• Clinical and Experimental Reproductive Medicine
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• 제31권1호
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• pp.1-7
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• 2004

연구 목적: 본 연구의 목적은 마우스에서 배양액의 용량이 배반포 배 형성과 세포수에 미치는 영향을 조사하기 위하여 실시하였다. 연구 재료 및 방법: $3{\sim}4$주령 ICR 암마우스에게 48시간 간격으로 5 IU PMSG와 hCG 주사 후 (hCG 주사 후 수컷과 동숙) $46{\sim}50$시간에 난관으로부터 총 138개의 2-세포기 배를 회수하여 2 ml (group I) 또는 $50{\mu}l$ (group II)의 배양액 (Dulbecco's Modified Eagle Medium + 20% human follicular fluid)에서 72시간 동안 배반포기까지 배양하였다. 배반포 배는 zona-intact (ZiB)와 zona-escape (ZeB)로 등급을 구분하고 나서, propidium iodide와 bisbenzimide를 이용한 differential staining 방법으로 염색하여 평균 세포수, 내세포괴(ICM) 세포수, 영양배엽(TE) 세포수, 총 세포수에 대한 ICM의 비율 (%ICM) 및 ICM:TE 비율을 조사하였다. 결과에 대한 유의성 검정은 $X^2$ test와 t-test를 이용하였으며, p<0.05일 때 통계적인 차이가 있는 것으로 하였다. 결 과: Group I과 II에서, 총 배반포 ($62.3{\pm}20.7%$ vs. $63.8{\pm}22.9%$), ZiB ($31.9{\pm}24.0%$ vs. $30.4{\pm}18.2%$)와 ZeB 형성율 ($30.4{\pm}20.8%$ vs. $33.3{\pm}22.3%$)은 차이가 없었다. 87개의 배반포 배를 염색 시도하였는데, 명확하게 differential staining된 41개의 배반포 배만을 대상으로 세포수를 조사하였다. 평균 세포수 ($61.6{\pm}19.5$ vs. $63.7{\pm}26.8$), ICM 세포수 ($13.0{\pm}10.6$ vs. $12.8{\pm}10.5$), TE 세포수 ($49.0{\pm}19.0$ vs. $47.8{\pm}18.7$), %ICM ($21.0{\pm}12.6%$ vs. $21.1{\pm}13.2%$) 및 ICM:TE 비율 (1:$3.77{\pm}4.9$ vs. 1:$3.72{\pm}4.8$)에서도 group I과 II에서 차이가 없었다. 결 론: 마우스에서 배 발생 능력의 척도로 쓰이는 배반포 배 형성율 배반포 배의 등급 세포수 및 %ICM 등이 20% 난포액을 첨가한 MEM 배양액의 용량에 따라 영향을 받지 않았다.

• Journal of the Korea Academia-Industrial cooperation Society
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• 제12권8호
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• pp.3541-3546
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• 2011

Autophagy is an evolutionarily conserved lysosomal pathway for degrading cytoplasmic proteins, macromolecules, and organelles in addition to recycling protein and ATP synthesis. Although autophagy is very important during embryogenesis, the mechanism underlying the dynamic development during this process remains largely unknown. In order to obtain insights into autophagy in early embryo development, we analyzed gene expression levels of autophagy-related genes (ATGs) in mouse embryos developing in vitro. Using real time RT-PCR technique, ATGs including Atg2a, Atg3, Atg4b, Atg5, Atg6, Atg7, Atg9a, and Wipi3, as maternal transcripts, were only up-regulated in 1-cell embryo stage before zygotic genomic activation (ZGA), and then expression decreased from 2-cell to blastocyst embryo stage. ATGs including Dram and Atg9b were expressed abundantly in 1-cell embryo state and in blastocyst embryo stage, athough Atg8 and Ulk1 were constantly expressed during preimplantation stage. However, Atg4d were only up-expressed from 4-cell to blastocyst stage. These results suggest that autophagy is related in mouse embryo, which possibly gives an important role for early development.

• Journal of Oral Medicine and Pain
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• 제40권3호
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• pp.110-114
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• 2015

