• BMB Reports
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• v.45 no.1
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• pp.20-25
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• 2012

The present study aimed to investigate the function of translationally controlled tumor protein (TCTP) in the regulation of Oct4 in mouse embryonic carcinoma P19 cells and mouse J1 embryonic stem (ES) cells. The mRNA level of endogenous TCTP in somatic cells was 2-4 folds higher than that in pluripotent P19 and J1 ES cells. Overexpression of TCTP in mouse pluripotent cells not only reduced the level of Oct4 transcription, but also decreased the pluripotency of stem cells. The N-terminal end of TCTP (amino acids 1-60) played an important role in suppressing the Oct4 promoter. Moreover, overexpression of TCTP in P19 cells suppressed the Oct4 promoter activity in a dose- and a time-dependent manner. In addition, knockdown of TCTP by small interfering RNA increased the expression of Oct4. Our study indicates that TCTP downregulates the Oct4 expression by binding the Sf1 site of Oct4 promoter in mouse pluripotent cells.

• The Journal of the Korean dental association
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• v.55 no.12
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• pp.828-840
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• 2017

In recent years, many researchers and clinicians found interest in regenerative medicine using induced pluripotent stem cells (iPSCs) with biomaterials due to their pluripotency, which is able to differentiate into any type of cells without human embryo, which of use is ethically controversial. However, there are limitations to make iPSCs from adult somatic cells due to their low stemness and donor site morbidity. Recently, to overcome above drawbacks, dental tissue-derived iPSCs have been highlighted as a type of alternative sources for their high stemness, easy gathering, and their complex (ectomesenchymal) origin, which easily differentiate them to various cell types for nerve, vessel, and other dental tissue regeneration. In other part, utilizing biomaterials for regenerative medicine using cell is recently highlighted because they can modulate cell adhesion, proliferation and (de)differentiation. Therefore, this paper will convey the overview of advantages and drawbacks of dental tissue-derived iPSCs and their future application with biomaterials.

• Reproductive and Developmental Biology
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• v.28 no.1
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• pp.65-70
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• 2004

In mammals, male and female germline stem cells are derived from primodial germ cells. Despite many efforts to identify stem cells from gonads, there has been little successe to identify germline stem cells yet. In this study, we isolate and characterized porcine germline stem cells using only stem cell markers that are prevalently expressed in various tissues. Gonadal cells derived from both male and female formed colonies and showed AP activities and different lectin binding properties. Pluripotency of germline stem cells was also identified by positive signals against putative stem cells markers such as SSEA-1 and SSEA-3. In addition, nestin was also found in primary gonad cells that have a similar morphology to the AP-positive cells. The nestin expression suggests that the germline stem cells may have similar expression of the prevalent stem cell markers found in other tissues. The demonstration of nestin expression together with pluripotent cell markers calls further investigation of the possible differentiation of nestin-positive cells into neurons.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.223-227
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• 2008

Human embryonic stem (ES) cells retain the capacity for self-renewal, are pluripotent and differentiate into the three embryonic germ layer cells. The regulatory transcription factors Oct4, Nanog and Sox2 play an important role in maintaining the pluripotency of human ES cells. The aim of this research was to identify unknown genes upregulated in human ES cells along with Oct4, Nanog, and Sox2. This study characterizes an unknown gene, named chromosome 1 open reading frame 31 (C1orf31) mapping to chromosome 1q42.2. The product of C1orf31 is the hypothetical protein LOC388753 having a cytochrome c oxidase subunit VIb (COX6b) motif. In order to compare expression levels of C1orf31 in human ES cells, human embryoid body cells, vascular angiogenic progenitor cells (VAPCs), cord-blood endothelial progenitor cells (CB-EPCs) and somatic cell lines, we performed RT-PCR analysis. Interestingly, C1orf31 was highly expressed in human ES cells, cancer cell lines and SV40-immortalized cells. It has a similar expression pattern to the Oct4 gene in human ES cells and cancer cells. Also, the expression level of C1orf31 was shown to be upregulated in the S phase and early G2 phase of synchronized HeLa cells, leading us to purpose that it may be involved in the S/G2 transition process. For these reasons, we assume that C1orf31 may play a role in on differentiation of human ES cells and carcinogenesis.

• Journal of Embryo Transfer
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• v.23 no.3
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• pp.133-139
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• 2008

DNA methylation is one of the reasons for poor survival of clone animals. The OCT-4 gene is essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that the 5'-promoter region of Oct-4 gene was a target of DNA methylation and the methylation status was changed variously during embryonic development in bovine. The study conducted to examine the expression and methylation pattern of tissue-dependent differentially methylated region (T-DMR) of Oct-4 gene in bovine somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF) blastocysts. The Oct-4 gene expression was evaluated by RT-PCR and fluorescence immunocytochemistry. The methylation pattern of T-DMR was analyzed using restriction mapping and bisulfite sequencing methods. The Oct-4 transcripts were highly expressed in IVF, while they were not expressed in SCNT. The Oct-4 protein was not detected or expressed at very low level in SCNT, the intensity of Oct-4 protein, however, was strong in IVF. On the other hand, the T-DMR of Oct-4 gene was hypermethylated in SCNT compared to that of IVF. These results suggested that expression and the failure of demethylation of Oct-4 gene was closely associated with incomplete development of SCNT embryos.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.2
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• pp.178-188
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• 2013

