• Reproductive and Developmental Biology
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• v.33 no.3
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• pp.171-177
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• 2009

Spermatogonial stem cells(SSCs) only are responsible for the generation of progeny and for the transmission of genetic information to the next generation in male. Other in vitro studies have cultured SSCs for proliferation, differentiation, and genetic modification in mouse and rat. Currently, information regarding in vitro culture of porcine Germline Stem Cell(GSC) such as gonocyte or SSC is limited and is in need of further studies. Therefore, in this study, we report development of a successful culture system for gonocytes of neonatal porcine testes. Testis cells were extracted from $10{\sim}14$-day-old pigs. These cells were harvested using enzymatic digestion, and the harvested cells were purified with combination of percoll, laminin, and gelatin selection techniques. The most effective culture system of porcine gonocytes was established through trial experiments which made a comparison between different feeder cells, medium, serum concentrations, temperatures, and $O_2$ tensions. Taken together, the optimal condition was established using C166 or Mouse Embryonic Fibroblast(MEF) feeder cell, Rat Serum Free Medium(RSFM), 0% serum concentration, $37^{\circ}C$ temperature, and $O_2$ 20% tension. Although we discovered the optimal culture condition for proliferation of porcine gonocytes, the gonocyte colonies ceased to expand after one month. These results suggest inadequate acquirement of ingredients essential for long term culture of porcine GSCs. Consequently, further study should be conducted to establish a successful long-term culture system for porcine GSCs by introducing various growth factors or nutrients.

• Analytical Science and Technology
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• v.31 no.5
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• pp.208-218
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• 2018

Autophagy is a cellular process whereby cytosolic materials or organelles are taken up in a double-membrane vesicle structure known as an autophagosome and transported into a lysosome for degradation. Although autophagy has been studied at the genetic, cellular, or biochemical level, systematic ultrastructural quantitative analysis of autophagosomes during the autophagy process by using transmission electron microscopy (TEM) has not yet been reported. In this study, we performed ultrastructural analysis of autophagosomes in wild-type (WT) mouse embryonic fibroblasts (MEFs) and autophagy essential gene (atg5) knockout (KO) MEFs. First, we performed ultrastructural analysis of autophagosomes in WT MEFs compared to atg5 KO MEFs in basal autophagy or starvation-induced autophagy. Although we observed phagopore, early, late autophagosomes, or autolysosomes in WT MEFs, atg5 KO MEFs had immature autophagosomes that showed incomplete closure. Upon starvation, late autophagosomes accumulated in WT MEFs while the number of immature autophagosomes significantly increased in atg5 KO MEF indicating that atg5 plays an important role in the maturation of autophagosomes. Next, we examined autophagosomes in the cell model expressing polyQ-expanded N-terminal fragment of huntingtin. Our TEM analysis indicates that the number of late autophagosomes was significantly increased in the cells expressing the mutant huntingtin, indicating that improving the fusion of autophagosome with lysosome may be effective to enhance autophagy for the treatment of Huntington's disease. Taken together, the results of our study indicate that ultrastructural and quantitative analysis of autophagosomes using TEM can be applied to various human cellular disease models, and that they will provide an important insight for cellular pathogenesis of human diseases associated with autophagy.

• Journal of the korean academy of Pediatric Dentistry
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• v.26 no.4
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• pp.652-663
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• 1999

