• Title/Summary/Keyword: Embryonic cell

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Effect of Antioxidants for Porcine Oocytes during In Vitro Maturation, Fertilization and Development (돼지 난포란으로부터 체외수정란의 생산에 있어서 항산화제의 첨가가 배 발달에 미치는 효과)

  • Park H.;Kim J. Y.;Kim J. Y.;Lee J. H.;Park H. D.;Kim J. M.
    • Journal of Embryo Transfer
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    • v.19 no.3
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    • pp.245-255
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    • 2004
  • In recent years, an increasing number of studies on pig in vitro maturation(IVM) and in vitro fertilization(IVF) have been separated. the wide range of new technologies, including that in applied molecular genetics, has increased this interest. the production of viable porcine embryos in vitro is a prerequisites for the successful production of transgenic pigs to date. The efficiency of IVM/IVF techniques in the porcine is lower than that obtained in other species such as cattle and mouse. The several problems are generally thought to be the cause of poor results: the low rate of MPN formation derived from inadequate IVM of oocytes, the high incidence of polyspermy after IVF and cell blocking at 4 cell during embryos culture. For there reasons overcoming, many studies have been conducted to improve in vitro embryo-genic competence of oocytes. In the last several years, many maturation culture media have been evaluated and various exogenous factors such as hormones and grows factors have been tested to improve the efficiency of porcine in vitro system. In the study several antioxidants have been examined to improve in vitro fertilization and development of porcine oocytes. In this study, several antioxidants were examined to determine the effects on the development of oocytes to the cleavage, morula and blastocyst stage when added at the maturation(IVM) or in vitro fertilization(IVF) or in vitro culture(IVC) of porcine embryos. Porcine oocytes were matured, fertilized and embryos were cultured in defind conditioned medium in vitro with or without supplementation with the antioxidents of cysteine, catalase and glutathione. 1. Significant improvement of blastocyst rate (27.2% versus 15.4%, p<0.05) were achieved when catalase(500U/$m\ell$) were added to TCM-199 medium and morula rate(72.0% versus 53.9%, p<0.05) were significantly higher when glutathione(1.0mM/$m\ell$) were added to TCM-199 medium than those of control. 2. In mTBM medium for oocytes fertilization, the addition of cysteine, catalase and glutathione had no positive effect on embryonic development. glutathione had no positive effect on embryonic development. In conclusion, this study shows that addition of catalase, gluththione during IVM improved the rate of porcine embryo development.

Production of Thrombopoietin Gene Targeted Clones by Homologous Recombination at $\beta$-casein Locus of Primary Bovine Ear Skin Fibroblasts

  • Mira Chang;Oh, Keon-Bong;Lee, Kyung-Kwang;Han, Yong-Mahn
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • 2003.10a
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    • pp.86-86
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    • 2003
  • Research has been in progress for more than a decade to production of useful proteins by genetic modification in cattle. However, the levels of protein production in transgenic cattle have been reported very low. To enhance protein production in transgenic animal, we tried homologous recombination to donor cells for production of transgenic clone cattle through nuclear transfer procedure. Thus, we constructed the two targeting vectors of human thrombopoietin (TPO) at bovine $\beta$-casein locus using homologous recombination with 13.6 kb and 9.6 kb homology. In two targeting vectors, positive selection was through the neomycin resistance gene and negative selection was by the diphtheria toxin (DT). Gene targeting was attempted in bovine embryonic fibroblasts (bEF) and bovine ear skin fibroblasts (bESF). To determine the most appropriate concentration of neomycin for bEF and bESF, G4l8 resistance was confirmed by culturing the cells in various concentrations of the drug and both of the cells were optimally selected at $900 \mu g/ml$ of neomycin. The transfected bEF and bESF by the targeting vectors were colonized efficiently at the ratio of DNA to transfection reagent such as $4 \mu g$:2 ${mu}ell$ and $1 \mu g$:$2 \mu l$. Comparing number of healthy clones from passage 4 to passage 8, bESF (17%) persist in culture for much longer than bEF (6%). The two gene-targeted bESF clones of 30 random-integrated clones with 9.6 kb homology length were confirmed, however, nothing was out of 72 random integration clones with 13.6 kb homology length, The DT also worked more efficiently in clones transfected with the vector of 9.6 kb homology length. Our data suggests that the choice of donor cell for long culture period should be considered to obtain targeted cell clone, and the gene-targeting frequency and the DT working efficiency are dependent on the length of target homology.

