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  • Title/Summary/Keyword: Embryonic developmental

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Gene Expression of Smad3 and Estrogen Receptor-related Receptorβ like 1 in Sea Urchin, Strongylocentrotus nudus (둥근성게(Strongylocentrotus nudus)의 Smad3와 Estrogen Receptor-related Receptorβ like 1 유전자 발현)

  • Jun, Yu-Jung;Sohn, Young-Chang
    • Development and Reproduction
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    • v.11 no.1
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    • pp.43-47
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    • 2007
  • Smad proteins mediate transforming growth factor(TGF)β signaling and play a pivotal role in embryonic development. The estrogen receptor-related receptors(ERRs), which are structurally similar to estrogen receptors, are members of orphan nuclear receptor in the nuclear receptor superfamily and their functions are known to be involved in the formation of extra-embryonic ectoderm. To investigate the involvement of Smad3 and ERRβ like 1 in reproductive activities and embryogenesis in marine invertebrate, we examined gene expression of Smad3 and ERRβ like 1 in Strongylocentrotus nudus during their seasonal changes and embryonic development using real-time polymerase chain reaction. The Smad3 mRNA levels in gonad showed an increasing pattern from February to June 2004 but decreased at August(spawning season) followed by an elevation of the levels at October and December 2004. The mRNA levels of the ERRβ like 1 significantly elevated during the spawning season. During embryonic development, Smad3 mRNA levels at 816 cell stages were significantly higher than those of other stages, whereas the mRNA of the ERRβ like 1 was significantly high levels at late development stages, i.e., blastular, gastrula and plutei stages. These results suggest that the Smad3 could be involved at least in part in the early cleavage stages and the ERRβ like 1 may play an important role in the spawning season and late developmental stage in the sea urchin.

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Vascular Endothelial Growth Factor Has Beneficial Effect Independent of Serum Components throughout Oocyte Maturation and Early Embryonic Development in Cattle

  • Luo, Hailing;Kimura, Koji;Hirako, Makoto
    • Asian-Australasian Journal of Animal Sciences
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    • v.19 no.4
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    • pp.495-499
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    • 2006
  • In our previous studies, we demonstrated that Vascular Endothelial Growth Factor (VEGF) enhances bovine oocyte maturation and early embryonic development in serum supplemented media. In this experiment, to determine the synergistic effect of VEGF with serum components on early embryonic development in vitro in cattle, 1 mg/ml polyvinyl-alcohol (PVA) was replaced with foetal bovine serum (FBS) in maturation and culture media. Bovine oocytes were matured in Synthetic Oviduct Fluid (SOF) supplemented with PVA, PVA+5 ng/ml of VEGF, FBS, or FBS+VEGF. Fertilized oocytes were cultured in the same conditions for 8 days. The development of embryos was examined at 48 h post- insemination and on days 6, 7 and 8. The results were analyzed using repeated measures two- factor ANOVA, in which the effects of VEGF and serum were assigned as two factors. The development rate to 4- to 8-cell embryos at 48 h was significantly higher in the PVA+VEGF group than in the PVA group (44.7% and 31.5%, respectively). However, the highest development rate to 4- to 8-cell embryos was obtained from the FBS+VEGF group (58.8%). On day 8, the blastocyst rates were higher in the PVA+VEGF (22.8%), FBS (32.1%, p<0.05) and FBS+VEGF (42.1%, p<0.05) groups than in the PVA group (17.1%). Two- factor ANOVA of the development rates indicates that VEGF had a significant effect, but had no synergistic effect with serum components on early embryonic development. The results of the present study demonstrate that VEGF improves the in vitro developmental competence of bovine oocytes and/or embryos independent of the effect of serum components.

Characterization of the Nanog 5'-flanking Region in Bovine

  • Choi, Don-Ho;Kim, Duk-Jung;Song, Ki-Duk;Park, Hwan-Hee;Ko, Tae Hyun;Pyao, Yuliya;Chung, Ku-Min;Cha, Seok Ho;Sin, Young-Su;Kim, Nam-Hyung;Lee, Woon-Kyu
    • 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.

