With respect to the effect of BME on the development of the murine-bovine iSCNT blastocyst, addition of BME produced a significant increase in blastocyst development (p<0.05). Karyotype analysis confirmed that the reconstructed embryos were derived from murine cells (40XX). The Xist gene was gradually increased from the 8-cell stage to the blastocyst stage. This is the first report of blastocyst development of iSCNT embryos derived from murine somatic cells and bovine oocytes. These results demonstrate that bovine cytoplasm can support the development of later stages of a preimplantation embryo from murine-bovine iSCNT.

• 한국환경보건학회지
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• 제24권2호
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• pp.126-133
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• 1998

Effects of B(a)P on preimplantation mouse embryos in vitro were studied. Preimplantation mouse embryos were exposed to a concentration of 0.3, 1, 3 and 10 $\mu$M B(a)P for 72 hrs. The toxicological effects of B(a)P were evaluated by morphological observation of embryos up to the blastocyst stage, and by measuring DNA, RNA and protein synthesis by radioactive precursor incorporation. At 1 $\mu$M B(a)P did not affect preimplantation development but interfered with hatching and ICM formation. Suppressing effect of ICM formation was dose dependent. At the eight cell stage, the developmental rate was decreased at above 3 $\mu$M of B(a)P. At the blastocyst stage, attachment and trophoblast outgrowth were diminished at the 10 $\mu$M of B(a)P and ICM formation was decreased at 1 $\mu$M of B(a)P. Inner cell number of blastocyst was decreased dose dependently. So, number of ICM was one of the most sensitive and toxicological end point. The RNA incorporation rate of 0.1 $\mu ^3$H-uridine was dosedependent and the protein incroporation of 0.5 $\mu Ci ^{35}$S-methionine showed a significant decrease after 48 hrs. But the DNA incorporation rate of methyl-$^3$H thymidine was not affected. Our results suggested that B(a)P did not affect the DNA replication but transcription was inhibited by dose dependent manner. There delay of development during the blastocyst stage was mainly due to the inhibition of RNA synthesis followed by protein synthesis.

• Reproductive and Developmental Biology
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• 제31권2호
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• pp.83-90
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• 2007

Insulin-like growth factor II (IGF2) and H19 genes are mutually imprinted genes which may be responsible for abnormalities in the cloned fetuses and offspring. This study was performed to identify putative differentially methylated regions (DMRs) of porcine H19 locus and to explore its genomic imprinting in in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) embryos. Based on mice genomic data, we identified DMRs on H19 and found porcine H19 DMRs that included three CTCF binding sites. Methylation patterns in IVF and SCNT embryos at the 2-, 4-, $8{\sim}16$-cells and blastocyst stages were analyzed by BS (Bisulfite Sequencing)-PCR. The CpGs in CTCF1 was significantly unmethylated in the 2-cell stage IVF embryos. However, the 4- (29.1%) and $8{\sim}16$-cell (68.2%) and blastocyst (48.2%) stages showed higher methylation levels (p<0.01). On the other hand, SCNT embryos were unmethylayted ($0{\sim}2%$) at all stages of development. The CpGs in CTCF2 showed almost unmethylation levels at the 2-,4- and $8{\sim}16$-cell and blastocyst stages of development in both IVF ($0{\sim}14.1%$) and SCNT ($0{\sim}6.4%$) embryos. At all stages of development, CTCF3 was unmethylated in IVF ($0{\sim}17.3%$) and SCNT ($0{\sim}1.2%$) embryos except at the blastocyst stage (54.5%) of IVF embryos. In conclusion, porcine SCNT embryos showed an aberrant methylation pattern comprised to IVF embryos. Therefore, we suggest that the aberrant methylation pattern of H19 loci may be a reason for increased abnormal fetus after embryo transfer of porcine SCNT embryos.