• Clinical and Experimental Reproductive Medicine
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• 제25권1호
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• pp.77-86
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• 1998

The uptake of glucose for metabolism and growth is essential to most animal cells and is mediated by glucose-transporter (GLUT) proteins. The aim of this study was to determine which class of glucose transporter molecules was responsible for uptake of glucose in the mouse early embryo and at which stage the corresponding genes were expressed. In addition, co-culture system with vero cell was used to investigate the effect of the system on GLUT expression. Two-cell stage embryos were collected from the superovulated ICR female and divided into 3 groups. As a control, embryos were cultured in 0.4% BSA-T6 medium which includes glucose. For the experimental groups, embryos were cultured in either co-culture system with vero cells or glucose-free T6 medium supplemented with 0.4% BSA and pyruvate as an energy substrate. 2-cell to blastocyst stage embryos in those groups were respectively collected into microtubes (50 embryos/tube). Total RNA was extracted and RT-PCR was performed. The products were analysed after staining ethidium bromide by 2% agarose gel electrophoresis. Blastocysts were collected from each group at l20hr after hCG injection. They were fixed in 2.5% glutaraldehyde, stained with hoechst, and mounted for observation. In control, GLUT1 was expressed from 4-cell to blastocyst. GLUT2 and GLUT3 were expressed in morula and blastocyst. GLUT4 was expressed in all stages. When embryos were cultured in glucose-free medium, no significant difference was shown in the expression of GLUT1, 2 and 3, compared to control. However GLUT4 was not expressed until morular stage. When embryos were co-cultured with vero cell, there was no significant difference in the expression of GLUT1, 2, 3 and 4 compared to control. To determine cell growth of embryos, the average cell number of blastocyst was counted. The cell number of co-culture ($93.8{\pm}3.1$, n=35) is significantly higher than that of control and glucose-free group ($76.6{\pm}3.8$, n=35 and $68.2{\pm}4.3$, n=30). This study shows that the GLUT genes are expressed differently according to embryo stage. GLUTs were detectable throughout mouse preimplantation development in control and co-culture groups. However, GLUT4 was not detected from 2- to 8-cell stage but detected from morula stage in glucose-free medium, suggested that GLUT genes are expressed autocrinally in the embryo regardless of the presence of glucose as an energy substrate. In addition, co-culture system can increase the cell count of blastocyst but not improve the expression of GLUT. In conclusion, expression of GLUT is dependent on embryo stage in preimplantation embryo development.

• 한국가축번식학회지
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• 제12권1호
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• pp.37-41
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• 1988

These studies were carried out to examine the sex of preimplantation mouse embryo. For the investigation of sex-ration of mouse embryos, morula and blastocysts stage embryos treated with H-Y antiserum (10%, v/v) and FITC anti-mouse-IgG were divided into the positive and negative embryos. Positive and negative identified embryos were observed the viability according to the in vitro cultured and the sex ratio was also investigated by chromosomal analysis. The results obtained in these studies were summarized as follows: 1. Two hundred sixty-seven recovered embryos of morula or blastocyst stage were incubated in medium containing H-Y antiserum and FITC anti-mouse-IgG. Positively or negatively identified embryos were 139 and 128. This trend indicated the approximal sex ratio was 1:1. 2. Sex ratio was measured using the embryos treated with indirect immunofluorescence assay to examine the relationship between embryo developmental stage and sex ratio. Sex ratio of morula stage embryos was 45.2% positive and 54.8% negative, on the other hand, the ratio switched to 56.4% positive and 43.6% negative embryo in blastocyst stage. 3. Fourty-seven positive and 57 negative embryos were obtained out of 104 morula stage embryos treated with indirect immunofluorescence assay. Survived positive or negative embryos during in vitro culture were 42 and 49, respectively out of 47 and 57 embryos. 4. The numbers of negative and positive embryos were 171 and 92 out of 163 blastocyst embryos which were incubated in the medium containing H-Y antiserum and FITC anti-mouse-IgG. The result of karyotype test showed the successful rate of sexing embryo is positive and negative embryos was63.0% (58/92) and 62.0% (44/71). The final female to male ratio within 58 positive embryos was 22.7:77.6, and the ratio of the 44 negative embryos was 77.3:22.7.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2003년도 제3회 발생공학 국제심포지움 및 학술대회
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• pp.81-81
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• 2003

