Production of somatic cell nuclear transfer embryos using in vitro-grown and in vitro-matured oocytes in rabbits

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 494-500 ◽  
Author(s):  
Hironobu Sugimoto ◽  
Yuta Kida ◽  
Noriyoshi Oh ◽  
Kensaku Kitada ◽  
Kazuya Matsumoto ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Histone Deacetylase Inhibitor ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Deacetylase Inhibitor ◽  
Number Of Cells

SummaryWe examined growing oocytes collected from follicles remaining in superovulated rabbit ovaries, that were grown (in vitro growth, IVG) and matured (in vitro maturation, IVM) in vitro. We produced somatic cell nuclear transfer (SCNT) embryos using the mature oocytes and examined whether these embryos have the ability to develop to the blastocyst stage. In addition, we examined the effects of trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), on the developmental competence of SCNT embryos derived from IVG–IVM oocytes. After growth for 7 days and maturation for 14–16 h in vitro, the growing oocytes reached the metaphase II stage (51.4%). After SCNT, these reconstructed embryos reached the blastocyst stage (20%). Furthermore, the rate of development to the blastocyst stage and the number of cells in the blastocysts in SCNT embryos derived from IVG–IVM oocytes were significantly higher for TSA-treated embryos compared with TSA-untreated embryos (40.6 versus 21.4% and 353.1 ± 59.1 versus 202.5 ± 54.6, P < 0.05). These results indicate that rabbit SCNT embryos using IVG–IVM oocytes have the developmental competence to reach the blastocyst stage.

Overexpression of MicroRNA-29b Decreases Expression of DNA Methyltransferases and Improves Quality of the Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

2018 ◽  
Vol 24 (1) ◽  
pp. 29-37 ◽  
Author(s):  
Shuang Liang ◽  
Zheng-Wen Nie ◽  
Jing Guo ◽  
Ying-Jie Niu ◽  
Kyung-Tae Shin ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cellular Proliferation ◽  
Dna Methyltransferases ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Somatic Cell Reprogramming

AbstractMicroRNA (miR)-29b plays a crucial role during somatic cell reprogramming. The aim of the current study was to explore the effects of miR-29b on the developmental competence of bovine somatic cell nuclear transfer (SCNT) embryos, as well as the underlying mechanisms of action. The expression level of miR-29b was lower in bovine SCNT embryos at the pronuclear, 8-cell, and blastocyst stages compared within vitrofertilized embryos. In addition, miR-29b regulates the expression of DNA methyltransferases (Dnmt3a/3bandDnmt1) in bovine SCNT embryos. We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency and downregulation inhibits developmental potency. Nevertheless, the quality of bovine SCNT embryos at the blastocyst stage improved significantly. The expression of pluripotency factors and cellular proliferation were significantly higher in blastocysts from the miR-29b overexpression group than the control and downregulation groups. In addition, outgrowth potential in blastocysts after miR-29b overexpression was also significantly greater in the miR-29b overexpression group than in the control and downregulation groups. Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

Development and spindle formation in rat somatic cell nuclear transfer (SCNT) embryos in vitro using porcine recipient oocytes

Zygote ◽  
2009 ◽  
Vol 17 (3) ◽  
pp. 195-202 ◽  
Author(s):  
Atsushi Sugawara ◽  
Satoshi Sugimura ◽  
Yumi Hoshino ◽  
Eimei Sato
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Fluorescent Protein ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Spindle Formation ◽  
Fetal Fibroblasts

SummaryCloning that uses somatic cell nuclear transfer (SCNT) technology with gene targeting could be a potential alternative approach to obtain valuable rat models. In the present study, we determined the developmental competence of rat SCNT embryos constructed using murine and porcine oocytes at metaphase II (MII). Further, we assessed the effects of certain factors, such as: (i) the donor cell type (fetal fibroblasts or cumulus cells); and (ii) premature chromosome condensation (PCC) with normal spindle formation, on the developmental competence of rat interspecies SCNT (iSCNT) embryos. iSCNT embryos that had been constructed using porcine oocytes developed to the blastocyst stage, while those embryos made using murine MII oocytes did not. Rat iSCNT embryos constructed with green fluorescent protein (GFP)-expressing fetal fibroblasts injected into porcine oocytes showed considerable PCC with a normal bipolar spindle formation. The total cell number of iSCNT blastocyst derived from GFP-expressing fetal fibroblasts was higher than the number derived from cumulus cells. In addition, these embryos expressed GFP at the blastocyst stage. This paper is the first report to show that rat SCNT embryos constructed using porcine MII oocytes have the potential to develop to the blastocyst stage in vitro. Thus the iSCNT technique, when performed using porcine MII oocytes, could provide a new bioassay system for the evaluatation of the developmental competence of rat somatic cells.

