scholarly journals Somatic cell nuclear transfer alters peri-implantation trophoblast differentiation in bovine embryos

Reproduction ◽  
2006 ◽  
Vol 132 (2) ◽  
pp. 279-290 ◽  
Author(s):  
Daniel R Arnold ◽  
Vilceu Bordignon ◽  
Réjean Lefebvre ◽  
Bruce D Murphy ◽  
Lawrence C Smith
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Placental Development ◽  
Trophoblast Differentiation ◽  
Expression Of Genes ◽  
And Function

Abnormal placental development limits success in ruminant pregnancies derived from somatic cell nuclear transfer (SCNT), due to reduction in placentome number and consequently, maternal/fetal exchange. In the primary stages of an epithelial–chorial association, the maternal/fetal interface is characterized by progressive endometrial invasion by specialized trophoblast binucleate/giant cells (TGC). We hypothesized that dysfunctional placentation in SCNT pregnancies results from aberration in expression of genes known to be necessary for trophoblast proliferation (Mash2), differentiation (Hand1), and function (IFN-τ and PAG-9). We, therefore, compared the expression of these factors in trophoblast from bovine embryos derived from artificial insemination (AI), in vitro fertilization (IVF), and SCNT prior to (day 17) and following (day 40 of gestation) implantation, as well as TGC densities and function. In preimplantation embryos, Mash2 mRNA was more abundant in SCNT embryos compared to AI, while Hand1 was highest in AI and IVF relative to SCNT embryos. IFN-τ mRNA abundance did not differ among groups. PAG-9 mRNA was undetectable in SCNT embryos, present in IVF embryos and highest in AI embryos. In postimplantation pregnancies, SCNT fetal cotyledons displayed higher Mash2 and Hand1 than AI and IVF tissues. Allelic expression of Mash2 was not different among the groups, which suggests that elevated mRNA expression was not due to altered imprinting status of Mash2. The day 40 SCNT cotyledons had the fewest number of TGC compared to IVF and AI controls. Thus, expression of genes critical to normal placental development is altered in SCNT bovine embryos, and this is expected to cause abnormal trophoblast differentiation and contribute to pregnancy loss.

Effects of VEGF on Porcine In Vitro Fertilized and Somatic Cell Nuclear Transfer (SCNT) Preimplantation Embryos.

2010 ◽  
Vol 83 (Suppl_1) ◽  
pp. 417-417
Author(s):  
Dibyendu Biswas ◽  
Yu-Byeol Jeon ◽  
Eui Man Jung ◽  
Eui Bae Jeung ◽  
Sang-Hwan Hyun
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Preimplantation Embryos

scholarly journals Expression Profile of Genes as Indicators of Developmental Competence and Quality of In Vitro Fertilization and Somatic Cell Nuclear Transfer Bovine Embryos

PLoS ONE ◽  
2014 ◽  
Vol 9 (9) ◽  
pp. e108139 ◽  
Author(s):  
Maria Jesús Cánepa ◽  
Nicolás Matías Ortega ◽  
Melisa Carolina Monteleone ◽  
Nicolas Mucci ◽  
German Gustavo Kaiser ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Somatic Cell ◽  
Expression Profile ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Vitro Fertilization

In vitro culture of stem-like cells derived from somatic cell nuclear transfer bovine embryos of the Korean beef cattle species, HanWoo

2016 ◽  
Vol 28 (11) ◽  
pp. 1762 ◽  
Author(s):  
Daehwan Kim ◽  
Sangkyu Park ◽  
Yeon-Gil Jung ◽  
Sangho Roh
Keyword(s):  
Beef Cattle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryoid Bodies ◽  
Marker Genes ◽  
Bovine Embryos ◽  
In Vivo Differentiation