• 한국발생생물학회지:발생과생식
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• 제2권2호
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• pp.197-203
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• 1998

포유류 배아발생 중 DNA 메틸화는 세포분화와 유전자발현에서 중요한 역할을 하는 것으로 알려져 있다. 그러나, 생쥐 착상전 초기배아 발생 중 메틸화효소에 의해 유지되는 DNA 메틸화의 중요성과 자세한 기작은 잘 이해되고 있지 않다. 이 연구에서 DNA 메틸화의 역할에 관하여 알아보기 위하여, 성숙난자와 착상전 초기배아에서 DNA 메틸전이효소의 발현양상을 조사하였다. 이를 위해, DNA 메틸전이효소를 암호화하고 있는 cDNA에서 primer를 고안하였다. Primer의 정확도와 PCR조건의 적합화를 통하여, DNA MTase 전사물이 성숙난자와 착상전 초기배아에서 검출되었다. DNA MTase의 mRNA량은 성숙난자에서 가장 높으며, 전핵시기까지 비슷한 정도로 유지되었다. 이후 8-세포기까지 지속적으로 감소하다 상실기 배아에서 다시 검출되어 포배기까지 증가하는 양상을 보였다. 그리고, RNA polymerase II 억제제를 전핵시기 배아에 처리하여, 난자와 전핵시기 배아에 다량 존재하는 전사물이 모계유래인 것을 확인하였다. 결국, 난자와 전핵시기 배아에 상대적으로 다량 존재하는 DNA 메틸전이효소의 전사물은 아마도 착상전 초기배아에서 DHA 메틸화의 유지에 필요하며, 착상전 초기배아 발생에 있어서 유전자발현과 세포분화에 영향을 줄 것임을 시사하고 있다.

• 한국발생생물학회지:발생과생식
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• 제3권1호
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• pp.69-74
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• 1999

인슐린 유사 성장 호르몬 1과 2 (IGF-1 & IGF-2)는 착상 전 초기배아 발생을 조절하는 중요한 요소이다. 생쥐 착상 전 초기배아에서 IGF-1의 역할에 관한 연구를 위해, IGF-1과 IGF-1 수용체의 전사물의 존재 여부를 난자와 착상 전 초기배아에서 조사하였다. 새로이 고안된 IGF-1 primer를 이용하여 난자에서 전사물을 검출하였다. 그리고, PCR 산물을 제한효소인 Msp I으로 절단하여 확인하였다. 이 실험에서 IGF-1과 IGF-1 수용체의 전사물이 난자와 착상 전 초기배아에서 모두 검출됨을 보였다. GV-난자에 다량 존재하는 mRNA는 4- 혹은 8-세포기까지 지속적으로 감소하다가 이후에 다시 증가하는 양상을 보였다. GV-난자에서 IGF-1과 IGF-lR 전사물이 존재한다는 것은 초기배아에 존재하는 전사물이 모계유래 산물임을 암시한다. 또한, 난자와 착상 전 초기배아에 IGF-1과 IGF-1 수용체 전사물이 존재하는 것으로 보아 착상 전 초기배아에서 IGF-1은 자가 분비되어 IGF-1 수용체의 신호전달 경로를 통하여 배아발생에 작용하는 것으로 사료된다.

• Clinical and Experimental Reproductive Medicine
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• 제38권3호
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• pp.126-134
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• 2011

Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence $in-situ$ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium.

• 한국수정란이식학회지
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• 제23권1호
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• pp.19-24
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• 2008

