• Reproductive and Developmental Biology
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• v.37 no.4
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• pp.269-279
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• 2013

Low efficiency of somatic cell nuclear transfer (SCNT) is attributed to incomplete reprogramming of transfered nuclei into oocytes. Trichostatin A (TSA), histone deacetylase inhibitor and 5-aza-2'deoxycytidine (5-aza-dC), DNA methylation inhibitor has been used to enhance nuclear reprogramming following SCNT. However, it was not known molecular mechanism by which TSA and 5-aza-dC improve preimplantation embryo and fetal development following SCNT. The present study investigates embryo viability and gene expression of cloned porcine preimplantation embryos in the presence and absence of TSA and 5-aza-dC as compared to embryos produced by parthenogenetic activation. Our results indicated that TSA treatment significantly improved development. However 5-aza-dC did not improve development. Presence of TSA and 5-aza-dC significantly improved total cell number, and also decreased the apoptotic and autophagic index. Three apoptotic-related genes, Bak, Bcl-xL, and Caspase 3 (Casp3), and three autophagic-related genes, ATG6, ATG8, and lysosomal-associated membrane protein 2 (LAMP2), were measured by real time RT-PCR. TSA and 5-aza-dC treatment resulted in high expression of anti-apoptotic gene Bcl-xL and low pro-apoptotic gene Bak expression compared to untreated NT embryos or parthenotes. Furthermore, LC3 protein expression was lower in NT-TSA and NT-5-aza-dC embryos than those of NT and parthenotes. In addition, TSA and 5-aza-dC treated embryos displayed a global acetylated histone H3 at lysine 9 and methylated DNA H3 at lysine 9 profile similar to the parthenogenetic blastocysts. Finally, we determined that several DNA methyltransferase genes Dnmt1, Dnmt3a and Dnmt3b. NT blastocysts showed higher levels Dnmt1 than those of the TSA and 5-aza-dC blastocysts. Dnmt3a is lower in 5-aza-dC than NT, NTTSA and parthenotes. However, Dnmt3b is higher in 5-aza-dC than NT and NTTSA. These results suggest that TSA and 5-aza-dC positively regulates nuclear reprogramming which result in modulation of apoptosis and autophagy related gene expression and then reduce apoptosis and autophagy. In addition, TSA and 5-aza-dC affects the acetylated and methylated status of the H3K9.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.105-113
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• 2011

The present study investigated the nuclear remodeling, development potential with telomerase activity and transcription level of X-linked genes (ANT3, HPRT, MeCP2, RPS4X, XIAP, XIST and ZFX) in the bovine somatic cell nuclear transfer (SCNT) embryos using two different fusion and activation methods. Female adult fibroblasts were injected into perivitelline space of in vitro matured oocytes. The oocyte-nucleus complexes were fused and followed by immediately either activated (Group 1), or activated at 1 h post-fusion (hpf) (Group 2), respectively. The incidence of normal premature chromosome condensation (PCC) at 1 hpf was slightly increased in the Group 2, compared to those of Group 1, but there was no significant (p<0.05) difference. The incidence of normal pronucleus (PN) and chromosome spread at 5 and 18 hpf were significantly (p<0.05) higher in the Group 2 than those of Group 1. The cleavage rate to 2-cell stage, developmental rate to blastocyst stage, and the mean number of total and ICM cell numbers were significantly (p<0.05) higher in the Group 2, compared to those of Group 1. Level of telomerase activity was significantly (p<0.05) higher in the SCNT blastocysts of Group 2, compared to those of Group 1. Transcript levels of HPRT, MeCP2 and XIST were not significantly (p<0.05) different between blastocysts of Group 1 and 2. However, transcript level of ANT3, RPS4X, XIAP and ZFX were significantly (p<0.05) up-regulated in the SCNT blastocysts of Group 2, compared to those of Group 1. Taken together, it is concluded that oocyte activation at 1 hpf induces the enhanced developmental potential by efficient nuclear remodeling and subsequent facilitation of the nuclear reprogramming of bovine SCNT embryos.

