Investigation of the Developmental Potential and Developmental Kinetics of Bovine Parthenogenetic and Somatic Cell Nuclear Transfer Embryos Using a Time-Lapse Monitoring System

2017 ◽  
Vol 19 (4) ◽  
pp. 245-254 ◽  
Author(s):  
Min-Jee Park ◽  
Eun-Young Kim ◽  
Man-Jong Kang ◽  
Jun-Beom Lee ◽  
Chang-Jin Jeong ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Monitoring System ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Time Lapse ◽  
Developmental Potential ◽  
Kinetics Of

24 BUFFALO (BUBALUS BUBALIS) SOMATIC CELL NUCLEAR TRANSFER EMBRYOS PRODUCED FROM FROZEN–THAWED SEMEN-DERIVED SOMATIC CELLS: EFFECT OF TRICHOSTATIN A ON THE IN VITRO AND IN VIVO DEVELOPMENTAL POTENTIAL, QUALITY, AND EPIGENETIC STATUS

2015 ◽  
Vol 27 (1) ◽  
pp. 104
Author(s):  
N. L. Selokar ◽  
M. Saini ◽  
H. Agrawal ◽  
P. Palta ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Somatic Cells ◽  
Developmental Potential ◽  
Reconstructed Embryos ◽  
Frozen Semen ◽  
Significant Difference

Cryopreservation of semen allows preservation of somatic cells, which can be used for the production of progeny through somatic cell nuclear transfer (SCNT). This approach could enable restoration of valuable high-genetic-merit progeny-tested bulls, which may be dead but the cryopreserved semen is available. We have successfully produced a live buffalo calf by SCNT using somatic cells isolated from >10 year old frozen semen (Selokar et al. 2014 PLoS One 9, e90755). However, the calf survived only for 12 h, which indicates faulty reprogramming of these cells. The present study was, therefore, carried out to study the effect of treatment with trichostatin A (TSA), an epigenetic modifier, on reprogramming of these cells. Production of cloned embryos and determination of quality and level of epigenetic markers in blastocysts were performed according to the methods described previously (Selokar et al. 2014 PLoS One 9, e90755). To examine the effects of TSA (0, 50, and 75 nM), 10 separate experiments were performed on 125, 175, and 207 reconstructed embryos, respectively. The percentage data were analysed using SYSTAT 12.0 (SPSS Inc., Chicago, IL, USA) after arcsine transformation. Differences between means were analysed by one-way ANOVA followed by Fisher's least significant difference test for significance at P < 0.05. When the reconstructed buffalo embryos produced by hand-made clones were treated with 0, 50, or 75 nM TSA post-electrofusion for 10 h, the cleavage percentage (100.0 ± 0, 94.5 ± 2.3, and 96.1 ± 1.2, respectively) and blastocyst percentage (50.6 ± 2.3, 48.4 ± 2.7, and 48.1 ± 2.6, respectively), total cell number (274.9 ± 17.4, 289.1 ± 30.1, and 317.0 ± 24.2, respectively), and apoptotic index (3.4 ± 0.9, 4.5 ± 1.4, and 5.6 ± 0.7, respectively) in Day 8 blastocysts were not significantly different among different groups. The TSA treatment increased (P < 0.05) the global level of H4K5ac but not that of H3K18a in embryos treated with 50 or 75 nM TSA compared with that in controls. In contrast, the level of H3K27me3 was significantly lower (P < 0.05) in cloned embryos treated with 75 nM TSA than in embryos treated with 50 nM TSA or controls. The ultimate test of the reprogramming potential of any donor cell type is its ability to produce live offspring. To examine the in vivo developmental potential of the 0, 50, or 75 nM TSA treated embryos, we transferred Day 8 blastocysts, 2 each to 5, 6, and 5 recipients, respectively, which resulted in 2 pregnancies from 75 nM TSA treated embryos. However, one pregnancy was aborted in the first trimester and the other in the third trimester. In conclusion, TSA treatment of reconstructed embryos produced from semen-derived somatic cells alters their epigenetic status but does not improve the live birth rate. We are currently optimizing an effective strategy to improve the cloning efficiency of semen-derived somatic cells.

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.