The purpose of this study was to compare the efficiency of slow freezing with that of vitrification method for the cryopreservation of human embryos. Human embryos were derived from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and the mixed solution of propanedial (1.5, 1.0, 0.5M PROH) and sucrose (0.1M), ethylene glycol (7.5, 15%), dimethyl sulfoxide (7.5, 15% DMSO), sucrose (0.5, 1.0M) and SPS (Serum Protein Substitute) was used for a cryoprotectant for slow freezing and vitrification solution, respectively. Rates of recovery after thawing, morphological normality, post-thaw viability, arrest, morphological abnormality and preimplantation development were compared between two protocols. After freezing-thawing, recovery and survial rate of slow freezing was (88.6% and 73.4%), whereas vitrification was (99.2% and 96.2%) (p<0.05). The arrest rate of slow freezing was significantly lower compared with those of vitrification(8.7% vs 29.9%) (p<0.05). Preimplantation development to the 2-cell (83.8% vs 67.7%), 4-cell (69.0% vs 47.2%) and 8-cell (62.4% vs 37.8%) stages 24, 48 and 72 h after thawing, respectively, were higher in the slow freezing than the vitrification. After slow freezing and vitrification of human embryo at 2-8cell stage, the rate of recovery rate, survival rate and partial damage rate were 92.0% vs 100%, 80.4% vs 96.2% and 52.2% vs 19.0%, respectively. And partial damage rate was significantly lower than those of slow freezing method (p<0.05). These results demonstrate that a slow freezing using PROH is more efficient than a vitrification for cryopreserving the human zygotes, although the vitrification yielded better recovery, survival and partial damage of frozen-thawed 2-8 cell stage embryos than slow freezing method.

• 한국수정란이식학회지
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• 제24권1호
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• pp.65-69
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• 2009

The aim of the present study was to investigate the cleavage pattern, its developmental ability and apoptosis of porcine embryo in vitro. Morphology data on a total of 919 embryos were analyzed retrospectively. Forty-eight hours after insemination, embryos were classified into five groups based on the cleavage state as follows; 1 cell, 2 cell, 4 cell, 5 to 8 cell and fragmentation. These groups were cultured another 120 hours and then evaluated for blastocyst formation. Blastocyst formation rates were significantly higher in 4 cell (42.5%) and 5 to 8 cell (48.6%) cleaving groups than in other groups (p<0.05). On the other hand, 2 cell and fragmentation groups produced 4.9% and 3,9% blastocysts, respectively. And we could verify that in the event of 2 cell block and fragmentation of embryo. To analyze the apoptotic frequency in preimplantation development of porcine IVF embryos, all cells of each blastocyst were performed by TUNEL assay. There were no significantly differences in the total cell numbers of embryos and apoptotic cell rate in blastocysts among the each classified groups. Data suggest that 4 cell and 5 to 8 cell cleaving embryos at 48 hour after insemination have high developmental competence, and may be an useful parameter to predict the development of preimplantation embryos and to study using preimplanation embryonic research.

• Clinical and Experimental Reproductive Medicine
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• 제44권3호
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• pp.119-125
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• 2017

Objective: In vitro culture of preimplantation embryos is improved by grouping embryos together in a drop of media. Individually cultured embryos are deprived of paracrine factors; with this in mind, we investigated whether the addition of a single embryo-secreted factor, interleukin-6 (IL-6), could improve the development of individually cultured embryos. Methods: Mouse embryos were cultured individually in $2{\mu}L$ of G1/G2 media in 5% oxygen and supplemented with a range of doses of recombinant mouse or human IL-6. Results: Mouse IL-6 increased hatching at doses of 0.01 and 10 ng/mL compared to the control (93% and 93% vs. 78%, p< 0.05) and increased the total number of cells at a dose of 0.1 ng/mL compared to the control ($101.95{\pm}3.36$ vs. $91.31{\pm}3.33$, p< 0.05). In contrast, the highest dose of 100 ng/mL reduced the total number of cells ($79.86{\pm}3.29$, p< 0.05). Supplementation with human IL-6 had a different effect, with no change in hatching or total cell numbers, but an increase in the percentage of inner cell mass per embryo at doses of 0.1, 1, and 100 ng/mL compared to the control ($22.9%{\pm}1.1%$, $23.3%{\pm}1.1%$, and $23.1%{\pm}1.1%$ vs. $19.5%{\pm}1.0%$, p< 0.05). Conclusion: These data show that IL-6 improved mouse embryo development when cultured individually in complex media; however, an excess of IL-6 may be detrimental. Additionally, these data indicate that there is some cross-species benefit of human IL-6 for mouse embryos, but possibly through a different mechanism than for mouse IL-6.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2005년도 제5회 발생공학 국제심포지엄 및 학술대회
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• pp.107-107
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• 2005