• Development and Reproduction
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• v.25 no.1
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• pp.43-53
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• 2021

We examine the effect of endoplasmic reticulum (ER) stress inhibitor treatment time on the in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine SCNT embryos were classified by four groups following treatment time of ER stress inhibitor, tauroursodeoxycholic acid (TUDCA; 100 µM); 1) non-treatment group (control), 2) treatment during micromanipulation process and for 3 h after fusion (NT+3 h group), 3) treatment only during in vitro culture after fusion (IVC group), and 4) treatment during micromanipulation process and in vitro culture (NT+IVC group). SCNT embryos were cultured for six days to examine the X-box binding protein 1 (Xbp1) splicing levels, the expression levels of ER stress-associated genes, oxidative stress-related genes, and apoptosis-related genes in blastocysts, and in vitro development. There was no significant difference in Xbp1 splicing level among all groups. Reduced expression of some ER stress-associated genes was observed in the treatment groups. The oxidative stress and apoptosis-related genes were significantly lower in all treatment groups than control (p<0.05). Although blastocyst development rates were not different among all groups (17.5% to 21.7%), the average cell number in blastocysts increased significantly in NT+3 h (48.5±2.3) and NT+IVC (47.7±2.4) groups compared to those of control and IVC groups (p<0.05). The result of this study suggests that the treatment of ER stress inhibitor on SCNT embryos from the micromanipulation process can improve the reprogramming efficiency of SCNT embryos by inhibiting the ER and oxidative stresses that may occur early in the SCNT process.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.82-82
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• 2003

Offspring have been produced from somatic cells in a number of species. This biotechnology introduced a new phenomenon in reprogramming and differentiation of somatic cell, namely totipotency. However, birth of oversized calves and perinatal abnormalities such as increased gestation length, lack of spontaneous parturition, higher incidence of dystocia, and reduced perinatal viability of offspring are frequently observed in pregnancies of cloned bovine fetuses. Disturbance of feto-placenta has been proposed as likely causes for abnomal growth. However. Little is known the mechanism responsible for the perinatal problems. Therefore, we focused on gestation length in somatic cell nuclear recipient cows. To solve this issues, placental tissues of control and cloned bovine were obtained by a cesarean section (C-section) before 5 days of paturition. Total RNA from control and cloned bovine placenta was extractd by TRIzol reagent. GeneFishing DEG kits (Seegene) were used to identify differentially expression genes. Total RNA (3 ug) were synthesized by M-MLV reverse transcriptase (200 u/ul) with 10 uM dT-annealing control primer (ACP1) at 42C for 90 min. Then, first-strand cDNA (50 ng) was amplified using the 5 uM arbitary ACP (1-20) and 10 uM dT-ACP2 primers. Some specific expression genes were amplified, Now, we are cloning and sequencing. These finding strongly can be support to solve the problems for parturition delay in nuclear transfer cows, suggest that placenta specific proteins are key indicators for the aberration of gestation and placental function in cows.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.3-6
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• 2003

The birth of the clone animals is influencing the frontier of research of animal biotechnology. It has effects on research of animal biotechnology itself by necessitating setting of new research subjects, modifications of the strategy of ongoing research projects, and challenges to schemes formerly considered impossible. In my talk, such topics including mass production of fertile ova and oocyte maturation will be discussed. (1) Oocytes are needed for the production of a clone by nuclear transplantation. Mitochondrial DNA inherited via the oocyte are involved also in the morphogenesis. Therefore, oocytes from the same animal must be used as recipients to produce genuine clones by nuclear transplantation. Experimenting on the assumption that selective oogenesis can be avoided, and apoptosis of oocytes can be prevented, by using ovarian angiogenic factos will be introduced. (2) It is important to clarify the factors of oocytes involving in reprogramming of somatic cells. Such factors are thought to be expressed in oocytes during oogenesis and oocyte maturation. Therefore, molecular mechanisms of oogenesis and oocyte maturation must be clarified extensively. Topics in this field including our recent advances will be discussed. (중략)

• Proceedings of the KSAR Conference
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• 2002.06a
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• pp.11-11
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• 2002

