scholarly journals Psammaplin A Improves Development and Quality of Somatic Cell Nuclear Transfer Mouse Embryos

2014 ◽  
Vol 16 (5) ◽  
pp. 392-406 ◽  
Author(s):  
Anna Mallol ◽  
Josep Santaló ◽  
Elena Ibáñez
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mouse Embryos ◽  
Psammaplin A

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.

Kdm6a overexpression improves the development of cloned mouse embryos

Zygote ◽  
2017 ◽  
Vol 26 (1) ◽  
pp. 24-32 ◽  
Author(s):  
Guang-yu Bai ◽  
Si-hang Song ◽  
Yu-wei Zhang ◽  
Xiang Huang ◽  
Xing-wei Huang ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Life Science ◽  
Cumulus Cell ◽  
Science Research ◽  
Mouse Embryos ◽  
Rdna Transcription ◽  
X Chromosomes ◽  
Blastocyst Rate

SummarySomatic cell nuclear transfer (SCNT) is an important technique for life science research. However, most SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we show that abnormal Xi occurs in somatic cell NT blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. H3K27me3 removal by Kdm6a mRNA overexpression could significantly improve preimplantation development of NT embryos, and even reached a 70.2% blastocyst rate of cleaved embryos compared with the 38.5% rate of the control. H3K27me3 levels were significantly reduced in blastomeres from cloned blastocysts after overexpression of Kdm6a. qPCR indicated that rDNA transcription increased in both NT embryos and 293T cells after overexpression of Kdm6a. Our findings demonstrate that overexpression of Kdm6a improved the development of cloned mouse embryos by reducing H3K27me3 and increasing rDNA transcription.

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

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.

Number of blastomeres and distribution of microvilli in cloned mouse embryos during compaction

Zygote ◽  
2010 ◽  
Vol 19 (3) ◽  
pp. 271-276 ◽  
Author(s):  
Chao-Bo Li ◽  
Zhen-Dong Wang ◽  
Zhong Zheng ◽  
Li-Li Hu ◽  
Shu-Qi Zhong ◽  
...  
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Time Course ◽  
Mouse Embryos ◽  
Apical Region ◽  
Blastocyst Formation ◽  
Cell Stage ◽  
Significant Difference ◽  
Before And After

SummaryThe events resulting in compaction have an important influence on the processes related to blastocyst formation. To analyse the quality of the embryos obtained by somatic cell nuclear transfer (SCNT) in aspects different from previous studies, not only the number of blastomeres of cloned embryos during the initiation of compaction, but also the distribution of microvilli in cloned, normal, parthenogenetic, and tetraploid embryos before and after compaction was preliminarily investigated in mouse. Our results showed that during compaction the number of blastomeres in SCNT embryos was fewer than that in intracytoplasmic sperm injection (ICSI) embryos and, before compaction, there was a uniform distribution of microvilli over the blastomere surface, but microvilli became restricted to an apical region after compaction in the four types of embryos. We also reported here that the time course of compaction in SCNT embryos was about 3 h delayed compared with that in ICSI embryos, while there was no significant difference between SCNT and ICSI embryos when developed to the 4-cell stage. We concluded that: (i) the cleavage of blastomeres in cloned embryos was slow at least before compaction; (ii) the distribution of microvilli in cloned, normal, parthenogenetic, and tetraploid embryos was coherent before and after compaction; and (iii) the initiation of compaction in SCNT embryos was delayed compared with that of ICSI embryos.

34 EVALUATION OF DIFFERENT SEQUENTIAL CULTURE SYSTEMS ON THE DEVELOPMENT AND QUALITY OF BOVINE EMBRYOS GENERATED BY SOMATIC CELL NUCLEAR TRANSFER

2011 ◽  
Vol 23 (1) ◽  
pp. 123
Author(s):  
R. F. Felmer ◽  
M. E. Arias ◽  
J. L. Riveros ◽  
G. A. Munoz ◽  
J. H. Rio
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Culture System ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Culture Systems