The glycosaminoglycans (GAGs) present in the female reproductive tract promote sperm capacitation. When bovine sperm were exposed to 10 ${\mu}g/ml$ of one of four GAGs (Chondroitin sulfate, CS; Dermatan sulfate, DS; Hyaluronic acid, HA; Heparin, HP) for 5 h, the total motility (TM), straight-line velocity (VSL), and curvilinear velocity (VCL) were higher in the HP- or HA-treated sperm, relative to control and CS- or DS-treated sperm. HP and HA treatments increased the levels of capacitated and acrosome-reacted sperm over time, compared to other treatment groups (p<0.05). In addition, sperm exposed to HP or HA for 1 h before IVF exhibited significantly improved fertilizing ability, as assessed by 2 pronucleus (PN) formation and cleavage rates at d 2. Exposure to these GAGs also enhanced in vitro embryo development rates and embryo quality, and increased the ICM and total blastocyst cell numbers at d 8 after IVF (p<0.05). A real-time PCR analysis showed that the expression levels of pluripotency (Oct 4), cell growth (Glut 5), and anti-apoptosis (Bax inhibitor) genes were significantly higher in embryos derived from HA- or HP-treated sperm than in control or other treatment groups, while pro-apoptotic gene expression (caspase-3) was significantly lower in all GAG treatment groups (p<0.05). These results demonstrated that exposure of bovine sperm to HP or HA positively correlates with in vitro fertilizing ability, in vitro embryo developmental potential, and embryonic gene expression.

• Korean Journal of Agricultural Science
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• v.43 no.1
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• pp.52-60
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• 2016

In animal reproduction, the quality of oocytes and embryos has been evaluated by the expression of specific molecules. Follistatin (FST), which was isolated from follicular fluid, binds and bio-neutralizes the TGF-${\beta}$ superfamily members. Previous studies using the bovine model showed FST could be an important molecular determinant of embryo developmental competence. However, the effect of FST treatment on porcine embryo developmental competence has not been established. In this study, the effect of exogenous FST on porcine embryo developmental competence was investigated during in vitro culture. FST (10 ng/ml) treatment induced a significant decrease in the rate of cell arrest at the 4-cell stage. The expression levels of DNA-methyltransferase 1 (DNMT1), histone deacetylase 1 (HDAC1), and histone deacetylase 2 (HDAC2) were decreased in 4-cell stage embryos. FST treatment also resulted in significant improvements in developmental competence of embryos in terms of blastocyst formation rate and OCT-4 mRNA levels, the latter being related to pluripotency. In conclusion, during in vitro culture, FST treatment significantly ameliorated 4-cell block during embryonic development and improved embryo developmental competence. Therefore, FST treatment may potentially have a functional role in porcine embryogenesis that is broadly applicable to enhance in vitro embryo development.

• Reproductive and Developmental Biology
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• v.37 no.3
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• pp.143-148
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• 2013

Spermatogenesis is initiated from spermatogonial stem cells (SSCs) that has an ability of self-renewal and unipotency to generate differentiating germ cells. The objective of this study is to develop the simple method for derivation of SSCs using non-sorting of both spermatogonia and feeder cells. Simply uncapsulated mouse testes were treated with enzymes followed by surgical mincing, and single cells were cultured in stempro-$34^{TM}$ cell culture media at $37^{\circ}C$. After 5 days of culture, aciniform of SSC colony was observed, and showed a strong alkaline phosphatase activity. Molecular characterization of mouse SSCs showed that most of the mouse SSC markers such as integrin ${\alpha}6$ and ${\beta}1$, CD9 and Stra8. In addition, pluripotency embryonic stem cell (ESC) marker Oct4 were expressed, however Sox2 expression was lowered. Interestingly, expression of SSC markers such as Vasa, Dazl and PLZF were stronger than mouse ESC (mESC). This data suggest that generated mouse SSCs (mSSCs) in this study has at least similar biomarkers expression to mESC and mSSCs derived from other study. Immunocytochemistry using whole mSSC colony also confirmed that mSSCs generated from this study expressed SSC specific biomarkers such as c-kit, Thy1, Vasa and Dazl. In conclusion, mSSCs from 5 days old mouse testes were successfully established without sorting of spermatogonia, and this cells expressed both mESC and SSC specific biomarkers. This simple derivation method for mSSCs may facilitate the study of spermatogenesis.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.1
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• pp.37-44
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• 2011

Parthenogenetic embryonic stem (pES) cells could provide a valuable model for research into genomic imprinting and X-linked diseases. In this study, pES cell lines were established from oocytes of hybrid offspring of Kunming and 129/Sv mice, and pluripotency of pES cells was evaluated. The pES cells maintained in the undifferentiated state for more than 50 passages had normal karyotypes with XX sex chromosomes and exhibited high activities of alkaline phosphatase (AKP) and telomerase. Meanwhile, these cells expressed ES cell molecular markers SSEA-1, Oct-4, Nanog, and GDF3 but not SSEA-3 detected by immunohistochemistry and RT-PCR. The pES cells could be differentiated into various types of cells from three germ layers in vitro by analysis of embryoid bodies (EBs) with immunohistochemistry and RT-PCR, and in vivo by observation of pES cell-derived teratoma sections. Therefore, the established pES cell lines contained all features of mouse ES cells. This work provides a new strategy for isolating pES cells from Kunming mice, and the pES cell lines could be applied as the cell model in research into genomic imprinting and epigenetic regulation of Kunming mice.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.10
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• pp.1383-1391
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• 2016

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5'-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the -134 to -19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.