Interspecies nuclear transfer has been interested to determine ability of oocyte cytoplasm to support reprogramming of somatic cell nuclei of different species. In this study, we investigated developmental ability and mRNA expression patterns of developmentally important genes in bovine reconstructed embryos using a mouse fibroblast cell nucleus. While 20% nuclear transferred embryos with bovine fibroblast developed to morulae/blastocysts, a few(2-5%) nuclear transferred bovine embryos with mouse fibroblast developed to morula. (omitted)

• Clinical and Experimental Pediatrics
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• v.53 no.8
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• pp.786-789
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• 2010

Induced pluripotent stem (iPS) cells are generated by epigenetic reprogramming of somatic cells through the exogenous expression of transcription factors. Recently, the generation of iPS cells from patients with a variety of genetic diseases was found to likely have a major impact on regenerative medicine, because these cells self-renew indefinitely in culture while retaining the capacity to differentiate into any cell type in the body, thereby enabling disease investigation and drug development. This review focuses on the current state of iPS cell technology and discusses the potential applications of these cells for disease modeling; drug discovery; and eventually, cell replacement therapy.

• Molecules and Cells
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• v.38 no.12
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• pp.1029-1036
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• 2015

Appropriate vessel development and its coordinated function is essential for proper embryogenesis and homeostasis in the adult. Defects in vessels cause birth defects and are an important etiology of diseases such as cardiovascular disease, tumor and diabetes retinopathy. The accumulative data indicate that ETV2, an ETS transcription factor, performs a potent and indispensable function in mediating vessel development. This review discusses the recent progress of the study of ETV2 with special focus on its regulatory mechanisms and cell fate determining role in developing mouse embryos as well as somatic cells.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.67-75
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• 2011

The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFR ${\alpha}$-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs ($11.2{\pm}0.8%$) and SSCs ($13.3{\pm}1.1%$). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.

• Reproductive and Developmental Biology
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• v.38 no.4
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• pp.147-158
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• 2014

Differentiated nuclei can experimentally be returned to an undifferentiated embryonic status after nuclear transfer (NT) to unfertilized metaphase II (MII) oocytes. Nuclear reprogramming is triggered immediately after somatic cell nucleus transfer (SCNT) into recipient cytoplasm and this period is regarded as a key stage for optimizing reprogramming. In a recent study (Dai et al., 2010), use of m-carboxycinnamic acid bishydroxamide (CBHA) as a histone deacetylase inhibitor during the in vitro early culture of murine cloned embryos modifies the acetylation status of somatic nuclei and increases the developmental competence of SCNT embryos. Thus, we examined the effects of CBHA treatment on the in vitro preimplantation development of porcine SCNT embryos and on the acetylated status of histone H3K9 on cloned embryos at the zygote stage. We performed the three groups SCNT: SCNT (NT), CBHA treatment at the porcine fetus fibroblast cells (PFFs) used as donor cells prior to SCNT (CBHA-C) and CBHA treatment at the porcine SCNT embryos during the in vitro early culture after oocyte activation (CBHA-Z). The PFFs were treated with a $15{\mu}M$ of CBHA (8 h) for the early culture and the porcine cloned embryos were treated with a $100{\mu}M$ concentration of CBHA during the in vitro early culture (10 h). Cleavage rates and development to the blastocyst stage were assessed. No significant difference was observed the cleavage rate among the groups (82.6%, 76.4% and 82.2%, respectively). However, the development competence to the blastocyst stage was significantly increased in CBHA-Z embryos (22.7%) as compared to SCNT and CBHA-C embryos (8.6% and 4.1%)(p<0.05). Total cell numbers and viable cell numbers at the blastocyst stage of porcine SCNT embryos were increased in CBHA-Z embryos as compared to those in CBHA-C embryos (p<0.05). Signal level of histone acetylation (H3K9ac) at the zygote stage of SCNT was increased in CBHA-Z embryos as compared to SCNT and CBHA-C embryos. The results of the present study suggested that treatment with CBHA during the in vitro early culture (10 h) had significantly increased the developmental competence and histone acetylation level at the zygote stage.