Craniosynostosis, the premature fusion of cranial sutures, presumably involves disturbance of the interactions between different tissues within the cranial sutures. Interestingly, point mutaions in the genes encoding for the fibroblast growth factor receptors(FGFRs), especially FGFR2, cause various types of human craniosynostosis syndromes. To elucidate the function of these genes in the early morphogenesis of mouse cranial sutures, we first analyzed by in situ hybridization the expression of FGFR2(BEK) and osteopontin, an early marker of osteogenic differentiation, in the sagittal suture of calvaria during embryonic(E15-E18) and postnatal stage(P1-P3). FGFR2(BEK) was intensely expressed in the osteogenic fronts, whose cells undergo differentiation into osteoprogenitor cells that ultimately lay down the bone matrix. Osteopontin was expressed throughout the parietal bones excluding the osteogenic fronts, the periphery of the parietal bones. To further examine the role of FGF-mediated FGFR signaling in cranial suture, we did in vitro experiments in E15.5 mouse calvarial explants. Interestingly, implantation of FGF2 soaked beads onto both the osteogenic fronts and mid-mesenchyme of sagittal suture after 36 hours organ culture resulted in the increase of the tissue thickness and cell number around FGF2 beads, moreover FGF4-soaked beads implanted onto the osteogenic fronts stimulated suture closure due to an accelerated bone growth, compared to FGF4 beads placed onto mid-mesenchyme of sagittal suture and BSA control beads. In addition FGF2 induced the ectopic expression of osteopontin and Msx1 genes. Taken together, these data indicate that FGF-mediated FGFR signaling has a important role in regulating the cranial bone growth and maintenance of cranial suture, and suggest that FGF-mediated FGFR signaling is involved in regulating the balance between the cell proliferation and differentiation through inducing the expression of osteopontin and Msx1 genes.

• Korean Journal of Animal Reproduction
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• v.24 no.3
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• pp.289-297
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• 2000

This study was conducted to investigate whether various protein sources and co-culture affect in vitro development of porcine zygotes derived from In vitro maturation/fertilization (IVM/IVF). These results obtained in these experiments are summarized as follows 1. When porcine oocytes matured and fertilized In vitro were cultured in NCSU 23 medium supplemented with various BSA concentrations (0.4, 0.8 and 3.2%), In vitro developmental rates of porcine zygotes to blastocyst stage were 22.9, 18.4 and 14.6%, respectively. High concentration of BSA (3.2%) showed a smaller nuclei number (36.1$\pm$11.8) of blastocysts than 0.4 and 0.8% BSA groups (53.2$\pm$27.4 and 61.2$\pm$22.5, respectively) (P<0.05). This result indicates that high concentration of BSA is detrimental on preimplantation development of IVF-derived porcine embryos. 2. No differences were detected in the developmental rate and mean nuclei number of porcine embryos between 10 and 20% FBS concentrations in culture medium. 3. IVF-derived porcine embryos co-cultured with mouse or porcine embryonic fibroblast cells showed a lower development to the blastocyst stage than those without co-culture system. Consequently, the present study suggests that high concentration of BSA as a protein source in culture medium suppresses development potential of porcine embryos produced In vitro. In addition, co-culture with somatic cells is not effective on in vitro development of IVF-derived porcine embryos to blastocyst stage.

• Journal of Life Science
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• v.15 no.4 s.71
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• pp.542-547
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• 2005

Hypersensitivity of cells lacking Brcal to DNA interstrand .ross-link (ICL) agents such as cisplatin and mitomycin C(MMC) implicates the important role of Brcal in cellular response following ICL treatment. Brca1 plays an essential role in DNA double-strand break (DSB) repair through homologous recombination (HR)-dependent and -independent process. Recently, our group has been reported that Brca1 involves in cellular ICL response through HR-dependent repair process (Yun J. et at., Oncogene 2005). In this report, the involvement of Brca1 protein in HR-independent repair process is examined using isogenic $p53^{-/-}\;and\;p53^{-/-}\;Brcal^{-/-}$ mouse embryonic fibroblast (MEF) and psoralen cross-linked reporter reactivation assay. Brcal-deficient MEFs showed significantly low HR-independent repair activity compare to Brca1-proficient MEFs. Hypersensitivity to MMC and ICL reporter repair activity were restored by the reconstitution of Brca1 expression. Interestingly, MEFs expressing exon 11-deleted isoform of Brca1 $(Brca1^{\Delta11/\Delta11})$ showed high resistance to MMC and ICL reporter repair activity comparable to Brca1-reconstituted MEFs. Taken together, these results suggest that Brca1 involves in ICL repair through not only HR-dependent process but also HR-independent process using N-terminal RINC finger domain or C-terminal BRCT domain rather than exon 11 region which mediate interaction with Rad50.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.4
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• pp.265-272
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• 2006