Different culture systems have been developed that support development of bovine embryos up to the blastocyst stage. However, the use of sequential culture systems has been studied less. The objective of the present study was therefore to examine the effect of 3 sequential culture systems, which involve the use of different culture media for early cleavage and later stage embryos, on the development and quality of bovine embryos generated by somatic cell nuclear transfer (SCNT). These systems were mainly based on different combinations of KSOM culture medium regularly used in our laboratory. Skin fibroblasts, at passage 5 to 6, were microsurgically placed into the perivitelline space evacuated during enucleation. Fusion was carried out by a single DC pulse of 1.7 kV cm–1 with an Electrocell Manipulator 830 (BTX Inc., San Diego, CA, USA) and activation by treatment of NT units in Ionomicin (5 μM, 4 min) and DMAP (1.9 mM) for 4 h. Embryo culture was carried out in 50-μL drops under mineral oil at 38.5°C and 5% CO2, 5% O2, and 90% N2, in a humidified atmosphere, according to the following sequential culture systems without co-culture: 1) KSOM + 0.4% BSA (FAF, A8806, Sigma, St. Louis, MO, USA) for 3 days and then KSOM + 5% FBS (characterized, Hyclone, Logan, UT, USA) to Day 7; 2) KSOM + 0.1% BSA for 3 days and then SOF + 0.8% BSA to Day 7 and 3) KSOM + 0.1% BSA for 3 days and then KSOM + 0.8% BSA to Day 7. Cleavage rate was evaluated on Day 3 and the number of blastocysts was recorded on Day 7. A total of 730 NT embryos randomly distributed were analysed for embryo development in 9 replicates and 5 blastocysts of each group were stained with bisbenzimide (Hoechst 33242) to assess the quality. ANOVA was used to test for statistically significant differences (P < 0.05) using Satgraphics Plus 2 Software. In cases where statistically significant differences were observed, a multiple comparison test was run using Tukey’s test. Sequential culture systems had no effect on cleavage rate (73, 76, and 73%, respectively). However, there was a significant difference (P < 0.01) in the rate of blastocysts. The sequential culture system consisting of KSOM + KSOM 5% FBS yielded a higher rate of blastocysts than other treatments (28, 18, and 16%, respectively). Despite this difference in embryo development, the quality of embryos as assessed by the total number of cells was not different (135 ± 6.5, 129 ± 7.5, and 128 ± 8.5, respectively). In conclusion, a sequential culture system consisting of KSOM + 0.4% BSA for 3 days and then KSOM + 5% FBS to Day 7 generated a higher number of cloned blastocyst than other treatments evaluated, although the quality of the embryos did not differ between treatments. Future studies are under way to establish the gene expression profile of NT embryos generated under these culture systems. The final aim is to evaluate the possibility of modulating the gene expression profile through changes in the culture medium composition. The provision of ovaries by our local slaughterhouse (Frigorifico Temuco) and funding support from FONDECYT 1080216 CONICYT, Chile, are gratefully acknowledged.

scholarly journals Advance in the Role of Epigenetic Reprogramming in Somatic Cell Nuclear Transfer-Mediated Embryonic Development

2021 ◽  
Vol 2021 ◽  
pp. 1-13
Author(s):  
Xiaolei Zhang ◽  
Shaorong Gao ◽  
Xiaoyu Liu
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Blastocyst Development ◽  
Low Efficiency ◽  
Nuclear Transfer Embryo

Somatic cell nuclear transfer (SCNT) enables terminally differentiated somatic cells to gain totipotency. Many species are successfully cloned up to date, including nonhuman primate. With this technology, not only the protection of endangered animals but also human therapeutics is going to be a reality. However, the low efficiency of the SCNT-mediated reprogramming and the defects of extraembryonic tissues as well as abnormalities of cloned individuals limit the application of reproductive cloning on animals. Also, due to the scarcity of human oocytes, low efficiency of blastocyst development and embryonic stem cell line derivation from nuclear transfer embryo (ntESCs), it is far away from the application of this technology on human therapeutics to date. In recent years, multiple epigenetic barriers are reported, which gives us clues to improve reprogramming efficiency. Here, we reviewed the reprogramming process and reprogramming defects of several important epigenetic marks and highlighted epigenetic barriers that may lead to the aberrant reprogramming. Finally, we give our insights into improving the efficiency and quality of SCNT-mediated reprogramming.