46 EFFECT OF SHORT-TERM TRICHOSTATIN A TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 123
Author(s):  
H. J. Oh ◽  
J. E. Park ◽  
M. J. Kim ◽  
S. G. Hong ◽  
J. T. Kang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Short Term ◽  
Total Cell Number

Epigenetic reprogramming such as acetylation in somatic cell nuclear transfer (SCNT) has been known as one of problems in cloned embryos. For resolving this acetylation reprogramming, many investigators recently have reported the effect of long-term culture of post-activated SCNT embryos using trichostatin A (TSA), a histone deacetylase inhibitor (HDACi). The objective of this study is to investigate the effect of short-term TSA treatment on in vitro developmental ability and the quality of bovine SCNT embryos. Immature oocytes were aspirated from abattoir-derived ovaries, matured in vitro for 22 h, and enucleated. A bovine fetal fibroblast was placed into the enucleated oocyte and fused by electrical stimulation. The fused couplets were activated by 4-min incubation in 10 μm ionomycin, followed by 4 h of culture in 1.9 mm 6-dimethylaminopurine with or without TSA (0, 50, or 100 nm). The SCNT embryos were subsequently cultured in modified synthetic oviduct fluid medium for 8 days. Developmental competence was assessed by blastocyst formation and total cell number. Total cell numbers were determined by staining with bisbenzimide 33342. As results, developmental competence to blastocysts was higher in 100 nm than control (36.7 v. 27.9%, P < 0.05). In blastocyst hatching rate, TSA 100 nm group (19.5%) at 8 days showed an increased pattern as opposed to control and TSA 50 nm group (11.1 and 12.7%; P < 0.05). No significant differences in two cell and morula stage were observed among treatment groups. In terms of development to hatching stage of blastocysts, TSA 100 nm group (19.5%) at 8 days has a significant effect compared to control and TSA 50 nm group (11.1 and 12.7%; P < 0.05). Total cell number of blastocysts derived from TSA 100 nm was significantly higher (P < 0.05) than that in TSA 50 nm (116 v. 100), whereas there was not significant difference between control and TSA 100 nm. In conclusion, short-term culture with high concentration of TSA improved the blastocysts formation however total cell number of blastocysts showed contradictory result. The epigenetic modification by TSA treatment on bovine SCNT needs further investigation. This study was financially supported by KOSEF (grant # M10625030005-08N250300510) and the Korean MEST, through the BK21 program for Veterinary Science.

Cotreatment with RepSox and LBH589 improves the in vitro developmental competence of porcine somatic cell nuclear transfer embryos

2018 ◽  
Vol 30 (10) ◽  
pp. 1342 ◽  
Author(s):  
Zhao-Bo Luo ◽  
Long Jin ◽  
Qing Guo ◽  
Jun-Xia Wang ◽  
Xiao-Xu Xing ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Abnormal Development ◽  
Control Group ◽  
Blastocyst Formation

Accumulating evidence suggests that aberrant epigenetic reprogramming and low pluripotency of donor nuclei lead to abnormal development of cloned embryos and underlie the inefficiency of mammalian somatic cell nuclear transfer (SCNT). The present study demonstrates that treatment with the small molecule RepSox alone upregulates the expression of pluripotency-related genes in porcine SCNT embryos. Treatment with the histone deacetylase inhibitor LBH589 significantly increased the blastocyst formation rate, whereas treatment with RepSox did not. Cotreatment with 12.5 μM RepSox and 50 nM LBH589 (RepSox + LBH589) for 24 h significantly increased the blastocyst formation rate compared with that of untreated embryos (26.9% vs 8.5% respectively; P < 0.05). Furthermore, the expression of pluripotency-related genes octamer-binding transcription factor 4 (NANOG) and SRY (sex determining region Y)-box 2 (SOX2) were found to significantly increased in the RepSox + LBH589 compared with control group at both the 4-cell and blastocyst stages. In particular, the expression of NANOG was 135-fold higher at the blastocyst stage in the RepSox + LBH589 group. Moreover, RepSox + LBH589 improved epigenetic reprogramming. In summary, RepSox + LBH589 increases the expression of developmentally important genes, optimises epigenetic reprogramming and improves the in vitro development of porcine SCNT embryos.

