Treatment of ovine oocytes with caffeine increases the accessibility of DNase I to the donor chromatin and reduces apoptosis in somatic cell nuclear transfer embryos

2010 ◽  
Vol 22 (6) ◽  
pp. 1000 ◽  
Author(s):  
Inchul Choi ◽  
Keith H. S. Campbell
Keyword(s):  
Dna Synthesis ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Chromatin Accessibility ◽  
Blastocyst Stage ◽  
Dnase I ◽  
Cell Numbers ◽  
Mapk Activity ◽  
Caffeine Treatment

Caffeine treatment of ovine oocytes increases the activity of maturation-promoting factor (MPF) and mitogen-activated protein kinases (MAPKs) and, in somatic cell nuclear transfer (SCNT) embryos, increases the frequency of nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). At the blastocyst stage, caffeine-treated SCNT embryos have increased cell numbers. One explanation for this is that NEBD and PCC release chromatin-bound somatic factors, allowing greater access of oocyte factors involved in DNA synthesis and nuclear reprogramming to donor chromatin. This could advance DNA replication and cleavage in the first cell cycle, resulting in increased cell numbers. Alternatively, increased MAPK activity may affect localisation of heat shock proteins (HSPs) and reduce apoptosis. To investigate these possibilities, we investigated chromatin accessibility, the timing of DNA synthesis and first cleavage, the localisation of HSP27 during early development and the frequency of apoptotic nuclei at the blastocyst stage. Compared with control SCNT (non-caffeine treatment), caffeine treatment (10 mM caffeine for 6 h prior to activation) increased the accessibility of DNase I to donor chromatin (P < 0.05 at 1.5 h post activation (h.p.a.)), advanced DNA synthesis (43.5% v. 67.6%, respectively; P < 0.01 at 6 h.p.a.) and first cleavage (27.3% v. 40.5% at 20 h.p.a., respectively) and increased nuclear localisation of HSP27. Although development to the blastocyst stage was not affected, caffeine increased total cell numbers (98.5 v. 76.6; P < 0.05) and reduced the frequency of apoptotic nuclei (11.27% v. 20.3%; P < 0.05) compared with control SCNT group.

Development of interspecies cloned embryos reconstructed with rabbit (Oryctolagus cuniculus) oocytes and cynomolgus monkey (Macaca fascicularis) fibroblast cell nuclei

Zygote ◽  
2012 ◽  
Vol 21 (4) ◽  
pp. 358-366 ◽  
Author(s):  
Takayuki Yamochi ◽  
Yuta Kida ◽  
Noriyoshi Oh ◽  
Sei Ohta ◽  
Tomoko Amano ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cynomolgus Monkey ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Oryctolagus Cuniculus ◽  
Macaca Fascicularis ◽  
Culture Medium ◽  
Blastocyst Stage ◽  
Cell Nuclei ◽  
Cell Numbers

SummaryInterspecies somatic cell nuclear transfer (ISCNT) has been proposed as a technique to produce cloned offspring of endangered species as well as to investigate nucleus–cytoplasm interactions in mammalian embryo. However, it is still not known which embryo culture medium is optimal for ISCNT embryos for the nuclear donor or the oocyte recipient. We assessed the effects of the culture medium on the developmental competence of the ISCNT embryos by introducing cynomolgus monkey (Macaca fascicularis) fibroblast nuclei into enucleated rabbit (Oryctolagus cuniculus) oocytes (monkey–rabbit embryo). The monkey–rabbit ISCNT embryos that were cultured in mCMRL-1066 developed to the blastocyst stage, although all monkey–rabbit ISCNT embryos cultured in M199 were arrested by the 4-cell stage. When monkey–rabbit ISCNT and rabbit–rabbit somatic cell nuclear transfer (SCNT) embryos were cultured in mCMRL-1066, the blastocyst cell numbers of the monkey–rabbit ISCNT embryos corresponded to the cell numbers of the control rabbit–rabbit SCNT embryos, which were produced from a rabbit fibroblast nucleus and an enucleated rabbit oocyte. In addition, the presence of mitochondria, which were introduced with monkey fibroblasts into rabbit recipient cytoplasm, was confirmed up to the blastocyst stage by polymerase chain reaction (PCR). This study demonstrated that: (1) rabbit oocytes can reprogramme cynomolgus monkey somatic cell nuclei, and support preimplantation development; (2) monkey–rabbit ISCNT embryos developed well in monkey culture medium at early embryonic developmental stages; (3) the cell number of monkey–rabbit ISCNT embryos is similar to that of rabbit–rabbit SCNT embryos; and (4) the mitochondrial fate of monkey–rabbit ISCNT embryos is heteroplasmic from the time just after injection to the blastocyst stage that has roots in both rabbit oocytes and monkey fibroblasts.

