32 MicroRNA-29b Improves the Quality and Developmental Potential of Blastocysts Derived from Somatic Cell Nuclear Transfer in Cattle

2018 ◽  
Vol 30 (1) ◽  
pp. 155
Author(s):  
W.-J. Zhou ◽  
S. Liang ◽  
X.-S. Cui
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cellular Proliferation ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Down Regulation ◽  
Developmental Potential ◽  
Somatic Cell Reprogramming ◽  
Underlying Mechanisms

MicroRNAs (miRNAs) are small non-coding RNAs with important roles in diverse cellular processes. miR-29b plays a crucial role during somatic cell reprogramming. However, studies of the function of miR-29b in embryogenesis are limited. 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 with IVF embryos (P < 0.05). To determine the function of miR-29b in the bovine SCNT embryo, we microinjected a miR-29b mimic and inhibitor into bovine SCNT zygotes. The results showed that miR-29b significantly decreased the expression of Dnmts (Dnmt3a/3b and Dnmt1) in bovine SCNT embryos (P < 0.05). We further investigated SCNT embryo developmental competence and found that miR-29b overexpression during bovine SCNT embryonic development does not improve developmental potency (P > 0.05) but down-regulation inhibits developmental potency (P < 0.05). Although miR-29b overexpression does not improve the developmental potency of bovine SCNT embryos, the quality of bovine SCNT embryos at the blastocyst stage improved significantly (P < 0.05). The expression of pluripotency factors (OCT4 and SOX2) and cellular proliferation rate were significantly higher in blastocysts from the miR-29b overexpression group than the control and down-regulation groups (P < 0.05). 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 down-regulation groups (P < 0.05). Taken together, these results demonstrated that miR-29b plays an important role in bovine SCNT embryo development.

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.

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.

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.

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.

scholarly journals Combined Chaetocin/Trichostatin A Treatment Improves the Epigenetic Modification and Developmental Competence of Porcine Somatic Cell Nuclear Transfer Embryos

2021 ◽  
Vol 9 ◽  
Author(s):  
Pil-Soo Jeong ◽  
Hae-Jun Yang ◽  
Soo-Hyun Park ◽  
Min Ah Gwon ◽  
Ye Eun Joo ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Histone Deacetylase Inhibitors ◽  
Epigenetic Modification ◽  
Trichostatin A ◽  
Combined Treatment ◽  
Epigenetic Reprogramming ◽  
Developmental Competence ◽  
Developmental Potential

Developmental defects in somatic cell nuclear transfer (SCNT) embryos are principally attributable to incomplete epigenetic reprogramming. Small-molecule inhibitors such as histone methyltransferase inhibitors (HMTi) and histone deacetylase inhibitors (HDACi) have been used to improve reprogramming efficiency of SCNT embryos. However, their possible synergistic effect on epigenetic reprogramming has not been studied. In this study, we explored whether combined treatment with an HMTi (chaetocin) and an HDACi (trichostatin A; TSA) synergistically enhanced epigenetic reprogramming and the developmental competence of porcine SCNT embryos. Chaetocin, TSA, and the combination significantly increased the cleavage and blastocyst formation rate, hatching/hatched blastocyst rate, and cell numbers and survival rate compared to control embryos. In particular, the combined treatment improved the rate of development to blastocysts more so than chaetocin or TSA alone. TSA and combined chaetocin/TSA significantly reduced the H3K9me3 levels and increased the H3K9ac levels in SCNT embryos, although chaetocin alone significantly reduced only the H3K9me3 levels. Moreover, these inhibitors also decreased global DNA methylation in SCNT embryos. In addition, the expression of zygotic genome activation- and imprinting-related genes was increased by chaetocin or TSA, and more so by the combination, to levels similar to those of in vitro-fertilized embryos. These results suggest that combined chaetocin/TSA have synergistic effects on improving the developmental competences by regulating epigenetic reprogramming and correcting developmental potential-related gene expression in porcine SCNT embryos. Therefore, these strategies may contribute to the generation of transgenic pigs for biomedical research.

38 Improved Cloning Efficiency and Developmental Potential in Bovine Somatic Cell Nuclear Transfer with the New Technology

2018 ◽  
Vol 30 (1) ◽  
pp. 158
Author(s):  
L. Xu ◽  
M.-D. Joo ◽  
A. Mesalam ◽  
S.-H. Song ◽  
S. Zhang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Reverse Transcription ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Total Cell ◽  
Developmental Competence ◽  
Mrna Levels ◽  
Methyl Transferase ◽  
Developmental Potential