scholarly journals Transcriptome Analyses Reveal Differential Transcriptional Profiles in Early- and Late-Dividing Porcine Somatic Cell Nuclear Transfer Embryos

Genes ◽  
2020 ◽  
Vol 11 (12) ◽  
pp. 1499
Author(s):  
Zhiguo Liu ◽  
Guangming Xiang ◽  
Kui Xu ◽  
Jingjing Che ◽  
Changjiang Xu ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Rna Processing ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Molecular Mechanisms ◽  
Differentially Expressed Gene ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Rna Seq ◽  
Transcriptional Profiles

Somatic cell nuclear transfer (SCNT) is not only a valuable tool for understanding nuclear reprogramming, but it also facilitates the generation of genetically modified animals. However, the development of SCNT embryos has remained an uncontrollable process. It was reported that the SCNT embryos that complete the first cell division sooner are more likely to develop to the blastocyst stage, suggesting their better developmental competence. Therefore, to better understand the underlying molecular mechanisms, RNA-seq of pig SCNT embryos that were early-dividing (24 h postactivation) and late-dividing (36 h postactivation) was performed. Our analysis revealed that early- and late-dividing embryos have distinct RNA profiles, and, in all, 3077 genes were differentially expressed. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that early-dividing embryos exhibited higher expression in genes that participated in the meiotic cell cycle, while enrichment of RNA processing- and translation-related genes was found in late-dividing embryos. There are also fewer somatic memory genes such as FLRT2, ADAMTS1, and FOXR1, which are abnormally activated or suppressed in early-dividing cloned embryos. These results show that early-dividing SCNT embryos have different transcriptional profiles than late-dividing embryos. Early division of SCNT embryos may be associated with their better reprogramming capacity, and somatic memory genes may act as a reprogramming barrier in pig SCNT reprogramming.

48 EFFECTS OF TRICHOSTATIN A ON DEVELOPMENT OF BOVINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 142 ◽  
Author(s):  
D. Iwamoto ◽  
K. Saeki ◽  
S. Kishigami ◽  
A. Kasamatsu ◽  
A. Tatemizo ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Mammalian Species ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Serum Starvation ◽  
Embryonic Genome ◽  
Blastocyst Rate

Although cloning by somatic cell nuclear transfer (SCNT) has been achieved in various mammalian species, its efficiency has been very low (Han et al. 2003 Theriogenology 59, 33–44). Successful cloning requires conversion from differentiated donor nuclei to embryonic nuclei after transfer of the somatic nuclei into enucleated oocytes. Reprogramming of the transferred somatic nuclei must be completed by the time when normal activation of the embryonic genome occurs (Solter 2000 Nat. Rev. Genet. 1, 199–207). Recently, both full-term development and pre-implantation development of mouse SCNT embryos were significantly enhanced by treatment with trichostatin A (TSA), an inhibitor of histone deacetylase (Kishigami et al. 2006 Biochem. Biophys. Res. Commun. 340, 183–189; Rybouchkin et al. 2006 Biol. Reprod. 74, 1083–1089). The objective of this study was to investigate the effects of TSA on the development of bovine SCNT embryos. Bovine fibroblasts were cultured under serum starvation (0.4% FCS) for 7 days and then used as donor cells. The cells were electro-fused with bovine enucleated matured oocytes, and activated with a calcium ionophore and cycloheximide. They were subsequently cultured in mSOF medium until 168 h post-activation (hpa). The NT embryos were exposed to 0 (control), 5, 50, and 500 nM TSA from the start of activation to 48 hpa. Experiments were repeated 3 times, and the data were analyzed with Fisher's PLSD test following ANOVA. The cleavage rates were the same among the groups (60 to 80&percnt;; P &gt;0.05). However, the blastocyst rate of NT embryos treated with 50 nM TSA was higher than that of control embryos (40&percnt; vs. 19&percnt;, respectively; P &lt; 0.05). On the other hand, the blastocyst rate was lower with 500 nM TSA than with 5 or 50 nM TSA (7&percnt; vs. 33&percnt; or 40&percnt;; P &lt; 0.05). These data suggest that proper TSA treatment after somatic cloning improves the rate of development of bovine cloned embryos to the blastocyst stage. Further research is needed to examine whether NT embryos derived from different cell lines or types have similar susceptibility to TSA.

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