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.

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.

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.

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.

43 PRODUCTION OF CLONED BLASTOCYSTS USING EPITHELIAL CELLS CULTURED FROM BOVINE SEMEN

2008 ◽  
Vol 20 (1) ◽  
pp. 102
Author(s):  
J. Liu ◽  
M. E. Westhusin ◽  
D. C. Kraemer
Keyword(s):  
Epithelial Cells ◽  
Somatic Cell ◽  
Cell Lines ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Bovine Semen ◽  
Cells Cultured

Somatic cells in semen could be a valuable source of nuclei for cloning animals by somatic cell nuclear transfer, especially when other ways of obtaining somatic cells are not available. The usefulness of the cells cultured from bovine semen for nuclear transfer was evaluated in the present study. Twelve ejaculates were collected from nine bulls representing three breeds: Charolais, Brahman, and a crossbreed rodeo bull. All of the samples were processed immediately, and somatic cells were isolated by centrifuging through 20%, 50%, and 90% percoll columns (Nel-Themaat et al. 2005 Reprod. Fertil. Dev. 17, 314–315). Somatic cell lines were obtained from 7 of the 12 ejaculates. These cell lines have classic epithelial morphology, express cytokeratin and vimentin, and proliferate well in the medium we previously designed for the epithelial cells in ovine semen (Jie Liu et al. 2007 Biol. Reprod. special issue, 177–178). Cell lines from three bulls that had been cultured in vitro for 1–2 months were used in the cloning experiments. Bovine ovaries were collected from a local slaughterhouse and transported to the laboratory in warm saline solution within 2–4 h. Compact cumulus–oocyte complexes with evenly distributed cytoplasm were selected and matured for 18 h at 38.5�C with 5% CO2 in humidified air. Cumulus cells were removed by pipetting in 0.3% hyaluronidase solution (Sigma Chemical Co., St. Louis, MO, USA) for 5 min. Oocytes were selected for the presence of a first polar body and stained in 5 µg mL–1 Hoechst 33342 (Sigma) and 5 µg mL–1 cytochalasin B (Sigma) for 10–15 min before enucleation. Successful enucleation was confirmed by brief exposure of the oocytes to ultraviolet light. Epithelial cell lines cultured to 90–100% confluence were trypsinized, and a single cell was inserted into the perivitelline space of an oocyte. Fusion was induced by applying two 1.8–1.9 kV cm–1, 20 µs direct-current pulses delivered by an Eppendorf Multiporator (Eppendorf, North America) in fusion medium comprising 0.28 m Mannitol (Sigma), 0.1 mm CaCl2 (Sigma), and 0.1 mm MgSO4 (Sigma). One and half to 2 h post fusion, activation was induced by applying two 0.3 kV cm–1, 55 µs direct-current pulses in the fusion medium, followed by incubation in 10 µg mL–1 cycloheximide (Sigma) and 5 µg mL–1 cytochalasin B for 5 h in a humidified 5% CO2, 5% O2, and 90% N2 gas mixture at 38.5�C. The embryos were washed three times and cultured in commercially available G1/G2 medium (Vitrolife, Inc., Englewood, CO, USA) for up to 10 days. Blastocyst development rates using somatic cells from three of the bulls, 1-year-old Charolais, 6-year-old Brahman, and 8-year-old Brahman, were 15.9% (18/113), 34.5% (29/84), and 14.4% (13/90) of the fused one-cell embryos, respectively. Of these blastocyst stage embryos, 38.9% (7/18), 72.4% (21/29), and 61.5% (8/13) hatched, respectively. The present study shows that epithelial cells cultured from bovine semen can be used to produce blastocyst-stage embryos by somatic cell nuclear transfer.