We established and maintained somatic cell nuclear transfer embryo-derived stem-like cells (SCNT-eSLCs) from the traditional Korean beef cattle species, HanWoo (Bos taurus coreanae). Each SCNT blastocyst was placed individually on a feeder layer with culture medium containing three inhibitors of differentiation (3i). Primary colonies formed after 2–3 days of culture and the intact colonies were passaged every 5–6 days. The cells in each colony showed embryonic stem cell-like morphologies with a distinct boundary and were positive to alkaline phosphatase staining. Immunofluorescence and reverse transcription–polymerase chain reaction analyses also confirmed that these colonies expressed pluripotent markers. The colonies were maintained over 50 passages for more than 270 days. The cells showed normal karyotypes consisting of 60 chromosomes at Passage 50. Embryoid bodies were formed by suspension culture to analyse in vitro differentiation capability. Marker genes representing the differentiation into three germ layers were expressed. Typical embryonal carcinoma was generated after injecting cells under the testis capsule of nude mice, suggesting that the cultured cells may also have the potential of in vivo differentiation. In conclusion, we generated eSLCs from SCNT bovine embryos, using a 3i system that sustained stemness, normal karyotype and pluripotency, which was confirmed by in vitro and in vivo differentiation.

scholarly journals 44 IMPROVED DEVELOPMENT OF BOVINE NUCLEAR TRANSFER EMBRYOS BY THE TREATMENT OF NUCLEAR DONOR CELLS WITH APOPTOSIS INHIBITORS DURING SERUM STARVATION

2005 ◽  
Vol 17 (2) ◽  
pp. 171
Author(s):  
C.-K. Lee ◽  
Bon-sik Koo ◽  
C.-H. Park ◽  
S.-G. Lee ◽  
D.-H. Choi ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Tunel Assay ◽  
Serum Starvation ◽  
Bovine Embryos ◽  
Embryonic Fibroblasts ◽  
Crown Rump Length ◽  
Donor Cells

In somatic cell nuclear transfer, serum starvation is a widely used method to synchronize donor cells at the quiescent stage (Go) of the cell cycle. However, it has been shown that serum starvation during culture of mammalian cells could induce cell death via apoptosis by removing growth factors and increasing intracellular stress. Therefore, apoptosis caused by serum starvation in somatic cells could induce damages to nuclear DNA contributing to a lower efficiency of nuclear transfer. This study was performed to characterize apoptosis during serum starvation of bovine embryonic fibroblasts (BEFs) and to determine the effects of BEFs treated with apoptosis inhibitors on the development of bovine embryos after nuclear transfer. BEFs, collected from a fetus with a 3–4-cm crown-rump length, were cultured for 7 days in starvation medium consisting of Dulbecco's modified Eagle's medium containing 0.5% fetal bovine serum to induce quiescence. Cells were also placed in starvation medium containing the apoptosis inhibitors, β2-macroglobulin (broad-range protease inhibitor: MAC; 1.4 pM) and glutathione (GSH: reactive oxygen species scavenger; 2.0 mM). Apoptosis of serum starved BEFs with or without apoptosis inhibitors were analyzed morphologically with light and electron microscope, and biochemically using a TUNEL assay. Somatic cell nuclear transfer was performed by our standard procedure as follows. Bovine oocytes were matured in vitro and enucleated after 22 h. Nuclear donor cells were collected randomly before injection. The reconstructed embryos were placed into the fusion chamber in a solution containing 0.28 M mannitol and aligned manually. A double pulse of 1.8 kV/cm for 15 μs was used to fuse the cells and activate the embryos simultaneously. The fused embryos were cultured for 4 min in 5 μ­M ionomycin and 4 h in 2 mM 6-DMAP. Then, embryos were moved to culture media and cultured in 5% CO2 and 39°C in 100% humidity. Development of NT embryos was recorded at 120 h post NT (morulae) and 168 h (blastocysts) with experiments being repeated three times. Serum starved BEFs showed typical morphology of apoptotic cells such as chromatin condensation and membrane blebbing. Also, when stained for DNA fragmentation by TUNEL assay, 22.6% ofBEFs showed apoptosis, in contrast to 0.1% in actively growing cells. However, when BEFs were cultured with MAC and GSH, the proportions of apoptotic BEFs were greatly reduced, 6.0% and 2.1%, respectively. After nuclear transfer with BEFs, embryos reconstructed with BEF treated with apoptosis inhibitors showed significant improvement in in vitro development compared to the controls (Table 1). In conclusion, while there are a number of factors affecting the nuclear transfer procedure, damage to the donor nuclei by serum starvation is likely to reduce the efficiency of the procedure; the addition of apoptosis inhibitors could reduce this unnecessary damage to donor nuclei and result in improvement in the development of nuclear transferred embryos. Further experiments are needed to assess the effect of apoptosis inhibitors on improvement of overall nuclear transfer efficiency. Table 1. Development of bovine embryos nuclear transferred with embryonic fibroblasts treated with or without apoptosis inhibitors