Objective: This study was carried out to evaluate the effect of the isolation methods of inner cell mass from mouse blastocyst, types of feeder cells and treatment time of mitomycin C on the formation rate of ICM colony. Methods: The inner cells were isolated by conventional immunosurgery, partial trophoblast dissection with syringe needles and whole blastocyst co-culture method. Commercially available STO and primary cultured mouse embryonic fibroblast (pMEF) feeder cells were used, and mitomycin C was treated for 1, 2 or 3 hours, respectively. The formation rate of ICM colony was observed after isolation of ICM and culture of ICM on the feeder cells for 7 days. Result: The ICM colony formation rate on STO were significantly higher in partial trophoblast dissection group (58%) than that in immunosurgery (12%) or whole blastocyst culture (16%) group (p<0.05). The formation rate on pMEF feeder layer was higher in partial trophoblast dissection (88%) and whole blastocyst culture (82%) group than that in immunosurgery (16%) group (p<0.05). When mitomycin C treated to pMEF for 2 hours, the formation rate of 88% was significantly higher than those of other conditions. Conclusion: Above results showed that the efficient isolation method of ICM from blastocyst was the partial trophoblast dissection and the appropriate treatment time of mitomycin C was 2 hours. However, the subculture of ICM colony and characterization of stem cells should be carried out to confirm the efficacy of the partial trophoblast dissection method.

• Journal of the korean academy of Pediatric Dentistry
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• v.30 no.4
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• pp.643-653
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• 2003

Fibroblast growth factor (FGF) / FGF receptor (FGFR) mediated signaling is required for skeletogenesis in cluding intramembranous and endochondral ossifications Runx2 ($Cbfa1/Pebp2{\alpha}A/AML3$) is an essential transcription factor for osteoblast differentiation and bone formation. Murine calvaria and mandible are concurrently undergoing both intramembranous bone and cartilage formations in the early developmental stage. However the mechanism by which these cartilage formations are regulated remains unclear. To elucidate the effect of FGF signaling on development of cranial sutural cartilage and Meckel's cartilage and to understand the role of Runx2 in these process, we have done both in vivo and in vitro experiments. Alcian blue staining showed that cartilage formation in sagittal suture begins from embryonic stage 16 (E16), Meckel's cartilage formation in mandible from E12. We analyzed by in situ hybridization the characteristics of cartilage cells that type II collagen, not type X collagen, was expressed in sagittal sutural cartilage and Meckel's cartilage. In addition, Runx2 was not expressed in Meckel's cartilage as well as sagittal sutural cartilage, except specific expression pattern only surrounding both cartilages. FGF signaling pathway was further examined in vitro. Beads soaked in FGF2 placed on the sagittal suture and mandible inhibited both sutural and Meckel's cartilage formations. We next examined whether Runx2 gene lies in FGF siganling pathway during regulation of cartilage formation. Beads soaked in FGF2 on sagittal suture induced Runx2 gene expression. These results suggest that FGF signaling inhibits formations of sagittal sutural and Meckel's cartilages, also propose that FGF siganling is involved in the proliferation and differentiation of chondroblasts through regulating the transcription factor Runx2.

• Korean Journal of Poultry Science
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• v.40 no.4
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• pp.299-304
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• 2013