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In Vitro Developmental Competence of Porcine SCNT Embryos is improved by m-Carboxycinnamic Acid Bishydroxamide, Histone Deacetylase Inhibitor

  • Park, Sang-Hoon;Lee, Mi-Ran;Kim, Tae-Suk;Baek, Sang-Ki;Jin, Sang-Jin;Kim, Jin-Wook;Jeon, Sang-Gon;Yoon, Ho-Baek;Lee, Joon-Hee
    • 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.

C-fos mRNA Expression in Rat Hippocampal Neurons by Antidepressant Drugs (배양한 흰쥐 해마신경세포에서 항우울제에 의한 c-fos mRNA의 발현)

  • Park, Eung-Chul;Cho, Yun-Gyoo;Yang, Byung-Hwan;Kim, Kwang-Iel;Yang, Bo-Gee;Chai, Young-Gyu
    • Korean Journal of Biological Psychiatry
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    • v.8 no.1
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    • pp.85-95
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    • 2001
  • This study was designed to examine the effects of two antidepressant drugs on the expression of c-fos mRNA in cultured embryonic rat hippocampal neurons. The drugs used were imipramine and amitriptyline. On the fourth day of culture, hippocampal neurons were treated with variable concentrations of each drug. Competitive RT-PCR(Reverse Transcriptase-PCR) analysis was used to quantify the c-fos mRNA expression induced by each drug. Experimental results showed that acute and direct treatment with imipramine and amitriptyline with relatively low concentrations(imipramine ${\leq}10{\mu}M$, amitriptylne ${\leq}10{\mu}M$) had no inductive effect on the expression of c-fos mRNA in the rat hippocampal neurons. However, after treatment with relatively high concentrations(imipramine ${\geq}100{\mu}M$, amitriptyline ${\geq}100{\mu}M$) c-fos mRNA was not detected. These findings suggest the followings. Firstly, the action mechanisms of these drugs on the hippocampal neurons might not be mediated by c-fos but by other immediate-early genes(IEGs). Secondly, their actions may be mediated indirectly via other areas of the brain. Thirdly, the expression of c-fos might be inhibited by high concentrations of these drugs, or the high concentrations could induce cell death. Finally, though cell death remains to be confirmed, the inhibition of c-fos induction or cell death could play a role in the cognitive impairments known to be adverse effects of some antidepressants. This study is believed to be a first step toward understanding the mechanisms of learning and memory. Further studies are needed to investigate the expression of various IEGs and changes in the hippocampal neurons of rat resulting from chronic treatment with antidepressant drugs.

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Effects of Several Cosmetic Preservatives on ROS-Dependent Apoptosis of Rat Neural Progenitor Cells

  • Ryu, Onjeon;Park, Bo Kyung;Bang, Minji;Cho, Kyu Suk;Lee, Sung Hoon;Gonzales, Edson Luck T.;Yang, Sung Min;Kim, Seonmin;Eun, Pyeong Hwa;Lee, Joo Young;Kim, Kyu-Bong;Shin, Chan Young;Kwon, Kyoung Ja
    • Biomolecules & Therapeutics
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    • v.26 no.6
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    • pp.608-615
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    • 2018
  • Benzalkonium chloride, diazolidinyl urea, and imidazolidinyl urea are commonly used preservatives in cosmetics. Recent reports suggested that these compounds may have cellular and systemic toxicity in high concentration. In addition, diazolidinyl urea and imidazolidinyl urea are known formaldehyde (FA) releasers, raising concerns for these cosmetic preservatives. In this study, we investigated the effects of benzalkonium chloride, diazolidinyl urea, and imidazolidinyl urea on ROS-dependent apoptosis of rat neural progenitor cells (NPCs) in vitro. Cells were isolated and cultured from embryonic day 14 rat cortices. Cultured cells were treated with 1-1,000 nM benzalkonium chloride, and $1-50{\mu}M$ diazolidinyl urea or imidazolidinyl urea at various time points to measure the reactive oxygen species (ROS). PI staining, MTT assay, and live-cell imaging were used for cell viability measurements. Western blot was carried out for cleaved caspase-3 and cleaved caspase-8 as apoptotic protein markers. In rat NPCs, ROS production and cleaved caspase-8 expression were increased while the cell viability was decreased in high concentrations of these substances. These results suggest that several cosmetic preservatives at high concentrations can induce neural toxicity in rat brains through ROS induction and apoptosis.