38 DNA METHYLATION IN PORCINE PREIMPLANTATION EMBRYOS DEVELOPED IN VIVO AND PRODUCED BY IN VITRO FERTILIZATION, PARTHENOGENETIC ACTIVATION, AND SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 125
Author(s):  
R. S. Deshmukh ◽  
O. Oestrup ◽  
E. Oestrup ◽  
M. Vejlsted ◽  
H. Niemann ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cell Mass ◽  
Epifluorescence Microscopy ◽  
Preimplantation Embryos ◽  
Parthenogenetic Activation

DNA de- and re-methylation are crucial for reprogramming of the differentiated parental/somatic genome in the ooplasm. The presented research was aimed at analysis of the DNA methylation dynamics in porcine preimplantation embryos developed in vivo (IV) and produced in vitro by IVF, somatic cell nuclear transfer (SCNT), and parthenogenetic activation (PA). Embryos of early and late 1-cell, 2-, 4-, and 8-cell, and early and late blastocysts stages obtained by the mentioned methods were fixed in 4% paraformaldehyde and subjected to immunocytochemistry using anti-5MetC (Mouse monoclonal, Abcam, Cambridge, MA, USA) antibody. DNA was labelled using Hoechst 33258 (Sigma, Copenhagen, Denmark). Epifluorescence microscopy (Leica Microsystems, Wetzlar, Germany) images were subjected to NIH imageJ software to measure the DNA methylation/DNA content signal by manually outlining the nuclei (n = 2003) of the embryos. The data were analysed using PROC-GLM statistical procedure in SAS 9.1 (SAS Institute Inc., Cary, NC, USA), least square means were compared and P-values were used to decide the significant differences within and between different groups of embryos. The 1-cell stages lacked active demethylation of paternal genome in IV and IVF embryos. Embryos produced under in vitro conditions presented higher levels of DNA methylation than IV. A lineage specific DNA methylation (hypermethylation of inner cell mass and hypomethylation of trophectoderm) observed in porcine IV late blastocysts was absent in PA and SCNT blastocysts despite the occurrence of de novo methylation in early blastocysts. SCNT early (50%) and late (14%) blastocysts presented DNA methylation pattern similar to IV early and late blastocysts, respectively. Concluding, DNA methylation patterns are strongly impaired under in vitro conditions in porcine preimplantation embryos.

Scriptaid Improves In Vitro Development and Nuclear Reprogramming of Somatic Cell Nuclear Transfer Bovine Embryos

2011 ◽  
Vol 13 (5) ◽  
pp. 431-439 ◽  
Author(s):  
Li-Jun Wang ◽  
Hui Zhang ◽  
Yong-Sheng Wang ◽  
Wen-Bing Xu ◽  
Xian-Rong Xiong ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Nuclear Reprogramming ◽  
Bovine Embryos ◽  
In Vitro Development ◽  
Vitro Development
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