• Development and Reproduction
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• v.24 no.1
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• pp.31-41
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• 2020

We investigated the effects of endoplasmic reticulum (ER) stress inhibitor and antioxidant treatments during the micromanipulation of somatic cell nuclear transfer (SCNT) on in vitro development of SCNT embryos. Tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor and vitamin C (Vit. C), an antioxidant, were treated by alone or in combination, then, the level of X-box binding protein 1 (Xbp1) splicing and the expressions of ER stress-associated genes, oxidative stress-related genes, and apoptotic genes were confirmed in the 1-cell and blastocyst stages. In the 1-cell stage, the levels of Xbp1 splicing were significantly decreased in TUDCA and Vit. C treatment groups compared to the control (p<0.05). In addition, the expression levels of most ER stress-associated genes and oxidative stress-related genes were significantly lower in all treatment groups than the control (p<0.05), and the transcript levels of apoptotic genes were also significantly lower in all treatment groups than the control (p<0.05). In the blastocyst stage, decreased expression of ER stress-, oxidative stress-, and apoptosis-related genes were observed only in some treatments. However, the blastocyst formation rates in TUDCA and Vit. C treatment groups (24.8% and 22.0%, respectively) and mean blastocyst cell number in all treatment groups (59.7±4.3 to 63.5±3.3) were significantly higher (p<0.05) than those of control. The results showed that the TUDCA or Vit. C treatment during micromanipulation inhibited both ER and oxidative stresses in the early stage of SCNT embryos, thereby reducing cell damage and promoting in vitro development.

• Journal of Embryo Transfer
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• v.24 no.3
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• pp.213-220
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• 2009

The objective of this study was to examine the effect of eCG and various concentrations (20, 40, and 80 ${\mu}g/ml$) of porcine FSH on nuclear maturation and intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (10 IU/ml hCG and 10 IU/ml eCG or $20{\sim}80{\mu}g/ml$ FSH) for the first 22 h and then further cultured in hormone-tree medium for an additional 22 h. Nuclear maturation of oocytes ($85{\sim}89%$) was not influencem foreCG and various concentrations FSH. Embryonic development to the cleavage stage ($86{\sim}94%$) and mean number of cells in blastocyst ($33{\sim}37$ cells) after PA were not altered but blastocyst formation e-treignificaddlor(p<0.05) improvem forthe supplementation eith 80 ${\mu}g/ml$ FSHr(64%) compared to 47%, io8%, iand 47% in oocytes that were treated with eCG, 20,i and 40 ${\mu}g/ml$ FSH,i numectivelo. In SCNT, fusion ($78{\sim}83%$) of cell-cytoplast couplets and siosequent embryo cleavage ($82{\sim}88%$) were not influencem fordifferent gonadotropins but blastocyst formation tended to increase forthe supplementation eith 80 ${\mu}g/ml$ FSHr(25% vs. $11{\sim}18%$). Our nuults demonstrated that oocyte maturation and embryonic development after PA and SCNT e-frinfluencem fortype of gcem fortype of gits concentration. In this study, supplementation of maturation medium eith 80 ${\mu}g/ml$ FSHrimproved preimplantation development of PA and SCNT pig embryos, probably by increasing intracellular GSH concentration of matured oocytes.

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.43-51
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• 2004

The present study was conducted to investigate the effects of fusion and/or activation protocol on in vitro development of porcine somatic cell nuclear transfer (SCNT) embryos. Porcine fetal fibroblast cells were transferred into the perivitelline space of enucleated in vitro matured oocytes. Cell fusion and activation were induced simultaneous fusion/activation (SA) or delayed activation (DA) with or without cytochalasin B (CB) treatment with electic pulses in 0.28 M mannitol-based medium. The SCNT embryos were cultured in vitro for 7 days and stained with Hoechst 33342 to determine the number of nuclei. After 7 days culture, cleavage and blastocyst formation rates were 72.4% and 7.6% in SCNT and 76.3% and 20.4% in parthenotes. To examine the effect of electric field strengths on development of SCNT embryos, oocytes were fused two pulses of 110 V/mm, 130 V/mm or 150 V/mm for 30 sec post-injection. The fusion and cleavage rates in 130 V/mm group (70.2% and 72.6%) and 150 V/mm group (72.6% and 70.5%) were higher (P<0.05) than 110 V/mm group (47.1% and 48.6%), respectively. However, the rate of embryos developing to the blastocyst stage (8.1%, 9.7% and 10.7%) were not different among three groups. The cleavage rates and the blastcyst formation rates were not different among three treatment groups (SA group, 71.4% and 9.7%; SA+CB treatment group, 74.7% and 8.0%; DA+CB treatment group, 70.8% and 11.2%, respectively). And, no different in the number of cells in blastocysts was observed among the three groups (22.5$\pm$12.8, 23.3$\pm$11.2 and 21.6$\pm$10.4, respectively). These result suggest that two pulses of 130 V/mm or 150 V/mm for 30 sec with SA treatment or DA treatment are enough for fusion/activation of porcine somatic cell nuclear transfer (SCNT) embryos to develop to the blastocyst stage.