27 PSAMMAPLIN A INCREASES DEVELOPMENT AND QUALITY OF MOUSE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 161
Author(s):  
A. Mallol ◽  
J. Santaló ◽  
E. Ibáñez
Keyword(s):  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Fold Increase ◽  
Full Term ◽  
Control Group ◽  
Latrunculin A ◽  
Psammaplin A ◽  
Mouse Somatic Cell

Among the many biological and technical factors affecting the success rate of mouse somatic cell nuclear transfer (SCNT), faulty reprogramming of the differentiated donor nucleus to a totipotent embryonic state by the recipient oocyte seems key. Accordingly, treatment of SCNT embryos with epigenetic modifiers such as valproic acid (VPA), a histone deacetylase inhibitor (HDACi), enhances cloning efficiency. Psammaplin A (PsA) is a natural and potent DNA methyltransferase inhibitor and HDACi that has never been used in nuclear reprogramming studies. The purpose of our study was to determine the effect of PsA on the development and quality of mouse SCNT embryos, and to compare it to that of VPA. To this aim, mechanically enucleated oocytes from B6CBAF1 female mice were reconstructed with cumulus cell nuclei, activated, and cultured in the presence of the epigenetic modifier. Embryos that reached the blastocyst stage were differentially stained for counting inner cell mass (ICM) and trophectoderm cells. Alternatively, 2-cell embryos were transferred to CD1 recipient females to assess full-term development. In a first set of experiments, embryos were exposed to different concentrations of PsA (5, 10, and 20 µM) or VPA (2 and 4 mM) for 1 to 2 h after reconstruction and 6 h of activation (total 8–9 h). We found that 10 µM PsA and 2 mM VPA significantly increased blastocyst rates (37.3 and 31 v. 23.3% for the control group), although no differences were found in blastocyst quality (10.4–13.6 ICM cells). In a second set of experiments, we studied the effect of treatment duration by incubating the embryos in 10 µM PsA or 2 mM VPA for 8 to 9, 16, or 24 h after reconstruction. With PsA, all treatments showed equivalent blastocyst rates (35.2–43.3%), which were significantly higher than in the control group (20%), but only treatments for 16 and 24 h yielded blastocysts with significantly higher numbers of ICM cells (16.3 and 18.5 v. 10 for the control group). With VPA, treatments for 8 to 9 h and 16 h were equivalent in terms of blastocyst rates (34.0 and 32.5%) and significantly higher than the control group, but only VPA 16 h yielded blastocysts with a significantly higher number of ICM cells (15.6). In a third set of experiments, we studied the full-term development of embryos treated with 10 µM PsA or 2 mM VPA for 16 h and we found that both treatments, but especially the PsA treatment, resulted in higher birth rates than those obtained in the control group, although the differences were not statistically significant (1.79 and 0.86 v. 0.46%). Finally, when the actin polymerization inhibitor latrunculin A was used instead of cytochalasin B in the SCNT protocol during oocyte micromanipulation and activation, we obtained a 3-fold increase in the birth rate of embryos treated with PsA (5.29%). In conclusion, PsA enhances development and quality of mouse SCNT embryos, to a greater extent than VPA, and when combined with the use of latrunculin A instead of cytochalasin B, it results in an 11.5-fold increase in full-term development. Support from MEC AGL-2011-23784, 2009-SGR-282, and PIF-UAB Fellowships is acknowledged.

Visualization of endogenous nuclear F-actin in mouse embryos reveals abnormal actin assembly after somatic cell nuclear transfer

2020 ◽  
Author(s):  
Taiki Shindo ◽  
Shunya Ihashi ◽  
Yuko Sakamoto ◽  
Tomomi Okuno ◽  
Junko Tomikawa ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Actin Filaments ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Mouse Embryos ◽  
Nuclear Actin ◽  
Actin Assembly ◽  
Filamentous Form ◽  
Nuclear Events ◽  
Dynamic Assembly

Abstract Actin in the nucleus, referred to as nuclear actin, is involved in a variety of nuclear events. Nuclear actin is present as a globular (G-actin) and filamentous form (F-actin), and dynamic assembly/disassembly of nuclear actin profoundly affects nuclear functions. However, it is still challenging to observe endogenous nuclear F-actin. Here, we present a condition to visualize endogenous nuclear F-actin of mouse zygotes using different fixation methods. Zygotes fixed with paraformaldehyde and treated with fluorescently conjugated phalloidin show both short and long actin filaments in their pronuclei. Short nuclear actin filaments are characteristic of phalloidin staining, rather than the consequence of severing actin filaments by the fixation process, since long nuclear actin filaments probed with the nuclear actin chromobody are not disassembled into short filaments after fixation with paraformaldehyde. Furthermore, we find that nuclear actin assembly is impaired after somatic cell nuclear transfer (SCNT), suggesting abnormal nucleoskeleton structures in SCNT embryos. Taken together, our presented method for visualizing nuclear F-actin with phalloidin can be used to observe the states of nuclear actin assembly, and revealed improper reprogramming of actin nucleoskeleton structures in cloned mouse embryos.

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