In vitro anticancer and antioxidant effects of acetone extract of Eucommia ulmoides oliver leaves (두충잎 아세톤 추출물의 in vitro 항암 및 항산화 효과)

  • In, Man-Jin;Kim, Eun Jeong;Kim, Dong Chung
    • Journal of Applied Biological Chemistry
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    • v.61 no.2
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    • pp.119-124
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    • 2018
  • In vitro anticancer and antioxidant effects of acetone extract from leaves of Eucommia ulmoides Oliver were investigated. The extraction yield and total phenolic content of the acetone extract were $1.13{\pm}0.033%$ (w/w) and $36.7{\pm}1.96mg$ gallic acid equivalents/g-extract, respectively. $GI_{50}$ values of the acetone extract for human non-small cell lung cancer cells (A549), human colon cancer cells (SNU-C4), human cervical cancer cells (HeLa), and human embryonic lung epithelial cell (L132) were 53.4, 53.8, 88.3, and $153.9{\mu}g/mL$, respectively. The acetone extract effectively inhibited the proliferation of human non-small cell lung cancer (A549) and colon cancer (SNU-C4) cells in a concentration-dependent manner, but was less cytotoxic with human normal cells (L132). $EC_{50}$ values of the acetone extract for free radical scavenging, reducing power, and lipid peroxidation inhibition were about 2,000, 275.8, and $257.9{\mu}g/mL$, respectively. The acetone extract showed a potent reducing power and lipid peroxidation inhibitory activity in a concentration-dependent manner.

Effects of Human Amniotic Fluid Supplemented to Whitten's Medium on Development and Outgrowth of Mouse Embryo (Whitten 배양액내 인간양수의 첨가가 생쥐 수정란의 체외발달 및 체외신장에 미치는 영향)

  • 김재환;승경록;최종현;정두용;이훈택;정길생
    • Korean Journal of Animal Reproduction
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    • v.18 no.2
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    • pp.87-94
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    • 1994
  • The objective of this study is to evaluate the developmental ability of mouse embryo in the presence of human amniotic fluid (hAF), The highest development rate was found in the culture media supplemented with 20% mid-term hAF but this rate was concomitantly reduced with more than 20% hAF. Furthermore, mouse two-cell embryos cultured in 20% mid-term hAF were developed more consistently to the expanded and hatched blastocyst stages compared to those cultured in simple medium. However, no significant differences in the embryo development rates were observed among the supplemented effects of 20% mid-term hAF, 0.3% bovine serum albumin (BSA), and 10% fetal calf serum (FCS), Development rates of two-ceiI mouse embryos cultured in 20% full-term hAF were declined compared to 20% mid-term hAF. Outgrowth of hatched blastocysts were observed when the embryos were cultured in medium containing 20% mid-term hAF or 10% FCS. But two-cell mouse embryos cultured in the presence of 20% full-term hAF or O.3% BSA was not observed their outgrowth. The kinetics of outgrowth processes in the presence of hAF were similar to those with 10% FCS. However, embryos with FCS showed a considerably greater extents of trophetodermal cell proliferation and outgrowth. Taken together, these data suggest that mid-term hAF may have a suitability for the mammalian embryos and induce embryonic outgrowth.

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DNA Methylation Change of Dnmt1o and Dnmt1s 5'-Region in the Early Porcine Embryo (돼지 초기수정란에서 Dnmt1o와 Dnmt1s 상류 영역의 DNA 메틸화 변화)

  • Kim, Hyun-Mi;Kim, Sung-Woo;Cho, Sang-Rae;Kim, Hyun;Park, Jae-Hong;Cho, Jae-Hyeon;Yang, Boh-Suk;Ko, Yeoung-Gyu
    • Reproductive and Developmental Biology
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    • v.35 no.3
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    • pp.281-285
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    • 2011
  • In the present study, we identified differentially methylated region (DMR) upstream of Dnmt1o and Dnmt1s gene in early porcine embryos. Porcine Dnmt1o had at least one DMR which was located between -530 bp to -30 bp upstream from transcription start site of the Dnmt1o gene. DNA methylation analyses of Dnmt1o revealed the DMR to be hypomethylated in oocytes, whereas it was highly methylated in sperm. Moreover, the DMR upstream of Dnmt1o was gradually hypermethylated from oocytes to two cells and dramatically changed in the methylation pattern from four cells to BL stages in an in vivo. In an IVF, the methylation status in the DMR upstream of Dnmt1o was hypermethylated from one cell to eight cells, but demethylated at the Morula and BL stages, indicating that the DNA methylation pattern in the Dnmt1o upstream ultimately changed from stage to stage before the implantation. Next, to elucidate whether DNA methylation status of Dnmt1s upstream is stage-by-stage changed in during porcine early development, we analyzed the dynamics of the DNA methylation status of the Dnmt1s locus in germ cell, or one cell to BL cells. The Dnmt1s upstream was highly methylated in one and eight cells, while less methylated in two, four, morula, and BL cells. Taken together, our data demonstrated that DNA methylation and demethylation events in upstream of Dnmt1o/Dnmt1s during early porcine embryos dramatically occurred, and this change may contribute to the maintenance of genomewide DNA methylation in early embryonic development.