Effects of (-)-Epicatechin Gallate on porcine oocyte in vitro maturation and subsequent embryonic development after parthenogenetic activation and in vitro fertilization

  • Seo, Min-Su;So, Kyoung-Ha;Hyun, Sang-Hwan
    • Journal of Embryo Transfer
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    • v.31 no.3
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    • pp.153-159
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    • 2016
  • (-)-Epicatechin gallate (ECG) is a polyphenol compound of green tea exhibiting biological activities, such as antioxidant and anticancer effects. To examine the effect of ECG on porcine oocytes during in vitro maturation (IVM), oocytes were treated with 0-, 5-, 15-, and 25μM ECG. After maturation, we investigated nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels and subsequent embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF). After 42 hours of IVM, the 5μM group exhibited significantly increased (p< 0.05) nuclear maturation (89.8%) compared with the control group (86.1%). However, the 25μM group observed significantly decreased (p< 0.05) nuclear maturation (83.5%). In intracellular maturation assessment the 5-, 15-, and 25μM groups had significantly increased (p< 0.05) GSH levels and decreased ROS levels compared with the controls. The 5- and 15μM group showed significantly increased (p< 0.05) embryo formation rates and total cell number of blastocysts after PA (18% and 68.9, 15% and 85.1 vs. 12% and 59.5, respectively) compared with controls. Although the 25μM group observed significantly lower blastocyst formation rates after PA (27.6% vs. 23.2%) than control group, the 5μM group showed significantly increased blastocyst formation rates after PA (37.2% vs. 23.2%) compared to the control group. Furthermore, the 5μM group measured significantly increased blastocyst formation rates (20.7% vs. 8.6%) and total cell number after IVF (88.3±1.5 vs. 58.0±3.6) compared to the control group. The treatment of 5μM ECG during IVM affectively improved the porcine embryonic developmental competence by regulating intracellular oxidative stress during IVM.

Comparison of In Vitro Development of Porcine Embryos Derived from Transfer of Embryonic Germ Cell Nuclei into Oocytes by Electrofusion and Piezo-Driven Microinjection

  • Ahn, Kwang-Sung;Won, Ji-Young;Heo, Soon-Young;Kang, Jee-Hyun;Shim, Ho-Sup
    • Reproductive and Developmental Biology
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    • v.31 no.2
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    • pp.127-131
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    • 2007
  • Embryonic germ (EG) cells are undifferentiated stern cells isolated from cultured primordial germ cells (PGC). These cells share many characteristics with embryonic stem cells including morphology and pluripotency. Undifferentiated porcine EG cell lines demonstrating capacities of differentiation both in vitro and in vivo have been established. Since EG cells can be cultured indefinitely in an undifferentiated state, whereas somatic cells in primary culture are often unstable and have limited lifespan, EG cells may provide inexhaustible source of karyoplasts in nuclear transfer (NT). In this study the efficiencies of NT using porcine EG and fetal fibroblast cells were compared. Two different techniques were used to perform NT. With conventional NT procedure (Roslin method) involving fusion of donor cells with enucleated oocytes, the rates of development to the blastocyst stage in EG and somatic cell NT were 16.8% (59/351) and 14.5% (98/677), respectively. In piezo-driven microinjection (Honolulu method) of donor nuclei into enucleated oocytes, the rates of blastocyst formation in EG and somatic cell NT were 11.9% (15/126) and 9.4% (9/96), respectively. Regardless of NT methods used in this study, EG cell NT gave rise to comparable rate of blastocyst development to somatic cell NT. Overall, EG cells can be used as karyoplast donor in NT procedure, and embryos can be produced by EG cell NT that may be used as an alternative to conventional somatic cell NT.

Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions

  • Jung, Juwon;Baek, Jin Ah;Seol, Hye Won;Choi, Young Min
    • Development and Reproduction
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    • v.20 no.1
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    • pp.63-71
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    • 2016
  • Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feeder layers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KO-SR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xeno-free conditions for clinical grade hESCs culture will be useful data in future clinical studies.

Directed Differentiation of Pancreatic Islets from Human Embryonic Stem Cells and Cell Therapy of Diabetes Mellitus (인간배아줄기세포를 이용한 췌장세포의 유도 분화 및 당뇨병의 세포치료)

  • Kim, Suel-Kee;Shim, Joong-Hyun;Woo, Dong-Hun;Kim, Jong-Hoon
    • Development and Reproduction
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    • v.11 no.2
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    • pp.67-77
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    • 2007
  • Replacement of insulin-producing cells represents an almost ideal treatment for patients with diabetes mellitus type 1. Transplantation of pancreatic islets of Langerhans is limited by the lack of donor organs. Therefore, generation of insulin-producing cells from human embryonic stem cells represents an attractive alternative. The present review summarizes the current knowledge on the differentiation of insulin-producing cells from human embryonic stem cells and their application to the cell therapy for treating diabetes mellitus.