Effect of volume of oocyte cytoplasm on embryo development after parthenogenetic activation, intracytoplasmic sperm injection, or somatic cell nuclear transfer

Zygote ◽  
2008 ◽  
Vol 16 (3) ◽  
pp. 211-222 ◽  
Author(s):  
Wakayama Sayaka ◽  
Kishigami Satoshi ◽  
Nguyen Van Thuan ◽  
Ohta Hiroshi ◽  
Hikichi Takafusa ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Intracytoplasmic Sperm Injection ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Subsequent Development ◽  
Developmental Potential ◽  
Parthenogenetic Activation ◽  
Somatic Cell Nucleus

SummaryAnimal cloning methods are now well described and are becoming routine. Yet, the frequency at which live cloned offspring are produced remains below 5%, irrespective of the nuclear donor species or cell type. One possible explanation is that the reprogramming factor(s) of each oocyte is insufficient or not properly adapted for the receipt of a somatic cell nucleus, because it is naturally prepared only for the receipt of a gamete. Here, we have increased the oocyte volume by oocyte fusion and examined its subsequent development. We constructed oocytes with volumes two to nine times greater than the normal volume by the electrofusion or mechanical fusion of intact and enucleated oocytes. We examined their in vitro and in vivo developmental potential after parthenogenetic activation, intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). When the fused oocytes were activated parthenogenetically, most developed to morulae or blastocysts, regardless of their original size. Diploid fused oocytes were fertilized by ICSI and developed normally and after embryo transfer, we obtained 12 (4–15%) healthy and fertile offspring. However, enucleated fused oocytes could not support the development of mice cloned by SCNT. These results suggest that double fused oocytes have normal potential for development after fertilization, but oocytes with extra cytoplasm do not have enhanced reprogramming potential.

Developmental potential of pig embryos reconstructed by use of sow versus pre-pubertal gilt oocytes after somatic cell nuclear transfer

Zygote ◽  
2013 ◽  
Vol 22 (3) ◽  
pp. 356-365 ◽  
Author(s):  
Juan Li ◽  
Hanne Skovsgaard Pedersen ◽  
Rong Li ◽  
Janne Adamsen ◽  
Ying Liu ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Follicular Fluid ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Control Experiment ◽  
Transfer Efficiency ◽  
Maturation Time ◽  
Developmental Potential ◽  
Routine Production ◽  
Cloning Experiment

SummaryIn this study, the developmental ability of cloned embryos using gilt versus sow oocytes was evaluated under the hypothesis that the efficiency of nuclear transfer using gilt oocytes was lower than that of sow oocytes, but that it could be optimized. Five experiments were performed with routine production of cloned embryos with sow oocytes serving as the control. Results showed that: Experiment 1: Blastocyst rates of cloned embryos with gilt oocytes was about half compared with control. Experiment 2: An extended maturation time of 48 h used for gilt oocytes resulted in lower blastocyst rates after cloning. Experiment 3: Development of cloned embryos with gilt oocytes was improved by co-culture with sow oocytes. Experiment 4: After maturation of gilt oocytes using follicular fluid from gilt instead of sow, the oocytes were sorted into large and small oocytes, and after cloning, blastocyst rates were higher using large gilt oocytes compared with small oocytes; however, the rate remained lower compared with control. Experiment 5: Six sow recipients received a total of 503 morulae and blastocysts cloned from gilt oocytes (four recipients) and 190 cloned from sow oocytes (two recipients). All recipients became pregnant and went to term, resulting in 26 (gilt oocytes) and six (sow oocytes) piglets. In conclusion, results confirmed that nuclear transfer efficiency was higher using sow versus gilt oocytes, but the use of gilt oocytes can be optimized by sorting after ooplasm size following maturation and by maturing gilt and sow oocytes together.

Effects of ovary storage temperature and embryo vitrification on somatic cell nuclear transfer outcomes in goats

2020 ◽  
Vol 32 (4) ◽  
pp. 419 ◽  
Author(s):  
Mehdi Hajian ◽  
Farnoosh Jafarpour ◽  
Sayed Morteza Aghamiri ◽  
Shiva Rouhollahi Varnosfaderani ◽  
Mohammad Hossein Nasr Esfahani
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Storage Temperature ◽  
Storage Conditions ◽  
Farm Animals ◽  
Blastocyst Formation ◽  
Term Pregnancy ◽  
Developmental Potential ◽  
Genetic Potential