Effect of Matrix Metalloproteinases-2 and -9 during IVC-2 on the Development Competence and Gene Expression Profile of Bovine In Vitro-Produced Embryos

  • Lee, Kyeong-Lim;Bang, Jae-Il;Ha, A-Na;Fakruzzaman, Md.;Min, Chan-Sik;Kong, Il-Keun
    • Journal of Embryo Transfer
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    • v.29 no.2
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    • pp.101-109
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    • 2014
  • Matrix Metalloproteinases (MMP)-2 and -9 are participated in embryo development, implantation, remodeling of epithelial cell and ovulation. The objective of this study is to evaluate an impact of MMP2 and MMP9 on embryonic developmental competence as well as gene expression profiles of in vitro-produced bovine embryos. After in vitro fertilization, embryos of all groups were transferred into IVC-2 medium treated with MMP2 and MMP9 to check the optimum concentration on the basis of embryo development competence and cell numbers. The optimum concentrations for MMP2 and 9 were 1,200 ng/ml and 300 ng/ml. The blastocyst development competence was not different among 1,200 ng/ml of MMP2 vs. 300 ng/ml of MMP9 vs. combined MMP2 + 9 vs. control groups ($41.46{\pm}10.66$ vs. $37.73{\pm}8.92$ vs. $45.11{\pm}11.41%$ vs. $41.59{\pm}11.88$, respectively). Furthermore, the developmental competences to hatching and hatched blastocysts were not also different among the same groups ($79.84{\pm}12.63$ vs. $83.3{\pm}17.46$ vs. $78.55{\pm}14.48%$ vs. $72.02{\pm}14.09$). In addition, total cell number was significantly (p<0.05) greater in blastocyst treated with MMP9 300 ng/ml among all treatment groups. On the other hand, there was no significant difference of ICM vs. TE ratio in all groups. The expression of five out of six genes (i.e., MMP2, MMP9, IFNt, SSLP1 and HNRNPA2B1) was different among the groups. The expression of IFNt and HNRNPA2B1 genes was significantly greater in MMP9 (p<0.05), but there was no difference of MMP9 expression between MMP2 and MMP9 group (p>0.05). The normalized expression of MMP2 and SSLP1 was greater in MMP2 than other groups (p<0.05). In conclusion, MMPs treatment during IVC-2 medium was remarkably effected on blastocyst developmental competence and gene expression profiles that are related to embryo quality and implantation.

Cellular Toxic Effects and Action Mechanisms Of 2,2', 4,6,6'-Pentachlorobiphenyl

  • Kim Sun-Hee;Shin Kum-Joo;Kim Dohan;Kim Yun-Hee;Ryu Sung Ho;Suh Pann-Ghill
    • 한국생물공학회:학술대회논문집
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    • 2004.07a
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    • pp.1-20
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    • 2004
  • Polychlorinated biphenyls (PCBs), one a group of persistent and widespread environmental pollutants, have been considered to be involved in immunotoxicity, carcinogenesis, and apoptosis. However, the toxic effects and physical properties of a PCB congener are dependent on the structure. In the present study, we investigate the toxic effects and action mechanisms of PCBs In cells. Among the various congeners tested, 2,2',4,6,6'-PeCB-pentachlorobiphenyl (PeCB), a highly ortho-substituted congener having negligible binding affinity for aryl hydrocarbon receptor (AhR), caused the most potent toxicity and specific effects in several cell types. 2,2',4,6,6'-PeCB induced apoptotic cell death of human monocytic cells, suggesting that PCB-induced apoptosis may be linked to immunotoxicity. In addition, 2,2',4,6,6'-PeCB induced mitotic arrest by interfering with mitotic spindle assembly in NIH3T3 fibroblasts, followed by genetic instability which triggers p53 activation. Which suggests that 2,2',4,6,6'-PeCB may be involved in cancer development by causing genetic instability through mitotic spindle damage. On the other hand, 2,2',4,6,6'-PeCB increased cyclooxygenase-2 (COX-2) involved in cell survival through ERK1/2 MAPK and p53 in Rat-1 fibroblasts and mouse embryonic fibroblasts, triggering compensatory mechanism for abating its toxicity. Taken together, these results demonstrate that PCB congeners of different structure have distinct mechanism of action and 2,2',4,6,6'-PeCB causes several toxicity as well as compensatory mechanism in cells.

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