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Teratogenic Effects of Phenytoin on Rat Embryos in Culture (랫드에 있어서 배양배자에 대한 Phenytoin의 최기형성 효과)

  • Kim, Jong-Choon;Lim, Kwang-Hyeon;Chung, Moon-Koo;Roh, Jung-Koo
    • Toxicological Research
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    • v.14 no.3
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    • pp.357-363
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    • 1998
  • The teratogenic potential of the anticonvulsant drug phenytoin (PHT) has been well documented both in the human and in the experimental animals. However there are few reports on the effects of PHT on embryonic development in rats in vitro. The present study was performed to evaluate the teratogenic effects of PHT using whole-embryo culture system in rats. Sprague-Dawley rat embryos were explanted on gestational day (GD) 9.5 and cultured for 48 hrs in the immediately centrifuged and heat-inactivated rat serum containing 0,25,50, or 100μg PHT/mL. At the end of culture period the embryos were scored for morphological development according to the procedure of Van Maele-Fabry, and their total protein contents were determined. At 100 μg/mL of culture medium. PHT caused significant reduction in developmental score and protein content of embryos and a high incidence morphological abnormalities (100%). Characteristic malformations included altered yolk and embryonic circulation, craniofacial hypoplasia, neural tube schisis, branchial arch defects, abnormal ratation, and limb bud hypoplasia, among others. There were no adverse effects on embryonic growth and development at concentrations of 25 and 50 μg /mL of culture medium. The results indicated that the dysmorphogenic effect of PHT on cultured embryos is due to a direct interference with embryonic development.

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Current Progress and Prospects of Reprogramming Factors - Stem Cells vs Germ Cells - (줄기세포와 생식세포에서 리프로그래밍 인자에 대한 최근 연구 동향과 전망)

  • Seo, You-Mi;Lee, Kyung-Ah
    • Development and Reproduction
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    • v.14 no.2
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    • pp.43-50
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    • 2010
  • Recently induced pluripotent stem (iPS) cells are derived from somatic cells by ectopic expression of several transcription factors (reprogramming factors) using technology of somatic cell reprogramming. iPS cells are able to selfrenew and differentiate into all type of cells in the body similarly to embryonic stem cells. Because iPS cells have advantages that can avoid immune rejection after transplantation and ethical issues unlike embryonic stem cells, research on iPS has made significant progress since the first report by Yamanaka in 2006. Nevertheless of many advantages of iPS, safer methods to introduce reprogramming factors into somatic cells must be developed due to safety concerns regarding viral vectors, and safer reprogramming factors to substitute the oncogenes should be evaluated for clinical application of iPS. Here we discuss the recent progress in reprogramming factors in embryonic stem cells, oocytes, and embryos, and discuss further research for finding new, more reliable and safer reprogramming factors.

Phosphorylation Status of RNA Polymerase II Carboxyl-terminal Domain in Porcine Oocytes and Early Embryos

  • Oqani, Reza K.;Zhang, Jin Yu;Lee, Min-Gu;Diao, Yun Fei;Jin, Dong-Il
    • Asian-Australasian Journal of Animal Sciences
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    • v.25 no.6
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    • pp.789-793
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    • 2012
  • Fertilization of the oocyte commences embryogenesis during which maternally inherited mRNAs are degraded and the embryonic genome is activated. Transcription of embryonic mRNA is initiated by embryonic genome activation (EGA). RNA polymerase II (RNA Pol II) is responsible for the synthesis of mRNAs and most small nuclear RNAs, and consists of 12 subunits, the largest of which characteristically harbors a unique C-terminal domain (CTD). Transcriptional activity of RNA Pol II is highly regulated, in particular, by phosphorylation of serine residues in the CTD. Here, we have shown the presence of RNA Pol II CTD phosphoisoforms in porcine oocytes and preimplantation embryos. The distribution pattern as well as phosphorylation dynamics in germinal vesicles and during embryogenesis differed in developmental stages with these isoforms, indicating a role of RNA Pol II CTD phosphorylation at the serine residue in transcriptional activation during both oocyte growth and embryonic genome activation. We additionally examined the effects of the RNA Pol II inhibitor, α-amanitin, on embryo development. Our results show that inhibition of polymerase, even at very early stages and for a short period of time, dramatically impaired blastocyst formation. These findings collectively suggest that the functionality of maternal RNA Pol II, and consequently, expression of early genes regulated by this enzyme are essential for proper embryo development.