Bovine somatic cell nuclear transfer (SCNT) embryos can develop to the blastocyst stage at a rate similar to that of embryos produced by IVF; however, its efficiency remains low. In this study, we examined the effects of cytoplasm restoration of enucleated oocyte, by injecting ~30% of the cytoplasm of a donor oocyte to restore the enucleated oocyte cytoplasm volume to normal, on the developmental competence and quality of bovine cloned embryos during pre-implantation using the TUNEL assay, quantitative reverse transcription PCR (RT-qPCR) and immunocytochemistry. The experiment was conducted in 6 replicates. The differences in embryo development and expression levels of the various genes between experimental groups were analysed by one-way ANOVA. The level of statistical significance was set at P < 0.05. The percentages of embryos that underwent cleavage and formed a blastocyst were significantly higher (P < 0.05) in the cytoplasmic injected group than in the traditional SCNT group (61.5 ± 1.3% v. 39.7 ± 2.1% and 28.9 ± 0.8% v. 20.2 ± 1.3%, respectively). Furthermore, the beneficial effects of cytoplasmic injection on the cloned embryos were associated with a significantly increased (P < 0.05) total cell number in Day 8 blastocysts compared with the traditional SCNT group (176.2 ± 6.5 v. 119.3 ± 7.7; P < 0.05); however, there was no difference (P > 0.05) between the number of apoptotic cells per blastocyst in the cytoplasmic injected group and in the traditional SCNT group (3.5 ± 1.1 v. 4.1 ± 0.8). Moreover, cytoplasm restoration of enucleated oocyte significantly increased (P < 0.05) mitochondrial activity, as identified by MitoTracker Green (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription-qPCR showed that the mRNA levels of DNA methyl-transferase 1 and DNA methyl-transferase 3a were significantly decreased (P < 0.05) in cytoplasmic injected group compared with the traditional SCNT group, but did not significantly differ (P > 0.05) between the cytoplasmic injected and IVF groups. Taken together, these data suggest that cytoplasm restoration of enucleated oocyte improves in vitro developmental competence and quality of bovine cloned embryos, as evidenced by increased total cell numbers, reprogramming efficiency, and mitochondria activity. This work was partly supported by grant from the Next-Generation BioGreen21 (No. PJ01107703), IPET (No. 315017-5 and 117029-3), Allergy free cat (Co. Felix Pets) and BK21plus.

Effect of melatonin treatment on developmental potential of somatic cell nuclear-transferred mouse oocytes in vitro

Zygote ◽  
2013 ◽  
Vol 22 (2) ◽  
pp. 213-217 ◽  
Author(s):  
Mohammad Salehi ◽  
Yoko Kato ◽  
Yukio Tsunoda
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Blastocyst Stage ◽  
Control Group ◽  
Developmental Potential ◽  
Significant Difference ◽  
Mouse Somatic Cell

SummaryThe beneficial effect of supplementing culture medium with melatonin has been reported during in vitro embryo development of species such as mouse, bovine and porcine. However, the effect of melatonin on mouse somatic cell nuclear transfer remains unknown. In this study, we assessed the effects of various concentrations of melatonin (10−6 to 10−12 M) on the in vitro development of mouse somatic cell nuclear transfer embryos for 96 h. Embryos cultured without melatonin were used as control. There was no significant difference in cleavage rates between the groups supplemented with melatonin, dimethyl sulphoxide (DMSO) and the control. The rate of development to blastocyst stage was significantly higher in the group supplemented with 10−12 M melatonin compared with the control group (P < 0.05). Thus, our data demonstrated that adding melatonin to pre-implantation mouse nuclear-transferred embryos can accelerate blastocyst formation.

332 EMBRYONIC DEVELOPMENT AFTER SOMATIC CELL NUCLEAR TRANSFER OF PORCINE OOCYTES MEIOTICALLY INHIBITED WITH ROSCOVITINE

2006 ◽  
Vol 18 (2) ◽  
pp. 273
Author(s):  
S. W. Kim ◽  
D. H. Kim ◽  
J. S. Seo ◽  
G. S. Im ◽  
B. C. Yang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Apoptotic Cell ◽  
Treated Group ◽  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Developmental Potential ◽  
Maturation Medium