Improving the genetic potential of farm animals is one of the primary aims in the field of assisted reproduction. In this regard, somatic cell nuclear transfer (SCNT) can be used to produce a large number of embryos from genetically elite animals. The aims of the present study were to assess the effects of: (1) ovary storage conditions on preimplantation development of recovered oocytes and the freezability of the derived blastocysts; and (2) vitrification of goat SCNT-derived blastocysts on postimplantation development. Goat oocytes were recovered from ovaries and stored under warm (25°C-27°C) or cold (11°C-12°C) conditions before being used to produce SCNT embryos. There were no differences in oocytes recovered from ovaries kept under cold versus warm storage conditions in terms of cleavage (mean (±s.d.) 95.68±1.67% vs 95.91±2.93% respectively) and blastocyst formation (10.69±1.17% vs 10.94±0.9% respectively) rates. The re-expansion rate of vitrified blastocysts was significantly lower for cold- than warm-stored ovaries (66.3±8.7% vs 90±11% respectively). To assess the effects of vitrification on postimplantation development, blastocysts from cold-stored ovaries only were transferred from fresh and vitrified–warmed groups. The pregnancy rate was comparable between the fresh and vitrified–warmed groups (41.65% and 45.45% respectively). In addition, established pregnancy in Day 28-38 and full-term pregnancy rates were similar between the two groups. In conclusion, this study shows similar invitro preimplantation developmental potential of warm- and cold-stored ovaries. This study introduces the vitrification technique as an appropriate approach to preserve embryos produced by SCNT for transfer to recipient goats at a suitable time.

19 OPTIMIZATION OF ENUCLEATION TIME AFTER IVM FOR SOMATIC CELL NUCLEAR TRANSFER IN GOAT

2009 ◽  
Vol 21 (1) ◽  
pp. 109 ◽  
Author(s):  
G. S. Ajithkumar ◽  
B. Krishnamohan ◽  
B. C. Sarkhel
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Proper Time ◽  
Polar Body ◽  
Fluorescent Microscopy ◽  
Developmental Potential ◽  
Randomized Design ◽  
Significant Difference ◽  
Arcsine Transformation

For cloning by somatic cell nuclear transfer (SCNT) in goat, there are conflicting reports about the proper time of enucleation after IVM of oocytes, which varied from 20 to 27 h (Das SK et al. 2003 Small Rumin. Res. 48, 217–225; Keefer CL et al. 2002 Biol. Reprod. 66, 199–203; Daniel SM et al. 2007 Small Rumin. Res. 77, 45–50). The present investigation has been undertaken to standardize the optimum time of enucleation after IVM of oocytes. The hypothesis behind the study was that enucleation performed during early stages of maturation maintains the MII plate and polar body (PB) in a closer position and therefore makes it easy to enucleate. To test this hypothesis, caprine COCs were aspirated from slaughterhouse ovaries of goats and good quality oocytes were matured in TCM 199 containing 7.5% FBS supplemented with FSH, LH, and estradiol. Enucleation was performed in four different interval groups after IVM (20–23 h, 23–26 h, 26–29 h and 29–32 h). The enucleation of oocytes was conducted as per method described by (Du F et al. 2006 Theriogenology 65, 642–665). Briefly, IVM oocytes were enucleated by squashing and compressing out the first PB along with 10 to 15% of its surrounding cytoplasm with an enucleation needle through a slit made on the zona pellucida. Successful enucleation was confirmed by fluorescent microscopy of removed ooplasm after staining with Hoechst 33342. The enucleation percentage values after arcsine transformation was analyzed by completely randomized design ANOVA. The result of enucleation at different intervals has been summarized in Table 1. There was no significant difference (P > 0.05) in number of PB observed among the four enucleation groups, however the enucleation percentage decreased significantly (P < 0.05) with increase in enucleation time (70.29% and 70.51% in G1 and G2 v. 59.52% and 55.61% in G3 and G4 respectively). With increase in time of enucleation after maturation the size of perivitelline space increases, causing deviation of PB from spindle, thus the success rate of enucleation is reduced (Song K et al. 2007 Repro. Fertil. Dev. 19, 293–294). In G1 and G2 groups the PB and MII chromosomes are located close together with stronger spindle force that requires minimum ooplasm to be removed with higher percentage of successful enucleation. Hence, it was concluded that G1 and G2 groups may be considered as most efficient for enucleation but the developmental potential of reconstructed oocytes after nuclear transfer in each group needs to be tested (study under progress). Table 1.Enucleation results at different intervals

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.

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