• Journal of Embryo Transfer
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• v.26 no.1
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• pp.79-84
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• 2011

This study was performed to identify the differentially methylated region (DMR) and to examine the mRNA expression of the imprinted H19 gene in day 35 of SCNT pig fetuses. The fetus and placenta at day 35 of gestation fetuses after natural mating (Control) or of cloned pig by somatic cell nuclear transfer (SCNT) were isolated from a uterus. To investigate the mRNA expression and methylation patterns of H19 gene, tissues from fetal liver and placenta including endometrial and extraembryonic tissues were collected. The mRNA expression was evaluated by real-time PCR and methylation pattern was analyzed by bisulfite sequencing method. Bisulfite analyses demonstrated that the differentially methylated region (DMR) was located between -1694 bp to -1338 bp upstream from translation start site of the H19 gene. H19 DMR (-1694 bp to -1338 bp) exhibits a normal mono allelic methylation pattern, and heavily methylated in sperm, but not in oocyte. In contrast to these finding, the analysis of the endometrium and/or extraembryonic tissues from SCNT embryos revealed a complex methylation pattern. The DNA methylation status of DMR Region In porcine H19 gene upstream was hypo methylated in SCNT tissues but hypermethylated in control tissues. Furthermore, the mRNA expression of H19 gene in liver, endometrium, and extraembryonic tissues was significantly higher in SCNT than those of control (p<0.05). These results suggest that the aberrant mRNA expression and the abnormal methylation pattern of imprinted H19 gene might be closely related to the inadequate fetal development of a cloned fetus, contributing to the low efficiency of genomic reprogramming.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.1-7
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• 2012

Somatic cell nuclear transfer (SCNT) for miniature pig has been developed for xenotransplantation and many other biomedical experiments. However, the efficiency of SCNT is still very low due to many factors. To optimize the surrogate mother condition for improvement of cloned miniature pigs efficiency, we investigated the effect of the status of surrogate mother on pregnancy, farrowed rate in SCNT pigs. After SCNT with mesenchymal stem cells as donor cells, the SCNT embryos were surgically transferred into the oviduct of surrogated pigs. To compare the effects of status of surrogate pigs on pregnancy, surrogate pigs were prepared by artificial abortion at day 20~29 (Group 1), 30~39 (Group 2), and 40~45 (Group 3) of gestation. After SCNT embryos transfer in three different status of surrogate pigs, Group 2 (56.3%) and 3 (55.6%) had significantly ($p$ <0.05) higher the pregnancy rate than group 1 (0%) at day 30 of gestation. The status of ovulation in surrogate pig also was investigated. Post-ovulation status (54.8%) had higher proportion than pre-ovulation status (38.7%) and ovulation status (6.5%). We obtained 19 cloned miniature piglets from seven surrogate gilts and five piglets are living healthy but fourteen piglets died soon after birth or stillbirth. The weights of piglets greatly differ from 254 to 1,296 g. Microsatellite analysis showed that cloned piglets were genetically different from the surrogate mother and cloned piglets were genetically equal to the donor cell. In conclusion, the present result indicates that artificially abortion method can improve the efficiency of pregnancy after SCNT in pigs. This study will provide available method for the further study and application in the field of xenotransplantation.

• Reproductive and Developmental Biology
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• v.33 no.2
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• pp.113-117
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• 2009

Animals produced by somatic cell nuclear transfer (SCNT) using genetically modified cells are almost always transgenic, implying that this method is more efficient than the traditional pronuclear microinjection method. Most somatic cells for SCNT in animals are fetus-derived primary cells and successful gene integration in somatic cells will depend on transfection condition. The objective of this study is to evaluate the efficiency of electroporation (Microporator) and liposome reagents (F-6, F-HD, W-EX, W-Q, W-M) for tissue-type plasminogen activator (tPA) gene transfection and to estimate the overall efficiency of transfection of Korean native pig fetal fibroblast cells (KNPFF). Electroporation showed significantly higher transfection efficiency than liposome reagents with regard to the transfection of in vitro cultures in the early stages of development (41.7% with Microporator vs. 18.3% with F-6, 20.0% with F-HD 18.5% with W-EX, 5.0% with W-M and 6.3% W-Q,). Colonies identified as tPA-positives were treated once more with G418 for 10 to 14 days and growing colonies were selected again. When the cells of newly selected colonies were subjected to single-cell PCR, reselection of colonies following second round of G418 selection increased the rate of transgene integration per each colony. These results suggest that transfection with electroporation is the most efficient and the second rounds of G418 selection may be an effective method for transfection of porcine fetal fibroblast cells.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.2
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• pp.194-200
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• 2009