Tracheal epithelial cells (TECs) are an important tool for studies of viral respiratory diseases. Primary TECs have been cultured from human, mouse and hamster. It is also necessary to diagnose viral respiratory disease and reveal infection mechanisms in chicken. In this study, we isolated tracheal epithelial layers from tracheal of 20-day-old chicks and cultured primary TECs from the isolated layers. Ciliated cells which were a typical morphology of TECs were observed in cultured primary TECs and maintained until cell passage 5 (15 to 20 days). When we analyzed expression patterns of epithelial marker genes (retinoic acid responder, FGF-binding protein, virus activating protease (VAP) in TECs compared to immortalized chicken embryonic fibroblast cell line (DF-1), all the marker genes are highly expressed in TECs than in DF-1. When TECs were cultured with 0.1 and 1 MOI of ND virus (rNDV-GFP strain) to test the susceptibility of TECs for ND virus, 12.6% and 48.2% of the incubated TECs were infected respectively. In addition, when DF-1 was incubated with 1 MOI of ND virus, the virus infection rate of DF-1 was three times lower than the virus infection rate of TECs. These data could contribute to study infection mechanisms of viral respiratory diseases and control them in chicken.

• Toxicological Research
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• v.24 no.3
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• pp.175-182
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• 2008

DNA-dependent protein kinase(DNA-PK) is involved in joining DNA double-strand breaks induced by ionizing radiation or V(D)J recombination and is activated by DNA ends and composed of a DNA binding subunit, Ku, and a catalytic subunit, DNA-PKcs. It has been suggested that DNA-PK might be $2^{nd}$ upstream kinase for protein kinase B(PKB). In this report, we showed that Ser473 phosphorylation in the hydrophobic-motif of PKB is blocked in DNA-PK knockout mouse embryonic fibroblast cells(MEFs) following insulin stimulation, while there is no effect on Ser473 phosphorylation in DNA-PK wild type MEF cells. The observation is further confirmed in human glioblastoma cells expressing a mutant form of DNA-PK(M059J) and a wild-type of DNA-PK(M059K), indicating that DNA-PK is indeed important for PKB activation. Furthermore, the treatment of cells with doxorubicin, DNA-damage inducing agent, leads to PKB phosphorylation on Ser473 in control MEF cells while there is no response in DNA-PK knockout MEF cells. Together, these results proposed that DNA-PK has a potential role in insulin signaling as well as DNA-repair signaling pathway.

• Food Science of Animal Resources
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• v.33 no.3
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• pp.411-416
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• 2013

This study aimed to examine the mechanisms underlying the effects of Garcinia cambogia extract on the adipogenic differentiation of 3T3-L1 cells and long-chain saturated fatty acid-induced lipotoxicity of HepG2 cells. 3T3-L1 preadipocytes, mouse embryonic fibroblast-adipose like cell line, were treated with MDI solution (0.5 mM IBMX, 1 ${\mu}M$ dexamethasone, 10 ${\mu}g/mL$ insulin) to generate a cellular model of adipocyte differentiation. Using this cellular model, the anti-obesity effect of Garcinia cambogia extract was evaluated. MDI-induced lipid accumulation and expression of adipogenesis-related genes were detected by Oil red O staining, Nile Red staining, and Western blot analysis. Effects Garcinia cambogia extract on palmitate-induced lipotoxicity was also analyzed by MTT assay, LDH release, and DAPI staining in HepG2 cells. Garcinia cambogia extract significantly suppressed the adipogenic differentiation of preadipocytes and intracellular lipid accumulation in the differentiating adipocytes. Garcinia cambogia extract also markedly inhibited the expression of peroxisome proliferator- activated receptor ${\gamma}2$ ($PPAR{\gamma}2$), CCAT/enhancer-binding protein ${\alpha}$ ($C/EBP{\alpha}$), and adipocyte protein aP2 (aP2). In addition, Garcinia cambogia extract significantly attenuated palmitate-induced lipotoxicity in HepG2 cells. Palmitateinduced cellular damage and reactive aldehydes were also significantly reduced in the presence of Garcinia cambogia extract. These findings suggest that the Garcinia cambogia extract inhibits the adipogenic differentiation of 3T3-L1 preadipocytes, probably by regulating the expression of multiple genes associated with adipogenesis such as $PPAR{\gamma}2$, $C/EBP{\alpha}$, aP2, and thereby modulating fatty acid-induced lipotoxicity to reduce cellular injury in hepatocytes.