62 REPROGRAMMING EVENTS AND DEVELOPMENTAL COMPETENCE OF RHESUS MONKEY EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. In this study, we examined the extent of nuclear remodeling in monkey somatic cell nuclear transfer (SCNT) embryos by monitoring the dynamics of lamin A/C appearance, as detected immunocytochemically, following fusion of donor cells with recipient cytoplasts. In the control, intracytoplasmic sperm injection (ICSI) fertilized embryos, lamin A/C was readily detected at the pronuclear stage but disappeared in early cleaving embryos only to reappear by the morula stage in association with the activation of the embryonic genome. We initially documented lack or incomplete NEBD and PCC in SCNT embryos in the form of retention of lamin A/C signal emanating from the donor nucleus. This observation was consistent with premature cytoplast activation due to the manipulation procedures. SCNT embryos produced by this approach typically arrested at the morula stage. Significant modifications in nuclear transfer protocols were then employed. Optimization of procedures resulted in robust NEBD and PCC, as indicated by loss of lamin A/C signal from the donor cell. Also, significant improvement of SCNT embryo development in vitro was observed, with a markedly improved blastocyst formation rate (21%). Several different fetal and adult somatic cell types screened as nuclear donors supported blastocyst development. SCNT blastocysts displayed a pattern of Oct-4 expression similar to that of sperm fertilized counterparts, indicative of efficient nuclear reprogramming. However, no pregnancies were established following a preliminary trial of 8 embryo transfers with 48 cloned embryos. Nevertheless, our results represent a breakthrough in efforts to produce cloned monkeys and should provide the resources required for the derivation of embryonic stem cells from SCNT blastocysts.

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

Reproduction Fertility and Development ◽

10.1071/rd17543 ◽

2018 ◽

Vol 30 (10) ◽

pp. 1342 ◽

Cited By ~ 4

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.

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Parthenogenetic activation and somatic cell nuclear transfer of porcine oocytes activated by an electric pulse and AZD5438 treatment

Zygote ◽

10.1017/s0967199417000272 ◽

2017 ◽

Vol 25 (4) ◽

pp. 453-461 ◽

Cited By ~ 5

Author(s):

Xiao-Chen Li ◽

Qing Guo ◽

Hai-Ying Zhu ◽

Long Jin ◽

Yu-Chen Zhang ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Electric Pulse ◽

Formation Rate ◽

Developmental Competence ◽

Oocyte Activation ◽

Blastocyst Formation ◽

Parthenogenetic Activation

SummaryWe examined the in vitro developmental competence of parthenogenetic activation (PA) oocytes activated by an electric pulse (EP) and treated with various concentrations of AZD5438 for 4 h. Treatment with 10 µM AZD5438 for 4 h significantly improved the blastocyst formation rate of PA oocytes in comparison with 0, 20, or 50 µM AZD5438 treatment (46.4% vs. 34.5%, 32.3%, and 24.0%, respectively; P < 0.05). The blastocyst formation rate was higher in the group treated with AZD5438 for 4 h than in the groups treated with AZD5438 for 2 or 6 h (42.8% vs. 38.6% and 37.2%, respectively; P > 0.05). Furthermore, 66.67% of blastocysts derived from these AZD5438-treated PA oocytes had a diploid karyotype. The blastocyst formation rate of PA and somatic cell nuclear transfer (SCNT) embryos was similar between oocytes activated by an EP and treated with 2 mM 6-dimethylaminopurine for 4 h and those activated by an EP and treated with 10 µM AZD5438 for 4 h (11.11% vs. 13.40%, P > 0.05). In addition, the level of maturation-promoting factor (MPF) was significantly decreased in oocytes activated by an EP and treated with 10 µM AZD5438 for 4 h. Finally, the mRNA expression levels of apoptosis-related genes (Bax and Bcl-2) and pluripotency-related genes (Oct4, Nanog, and Sox2) were checked by RT-PCR; however, there were no differences between the AZD5438-treated and non-treated control groups. Our results demonstrate that porcine oocyte activation via an EP in combination with AZD5438 treatment can lead to a high blastocyst formation rate in PA and SCNT experiments.

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Effect of cytochalasins B and D on the developmental competence of somatic cell nuclear transfer embryos in miniature pigs

Zygote ◽

10.1017/s0967199407004480 ◽

2008 ◽

Vol 16 (2) ◽

pp. 153-159 ◽

Cited By ~ 14

Author(s):

Satoshi Sugimura ◽

Manabu Kawahara ◽

Takuya Wakai ◽

Ken-ichi Yamanaka ◽

Hiroshi Sasada ◽

...