The aim of this study was to investigate the effects of donor cell passage, size and type on the development of nuclear transfer embryos. Porcine cumulus cells, fetal fibroblasts and oviductal epithelial cells from 1-2, 3-6 and 7-10 passages were used for the nuclear transfer. In the oocytes with the cumulus donor cells, fusion and cleavage rates of oocytes and cell numbers per blastocyst among the three different passage groups did not show any differences, but the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher than those from 7-10 passage group. The rates of fusion, cleavage and blastocyst formation, and the cell numbers per blastocyst were higher in the embryos with the sizes of <20 and 20 ${\mu}m$ cumulus donor cells compared to the >20 ${\mu}m$ cumulus donor cell. In the oocytes with the fetal fibroblast donor cells, the rate of blastocyst formation from the 3-6 passage group was higher than from 1-2 and 7-10 passage groups. The embryos with the size of 20 $\mu{m}$ fetal fibroblast donor cell showed higher rate of blastocyst formation compared to those with <20 and >20 ${\mu}m$ donor cells. In the oocytes with the oviductal epithelial cells, the rates of blastocyst formation from 1-2 and 3-6 passage groups were higher compared to those from 7-10 passage group. The embryos with the sizes of <20 and 20 ${\mu}m$ oviductal epithelial donor cells had a higher rate of blastocyst formation compared to those with >20 ${\mu}m$ donor cell. Fusion and cleavage rates of oocytes, and cell numbers per blastocyst among the three different donor cell types from the 3-6 passage did not show any differences. However, the rate of blastocyst formation of somatic cell nuclear transfer (SCNT) embryos with the fetal fibroblast donor cell was higher than that of blastocyst formation of SCNT embryos with the cumulus and oviductal epithelial donor cells.

• 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.

• Journal of Veterinary Clinics
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• v.36 no.5
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• pp.253-258
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• 2019

The objective of this study was to analyse the effects of MS-275 (Class I and II histone deacetylase inhibitor) supplementation on the development of porcine in-vitro somatic nuclear transfer embryo production. During in-vitro development, early embryos were exposed to different concentrations of MS-275 (0, $5{\mu}M$, $10{\mu}M$, and $20{\mu}M$). In in-vitro culture supplemented group, the blastocyst development rate was significantly enhanced by $10{\mu}M$ concentration than other groups (24.0% vs. 19.3%, 21.8%, 11.5%; P < 0.05). Additionally, the 6 h supplementation group, significantly improved the blastocysts production than 24 h, 48 h and control groups (26.1% vs. 17.0%, 15.2%, 2.8%; P < 0.05). Following supplementation with optimal concentrations and time ($10{\mu}M$-6 h group), the blastocyst production was significantly higher than control (25.7% vs 15.8%; P < 0.05). The optimal concentrations of MS-275 significantly enhanced the percentages of ICM:TE than control (43.6% vs. 38.4%; P < 0.05) accompanied with significantly higher expression levels of reprogramming related genes (POU5F1, Naong, and SOX2). In conclusion, the optimal concentrations of $10{\mu}M$ MS-275 and 6 h supplementation during in-vitro culture can significantly improve the quality of porcine in-vitro somatic nuclear transfer embryos through histone acetylation and epigenetic modification. Increasing the efficiency of clonal animal production will greatly promote the development of animal disease models and xenotransplantation.

• Journal of Embryo Transfer
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• v.31 no.1
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• pp.97-107
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• 2016

Although many diseases could be treated by the development of modern medicine, there are some incurable diseases including brain cancer, Alzheimer disease, etc. To study human brain cancer, various animal models were reported. Among these animal models, mouse models are valuable tools for understanding brain cancer characteristics. In spite of many mouse brain cancer models, it has been difficult to find a new target molecule for the treatment of brain cancer. One of the reasons is absence of large animal model which makes conducting preclinical trials. In this article, we review a recent study of molecular characteristics of human brain cancer, their genetic mutation and comparative analysis of the mouse brain cancer model. Finally, we suggest the need for development of large animal models using somatic cell nuclear transfer in translational research.