Purpose: Growth and differentiation factor (GDF)-11 is a transforming growth factor-${\beta}$ family member that plays important regulatory roles in development of multiple tissues which include axial skeletal patterning, palatal closure, and tooth formation. Proteins that have been identified as GDF-11 inhibitors include GDF-associated serum protein (GASP)-1 and GASP-2. Recently, we found that mice genetically engineered to lack both Gasp1 and Gdf11 have an increased frequency of cleft palate. The goal of this study was to investigate the roles of GDF-11 and its inhibitors, GASP-1 and GASP-2, during dental and craniofacial development and growth. Methods: Mouse genetic studies were used in this study. Homozygous knockout mice for Gasp1 ($Gasp1^{-/-}$) and Gasp2 ($Gasp2^{-/-}$) were viable and fertile, but Gdf11 homozygous knockout ($Gdf11^{-/-}$) mice died within 24 hours after birth. The effect of either Gasp1 or Gasp2 deletion in $Gdf11^{-/-}$ mice during embryogenesis was evaluated in $Gasp1^{-/-}$;$Gdf11^{-/-}$ and $Gasp2^{-/-}$;$Gdf11^{-/-}$ mouse embryos at 18.5 days post-coitum (E18.5). For the analysis of adult tissues, we used $Gasp1^{-/-}$;$Gdf11^{+/-}$ and $Gasp2^{-/-}$;$Gdf11^{+/-}$ mice to evaluate the potential haploinsufficiency of Gdf11 in $Gasp1^{-/-}$ and $Gasp2^{-/-}$ mice. Results: Although Gasp2 expression decreased after E10.5, Gasp1 expression was readily detected in various ectodermal tissues at E17.5, including hair follicles, epithelium in nasal cavity, retina, and developing tooth buds. Interestingly, $Gasp1^{-/-}$;$Gdf11^{-/-}$ mice had abnormal formation of lower incisors: tooth buds for lower incisors were under-developed or missing. Although $Gdf11^{+/-}$ mice were viable and had mild transformations of the axial skeleton, no specific defects in the craniofacial development have been observed in $Gdf11^{+/-}$ mice. However, loss of Gasp1 in $Gdf11^{+/-}$ mice occasionally resulted in small and abnormally shaped auricles. Conclusions: These findings suggest that both GASP-1 and GDF-11 play important roles in dental and craniofacial development both during embryogenesis and in adult tissues.

• Journal of Life Science
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• 제25권11호
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• pp.1230-1234
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• 2015

GATA binding protein 6 (GATA6) is a transcription factor that is expressed in the early blastocyst stage and controls the expression of important genes in the differentiation and development of the heart, pancreas, and intestine. This study confirmed the role of GATA6 in cell differentiation and organ development using mouse embryonic stem cells and zebrafish, respectively. First, the mouse embryonic stem cells were differentiated into pacemaker cardiomyocytes. An RT-PCR analysis revealed that the expression of the GATA6 gene was greatly increased from day 4 of differentiation. The expression of GATA6 was upregulated prior to increased expression of NK2 homeobox 5 (Nkx2.5) and myocyte enhancer factor 2C (MEF2C), which are critical transcription factors involved in regulating heart formation. To examine the role of GATA6 in development, GATA6 morpholino was microinjected into zebrafish embryos. Knockdown of GATA6 expression significantly decreased the heart size and heart rate in the zebrafish compared to a control. In addition, the brains were degenerated in the GATA6 morpholino-injected zebrafish. Acridine orange staining showed that knockdown of GATA6 expression increased apoptotic cells in the brain. Interestingly, knockdown of GATA6 expression decreased apoptotic cells in the early bud stage. This study points to the importance of the GATA6 gene in heart and brain development.

• Clinical and Experimental Reproductive Medicine
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• 제25권2호
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• pp.109-113
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• 1998

Objective: To ananlyze the direct effect of nitric oxide (NO), generated from sodium prusside (SNP) on the embryo developments in reproductive process. Design: Ova from mouse were treated to allow fertilization in in vitro culture. And the samples of fertilized ova were alloted into five alliqutos. Each alliquot was cultured in media treated with either concentration at 0 (n=92), $25{\mu}M$ (n=84), $50{\mu}M$ (n=80), $100{\mu}M$ (n=77), $500{\mu}M$ (n=54) of SNP. Main Outcome Measure: Rates of embryonal cell cleavages, viability and cell morphology were assessed during in vitro fertilization and culture. Results: As analyse the cell cleavage at 24 hours after in vitro culture of fertilised egg in variuos NO concentration, all of egg cells of each alliquot were developed to $2\sim4$ cell stage. But the alliquot of egg cells treated with $50{\mu}M$, which were totally degenerated. And also all embryonal cells of each alliquot were developed to 8 cell stage and morula stage on culture continuosly. And the embryonal cells of each alliquot were analysed at 24 and 48 hours following the in vitro culture. The rates of cell fragmentation and fusion were $4.2{\pm}3.4%$ in control group which is not treated with NO, while experimental groups was high, as rated $23.4{\pm}6.2%$ in $25{\mu}M$, $28.2{\pm}5.7%$ in $50{\mu}M$ and $32.1{\pm}6.4%$ in $100{\mu}M$ concentration of NO. Accordingly the rate of abnormal morphology of embryonal cell in control was lower significantly than that in each alliquot of experimental groups (p<0.05). And the degenerated rates of embryonal cells were 0% in control, $17.8{\pm}6.7%$ in $25{\mu}M$, $23.6{\pm}4.7%$ in $50{\mu}M$ and $26.8{\pm}11.2%$ in $100{\mu}M$ at 8 cells and morula on culture of 48 and 72 hours. On the examination of embryonal cells developed to blastocyst through in vitro culture, the rates of degenerated cells were $16.8{\pm}7.2%$ in control, $37.5{\pm}6.2%$ in $25{\mu}M$, $73.4{\pm}4.6%$ in $50{\mu}M$, 100% in $100{\mu}M$. Conclusion: This results suggeted that the NO in any concentrations is harmful on embryos in view of morphology as well as viability of cell, and the toxicity of NO on embryo is stronger at condition in higher concentration of NO.