Keyword(s):

Somatic Cell ◽

Uniform Distribution ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Polar Body ◽

Formation Rate ◽

Developmental Competence ◽

Miniature Pigs ◽

Artificial Activation

SummaryIn many animals, cytochalasins have generally been used as cytoskeletal inhibitors for the diploid complement retention of somatic cell nuclear transfer (SCNT) embryos. However, limited information is available on the effects of cytochalasins on the in vitro development of SCNT embryos. Hence, we compared the effects of cytochalasin B (CB) and cytochalasin D (CD) on pseudo-polar body (pPB) extrusion, cortical actin filament (F-actin) distribution in porcine parthenogenetic oocytes and in vitro development of SCNT embryos that were reconstructed using foetal fibroblasts in the G0/G1 phase derived from miniature pigs. CB (7.5 μg/ml) and CD (2.5 μg/ml) treatments effectively inhibited pPB extrusion in SCNT embryos. CB (2.5 μg/ml) treatment could not inhibit pPB extrusion and insufficiently destabilized F-actin immediately following artificial activation. In parthenogenetic oocytes treated with 2.5 μg/ml CD, normal reorganization and uniform distribution of cortical F-actin at the cytoplasmic membrane were observed at 8 h after artificial activation; this finding was similar to that of control oocytes. In contrast, parthenogenetic oocytes treated with 7.5 μg/ml CB showed non-uniform distribution of F-actin at 8 h after artificial activation. On day 5 after in vitro cultivation, the blastocyst formation rate of SCNT embryos treated with 2.5 μg/ml CD was significantly higher than that of SCNT embryos treated with 2.5 and 7.5 μg/ml CB (p < 0.05). Hence, the present findings suggest that CD is more effective than CB as the cytoskeletal inhibitor for the production of SCNT embryos in miniature pigs.

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Effects of treatment with a microRNA mimic or inhibitor on the developmental competence, quality, epigenetic status and gene expression of buffalo (Bubalus bubalis) somatic cell nuclear transfer embryos

Reproduction Fertility and Development ◽

10.1071/rd19084 ◽

2020 ◽

Vol 32 (5) ◽

pp. 508

Author(s):

S. Sah ◽

A. K. Sharma ◽

S. K. Singla ◽

M. K. Singh ◽

M. S. Chauhan ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Inner Cell Mass ◽

Cell Mass ◽

Bubalus Bubalis ◽

Cell Number ◽

Developmental Competence ◽

Cell Stage ◽

Morula Stage

Expression levels of 13 microRNAs (miRNAs) were compared between buffalo blastocysts produced by somatic cell nuclear transfer through hand-made cloning and IVF to improve cloning efficiency. Expression of miR-22, miR-145, miR-374a and miR-30c was higher, whereas that of miR-29b, miR-101, miR-302b, miR-34a, miR-21 and miR-25 was lower, in nuclear transferred (NT) than IVF embryos; the expression of miR-200b, miR-26a and miR-128 was similar between the two groups. Based on these, miR-145, which is involved in the regulation of pluripotency, was selected for further investigation of NT embryos. miR-145 expression was lowest at the 2-cell stage, increased through the 4-cell stage and was highest at the 8-cell or morula stage in a pattern that was similar between NT and IVF embryos. miR-145 expression was higher in NT than IVF embryos at all stages examined. Treatment of reconstructed embryos 1h after electrofusion with an inhibitor of miR-145 for 1h decreased the apoptotic index and increased the blastocyst rate, total cell number, ratio of cells in the inner cell mass to trophectoderm, global levels of acetylation of histone 3 at lysine 18 and expression of Krueppel-like factor 4 (KLF4), octamer-binding transcription factor 4 (OCT4) and SRY (sex determining region Y)-box 2 (SOX2) in blastocysts. Treatment with an miR-145 mimic had the opposite effects. In conclusion, treatment of NT embryos with an miR-145 inhibitor improves the developmental competence and quality, and increases histone acetylation and expression of pluripotency-related genes.