Numerous factors affect on the developmental competence of cloned embryos, and one of the factors might be the disturbed synchronization of nuclear and cytoplasm maturation. Roscovitine, a purine known to specifically inhibit M-phase promoting factor (MPF) kinase activity by blocking the ATP in numerous cell systems, has been successfully used in maintaining porcine oocytes at GV stage without affecting their developmental potential. However, developmental ability of roscovitine treated porcine oocytes after nuclear transfer has not been evaluated. The purpose of this study was to examine the development of nuclear transferred porcine embryos after meiotic inhibition with roscovitine (ROS). Cumulus-oocyte complexes (COCs) were collected from antral follicles of slaughtered prepubertal gilts. COCs were cultured in pre-maturation medium (TCM-199 containing 50 �M Roscovitine) for 24 h, and then further cultured in conventional maturation medium for 44 h. A control group was cultured in the maturation medium for 44 h. Matured oocytes were enucleated and a porcine fetus cell was inserted into each enucleated oocyte. Couplets were simultaneously fused and activated with electric pulse of two 1.2 kV/cm for 30 �s. Nuclear transferred (NT) embryos were cultured in PZM-1 medium for 6 days (five replicates). Apoptotic cell death was analyzed by using a TUNEL assay and total cell number was examined by Hoechest 33342 counterstaining. At 3 h after fusion, NT embryos were fixed for microfilament staining. Data were analyzed by ANOVA and Student's t-test. The rates of fusion, cleavage, and blastocyst formation of the ROS-treated group (85, 68, and 18%, respectively) after nuclear transfer did not differ from control (78, 76, and 16%, respectively). The cell number in blastocysts of the ROS-treated group (30.8 � 10.6) was significantly lower than that of the control (42.3 � 13.7) (P < 0.01), but the mean proportion of apoptotic cells was not different between the two groups (6.9 � 7.1 and 4.8 � 4.9% for control and ROS group, respectively). Recovery of microfilaments after fusion was delayed in NT embryos derived from ROS-treated oocytes. This study demonstrated that porcine oocytes pre-cultured for 24 h in presence of roscovitine can be developed to blastocysts after somatic cell nuclear transfer. This could provide flexibility for studying porcine oocyte development and embryo cloning.

Developmental competence of 8–16-cell stage bison embryos produced by interspecies somatic cell nuclear transfer

2016 ◽  
Vol 28 (9) ◽  
pp. 1360 ◽  
Author(s):  
L. Antonio González-Grajales ◽  
Laura A. Favetta ◽  
W. Allan King ◽  
Gabriela F. Mastromonaco
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Bos Taurus ◽  
Developmental Competence ◽  
Bison Bison ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Cell Stage ◽  
Developmental Potential

Altered communication between nuclear and cytoplasmic components has been linked to impaired development in interspecies somatic cell nuclear transfer (iSCNT) embryos as a result of genetic divergence between the two species. This study investigated the developmental potential and mitochondrial function of cattle (Bos taurus), plains bison (Bison bison bison) and wood bison (Bison bison athabascae) embryos produced by iSCNT using domestic cattle oocytes as cytoplasts. Embryos in all groups were analysed for development, accumulation of ATP, apoptosis and gene expression of nuclear- and mitochondrial-encoded genes at the 8–16-cell stage. The results of this study showed no significant differences in the proportion of developed embryos at the 2-, 4- and 8–16-cell stages between groups. However, significantly higher ATP levels were observed in cattle SCNT embryos compared with bison iSCNT embryos. Significantly more condensed and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL)-positive nuclei were found in plains bison iSCNT embryos. No significant differences in the expression levels of nuclear respiratory factor 2 (NRF2) or mitochondrial subunit 2 of cytochrome c oxidase (mt-COX2) were found in any of the groups. However, mitochondrial transcription factor A (TFAM) expression significantly differed between groups. The results of this study provide insights into the potential causes that might lead to embryonic arrest in bison iSCNT embryos, including mitochondrial dysfunction, increased apoptosis and abnormal gene expression.

Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions

Zygote ◽  
2003 ◽  
Vol 11 (2) ◽  
pp. 167-174 ◽  
Author(s):  
Xi-Jun Yin ◽  
Seong-Keun Cho ◽  
Mi-Ryeung Park ◽  
Yeo-Jeoung Im ◽  
Joung-Ju Park ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Blastocyst Stage ◽  
Developmental Potential ◽  
Nuclear Envelope Breakdown ◽  
Donor Nucleus ◽  
Pcr Rflp ◽  
Decondensed Chromatin ◽  
Microtubule Aster

It is still unclear whether nuclear envelope breakdown and premature chromosome condensation are essential for the reprogramming of the donor nucleus following somatic nuclear transfer. To address this, we determined the ability of delayed-activated or simultaneously activated porcine oocytes to undergo nuclear remodelling and development following somatic cell nuclear transfer. A small microtubule aster was observed in association with decondensed chromatin following nuclear transfer, suggesting the introduction of a somatic cell centrosome. In the delayed-activated condition, most fibroblast nuclei divided into two chromosome masses and two pronuclear-like structures following transfer into oocytes. In contrast, fibroblast nuclei in the simultaneously activated condition formed a large, swollen, pronuclear-like structure. Microtubule asters were organised in the vicinity of the nucleus regardless of the number of nuclei. More reconstructed oocytes developed to the blastocyst stage in the delayed-activated condition than in the simultaneously activated condition (p < 0.05). Nine piglets were born from two recipient sows following transfer of delayed-activated reconstructed oocytes, while none developed to full term in the simultaneously activated condition. Fingerprint analysis showed that the PCR-RFLP patterns of the nine offspring were identical to that of the donor pig. These results suggest that the activation of recipient oocytes during nuclear transfer probably relates to the nuclear remodelling process, which can affect the ability of embryos created by somatic cell nuclear transfer to develop.

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