• Journal of Periodontal and Implant Science
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• 제32권1호
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• pp.1-12
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• 2002

The periodontal ligament(PDL) is a unique tissue that is crucial for tooth function. However, little is known of the molecular mechanisms controlling PDL function. PDL-specific protein;PDLs22 had been previously identified as a novel protein isolated from cultured human PDL fibroblasts using subtraction hybridization between human gingival fibroblasts and PDL fibroblasts. The aim of this study was to examine the expression pattern and tissue localization of PDLs22 protein in embryonic and various postnatal stages of developing mouse using immunohistochemical staining. Embryos (E18) and postnatal (P1, P4, P5, P15, P18) were decapitated and the heads were fixed overnight in a freshly prepared solution of 4% paraformaldehyde. Some specimens were decalcified for $2{\sim}4$ weeks in a solution containing 10% of the disodium salt of ethylenediamine-tetraacetic acid (EDTA). Next, tissues were dehydrated, embedded in paraffin and sectioned serially at $6{\mu}m$ in thickness. Polyclonal antiserum raised against PDLs22 peptides, ISNKYLVKRQSRD, were made. The localization of PDLs22 in tissues was detected by polyclonal antibody against PDLs22 by means of immunohistochemical staining. The results were as follows; 1. Expression of PDLs22 protein was not detected in the tooth germ of bud and cap stage. 2. At the late bell stage and root formation stage, strong expression of PDLs22 protein was observed in developing tooth follicle, osteoblast-like cells, and subodontoblastic cells in the tooth pulp, but not in gingival fibroblasts, ameloblasts and odontoblasts of tooth germ 3. In erupted tooth, PDLs22 protein was intensely expressed in PDL and osteoblast-like cells of alveolar bone, but not in gingival fibroblasts, mature osteocytes and adjacent salivary glands. 4. In the developing alveolar bone and mid-palatal suture, expression of PDLs22 protein was seen in undifferentiated mesenchymal cells and osteoblast-like cells of developing mid-palatal suture, but not in mature osteocytes and chondrocytes. These results suggest that PDLs22 protein may play an important role in the differentiation of undifferentiated mesenchymal cells in the bone marrow and PDL cells, which can differentiate into multiple cell types including osteoblasts, cementoblasts, and PDL fibroblasts. However, more researches should be performed to gain a better understanding of the exact function of PDLs22 protein which related to the PDL cell differentiation.

• Korean Journal of Veterinary Research
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• 제42권1호
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• pp.35-43
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• 2002

Cryopreservation is commonly used for an efficient utilization of semen, oocytes and embryos but has disadvantage in the survival, development of the post-thawed eggs. The high risk in the survival, development of eggs after thawing is thought to be caused by inappropriate internal regulation of $Ca^{2+}$ and/or formation of intracellular ice crystals. In this experiment, we tested whether the $Ca^{2+}$ current (iCa), a decisive factor to $Ca^{2+}$ entry, was altered in post-thawed oocytes by using whole cell voltage clamp technique. The quality and survival rates of the oocytes derived from both fresh and frozen groups were examined by morphology and FDA-test. Vitrified oocytes (VOs) were incubated for 4 hr after thawing and then donated to this experiment. Ethyleneglycol-ficoll-galactose (EFG) was used as a cryoprotectant for vitrification. The membrane potential was held at -80 mV and step depolarizations of 250 ms were applied from -50 mV to 50 mV in 10 mV increments. The survival rates showed a higher in VOs vitrified with EFG containing $Ca^{2+}$ than in VOs vitrified with EFG under the $Ca^{2+}$-free condition (82.0% vs 14%). In group with/without $Ca^{2+}$, the survival rates were significantly (P<0.01) difference. In the fresh metaphase II oocytes (FOs), current-voltage (I-V) relationship showed that iCa began to activate at -40 mV and reached its maximum at -10 mV. With same voltage pulses, inward currents were elicited in VOs. I-V relationships observed in VOs were similar to those in FOs. Time constants of activation and inactivation of the inward current shown in VOs were not different to those in FOs. This accordance in I-V relations and time constants in FOs with those in VOs indicates that the inward currents in FOs are unaltered by vitrification and thawing. Therefore, vitrification with EFG does not play as a factor to deteriorate $Ca^{2+}$ entry across the membrane of the oocytes.