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Somatic cell nuclear transfer in horses: effect of oocyte morphology, embryo reconstruction method and donor cell type

Reproduction ◽

10.1530/rep.1.00772 ◽

2005 ◽

Vol 130 (4) ◽

pp. 559-567 ◽

Cited By ~ 75

Author(s):

Irina Lagutina ◽

Giovanna Lazzari ◽

Roberto Duchi ◽

Silvia Colleoni ◽

Nunzia Ponderato ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Formation Rate ◽

Donor Cell ◽

Cumulus Cells ◽

Reconstruction Method ◽

Blastocyst Development ◽

Adult Fibroblasts

The objective of the present work was to investigate and clarify the factors affecting the efficiency of somatic cell nuclear transfer (NT) in the horse, including embryo reconstruction, in vitro culture to the blastocyst stage, embryo transfer, pregnancy monitoring and production of offspring. Matured oocytes, with zona pellucida or after zona removal, were fused to cumulus cells, granulosa cells, and fetal and adult fibroblasts, and fused couplets were cultured in vitro. Blastocyst development to Day 8 varied significantly among donor cells (from 1.3% to 16%, P < 0.05). In total, 137 nuclear transfer-embryos were transferred nonsurgically to 58 recipient mares. Pregnancy rate after transfer of NT-embryos derived from adult fibroblasts from three donor animals was 24.3% (9/37 mares transferred corresponding to 9/101 blastocysts transferred), while only 1/18 (5.6%) of NT-blastocysts derived from one fetal cell line gave rise to a pregnancy (corresponding to 1/33 blastocysts transferred). Overall, seven pregnancies were confirmed at 35 days, and two went to term delivering two live foals. One foal died 40 h after birth of acute septicemia while the other foal was healthy and is currently 2 months old. These results indicate that (a) the zona-free method allows high fusion rate and optimal use of equine oocytes, (b) different donor cell cultures have different abilities to support blastocyst development, (c) blastocyst formation rate does not correlate with pregnancy fate and (d) healthy offspring can be obtained by somatic cell nuclear transfer in the horse.

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60 EFFECTS OF REPETITIVE IONOMYCIN TREATMENT ON BOVINE SOMATIC CELL NUCLEAR TRANSFER (SCNT) EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab60 ◽

2010 ◽

Vol 22 (1) ◽

pp. 188

Author(s):

S. H. Jeong ◽

S. Kim ◽

M. K. Choi ◽

J. M. Na ◽

J. Choi ◽

...

Keyword(s):

Somatic Cell ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Formation Rate ◽

Essential Element ◽

Developmental Competence ◽

P Value ◽

Oocyte Activation ◽

Reconstructed Embryos ◽

Group 3

In bovine somatic cell nuclear transfer (SCNT), oocyte activation is an essential element for the successful development of cloned embryos. Chemical treatment with ionomycin induces transient levels of Ca2+, and this has been used in the activation of reconstructed embryos. In vitro-fertilized oocytes are naturally activated by sperm following several Ca2+ transients known collectively as Ca2+ oscillations. We aimed to improve the developmental efficiency of embryos produced by SCNT by mimicking this Ca2+ oscillation artificially. SCNT was performed as follows; bovine calf skin fibroblast cells were transferred into the perivitelline space of IVM oocytes, and then these NT couplets were treated with electrical fusion (2 pulses, 1.75 kV cm-1, 15 μs). Reconstructed embryos were subsequently cultured in SOF medium (5% CO2, 5% O2, and 38°C). To mimic Ca2+ oscillation, we carried out 3 different repetitive ionomycin (10 μM) treatments at 1-h intervals. There were 3 groups: group 1 (4 min, 1 time), group 2 (30 s, 4 times), and group 3 (1 min, 4 times). The difference in embryo development amongst these experimental groups was then analyzed using the one-way ANOVA test after arcsine transformation to maintain homogeneity of variance. All analyses were performed using SAS (version 8.1, SAS Institute, Cary, NC, USA). Significant differences among the treatments were determined when the P-value was <0.05. In experiment 1, to assess developmental efficiency, the cleavage rate was investigated on Day 2 and the formation rate of blastocysts (BL) was examined on Day 7. In group 3, a significant increase in BL formation was observed [47/263 (17.8%), 50/259 (19.3%), and 67/258 (26.0%), respectively]. In experiment 2, culturing each group of embryos with different ionomycin treatments caused no significant differences among the groups in terms of the total number of BL (164.3, 158.5, and 145.1, respectively). In experiment 3, expression of apoptosis-related genes in each group was evaluated by real-time PCR and the TUNEL assay. The 3 BL within each group were analyzed for the expression of apotosis-related genes. Expression of the anti-apoptotic Bcl-2 gene was increased in group 3, whereas the expression of the pro-apoptotic Bax was decreased. A decrease in the number of apoptotic nuclei was also observed in group 3. In conclusion, the present study demonstrated that repetitive ionomycin treatment is an improved activation method that can increase the developmental competence of SCNT embryos by decreasing the incidence of apoptosis.

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

Stem Cells International ◽

10.1155/2021/6681337 ◽

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.

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