• Asian-Australasian Journal of Animal Sciences
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• 제21권5호
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• pp.745-753
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• 2008

Gene-manipulated mice were discovered for the first time about a quarter century ago. Since then, numerous sophisticated technologies have been developed and applied to answer key questions about the fundamental roles of the genes of interest. Functional genomics can be characterized into gain-of-function and loss-of-function, which are called transgenic and knock-out studies, respectively. To make transgenic mice, the most widely used technique is the microinjection of transgene-containing vectors into the embryonic pronucleus. However, there are critical drawbacks: namely position effects, integration of unknown copies of a foreign gene, and instability of the foreign DNA within the host genome. To overcome these problems, the ROSA26 locus was used for the knock-in site of a transgene. Usage of this locus is discussed for the gain of function study as well as for several brilliant approaches such as conditional/inducible transgenic system, reproducible/inducible knockdown system, specific cell ablation by Cre-mediated expression of DTA, Cre-ERTM mice as a useful tool for temporal gene regulation, MORE mice as a germ line delete and site specific recombinase system. Techniques to make null mutant mice include complicated steps: vector design and construction, colony selection of embryonic stem (ES) cells, production of chimera mice, confirmation of germ line transmission, and so forth. It is tedious and labor intensive work and difficult to approach. Thus, it is not readily accessible by most researchers. In order to overcome such limitations, technical breakthroughs such as reporter knock-in and gene knock-out system, production of homozygous mutant ES cells from a single targeting vector, and production of mutant mice from tetraploid embryos are developed. With these upcoming progresses, it is important to consider how we could develop these systems further and expand to other animal models such as pigs and monkeys that have more physiological similarities to humans.

• Journal of Animal Science and Technology
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• 제56권4호
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• pp.15.1-15.10
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• 2014

Background: Morphologically classifying embryos is important for numerous laboratory techniques, which range from basic methods to methods for assisted reproduction. However, the standard method currently used for classification is subjective and depends on an embryologist's prior training. Thus, our work was aimed at developing software to classify morphological quality for blastocysts based on digital images. Methods: The developed methodology is suitable for the assistance of the embryologist on the task of analyzing blastocysts. The software uses artificial neural network techniques as a machine learning technique. These networks analyze both visual variables extracted from an image and biological features for an embryo. Results: After the training process the final accuracy of the system using this method was 95%. To aid the end-users in operating this system, we developed a graphical user interface that can be used to produce a quality assessment based on a previously trained artificial neural network. Conclusions: This process has a high potential for applicability because it can be adapted to additional species with greater economic appeal (human beings and cattle). Based on an objective assessment (without personal bias from the embryologist) and with high reproducibility between samples or different clinics and laboratories, this method will facilitate such classification in the future as an alternative practice for assessing embryo morphologies.

• Journal of Embryo Transfer
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• 제8권2호
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• pp.75-82
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• 1993

To investigate the effect of EDTA on the in vitro development of blastomeres isolated from 2, 4, and 8-cell embryos(termed 1 /2, 1 /4 and 1 /8 blastomeres, respectively) of ICR strain mice, those were cultured in vitro in 35 mm culture dishes containing NaHCO$_3$-BMOC-3 medium supplemented with 10, 50, 100, or 500 $\mu$M of EDTA at 37$^{\circ}C$ for 72hrs. under the atmosphere of 5% $CO_2$and 95% air. EDTA supplementation of 10, 50, or 100 $\mu$M to medium significantly(P<0.01) increased blastocyst formation rate compared with controls in 1 /2(58.3, 63.7, and 61.3% vs 21.6%), 1 /4(54.7, 57.5 and 62.2% vs. 2L3%), and 1 /8 blastomeres(46.2, 48.7, and 57.7% vs. 19.1%). Whereas, it was significantly(P<0.01) decreased to 4.5, 2.3, and 2.0% for 1 /2, 1 /4 and 1 /8 blastomeres, respectively by the EDTA supplementation of 500 M Both the nuclear number(P<0.05) and diameter of blastocysts(P<0.01) developed from balstomeres were significantly affected by the origin of blastomeres. The nuclear number of blastocysrs developed from 1/2, 1/4, and 1/8 blastomeres ranged 28.3i1.3, 24.18i1.2, and 19.84$\pm$0.9, respectively. And the diameter of those blastocysts was 87.2$\pm$1.1, 56.4$\pm$0.9, and 39.2$\geq$0.8 $\mu$M, respectively.