62 DIFFERENT EFFECT OF TRICHOSTATIN A ON IN VITRO DEVELOPMENT OF PORCINE TRANSGENIC SOMATIC CELL NUCLEAR TRANSFER AND PARTHENOGENETICALLY ACTIVATED EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 112 ◽  
Author(s):  
H. X. Wei ◽  
K. Zhang ◽  
Y. F. Ma ◽  
Y. Li ◽  
Q. Y. Li ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Trichostatin A ◽  
Polar Body ◽  
Cell Number ◽  
Cell Numbers ◽  
First Polar Body ◽  
Electrical Fusion

Accumulating evidence suggests that trichostatin A (TSA), a histone deacetylase inhibitor, can increase the success rate of somatic cloning. The objective of this study was to investigate the effect of 50 nm TSA treatment on the development of porcine somatic cell nuclear transfer (SCNT) and parthenogenically activated (PA) embryos. Cumulus-oocyte complexes were matured in vitro. The oocytes with the first polar body (PB1) were chosen for SCNT, and the rest with PB1 or good morphology were selected for PA by a single 100-μs direct current pulse of 1.6 kV cm–1, the same parameter as for electrical fusion. GFP transgenic fetal fibroblast cells were used as nuclear donors. Data were analyzed using SPSS (13.0; SPSS, Inc., Chicago, IL, USA) with one-way ANOVA. In Experiment 1, immediately after electrical fusion and activation, the reconstructed embryos were randomly cultured in porcine zygote medium 3 (PZM3) with 10 μg mL–1 cytochalasin B (CB) and 10 μg mL–1 cycloheximide (CHX), with either 0 nm (control) or 50 nm TSA for the first 4 h, before being cultured for another 20 h in PZM3 without CB and CHX. After being washed, the embryos were cultured in PZM3 medium without TSA until Day 6 at 39.0°C, 5% CO2, 5%O2, 90% N2, and 100% humidity. The same experimental design was used for PA embryos concurrently. The results showed that there were no significant differences in blastocyst rates for SCNT or PA between control and TSA groups (23.0 ± 6.1% v. 27.9 ± 6.3%; 21.0 ± 1.0% v. 17.5 ± 3.2%, respectively). Neither were there differences in the cell numbers of blastocysts (38.3 ± 5.7 v. 32.2 ± 3.4; 42.2 ± 3.5 v. 39.0 ± 1.9, respectively). In Experiment 2, TSA treatment was prolonged to either 36 or 40 h. The blastocyst rates of SCNT were increased (7.3 ± 1.2% (0 h), 13.3 ± 2.6% (36 h), and 20.0 ± 3.3% (40 h)), whereas those of PA were decreased (46.7 ± 5.0% (0 h), 27.7 ± 6.5% (36 h), and 30.8 ± 6.3% (40 h)). The cell numbers of blastocysts from either SCNT or PA were also decreased (SCNT: 47.5 ± 3.8, 37.5 ± 2.0, and 37.1 ± 3.3; PA: 46.1 ± 1.9, 37.5 ± 1.9, and 39.3 ± 2.2; P < 0.05). In Experiment 3, the cell number and the apoptotic index of Day 5, 6, and 7 PA blastocysts treated with 0 or 50 nm TSA were determined by the terminal deoxynucleotide-mediated nick end labeling (TUNEL) assay (Table 1). The results suggested that TSA treatment probably delayed embryo development, which may be one of the reasons for the lower cell numbers in the TSA-treated group. Table 1. Cell apoptosis of PA blastocyst by TUNEL

27 SOMATIC CELL NUCLEAR TRANSFER CLONING AND EMBRYO AGGREGATION IN PIGS

2014 ◽  
Vol 26 (1) ◽  
pp. 128
Author(s):  
C. P. Buemo ◽  
A. Gambini ◽  
I. Hiriart ◽  
D. Salamone
Keyword(s):  
In Vitro Culture ◽  
Somatic Cell ◽  
Embryo Development ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Electric Pulse ◽  
Blastocyst Stage ◽  
Control Group

Somatic cell nuclear transfer (SCNT) derived blastocysts have lower cell number than IVF-derived blastocysts and their in vivo counterparts. The aim of this study was to improve the blastocyst rates and quality of SCNT blastocysts by the aggregation of genetically identical free zona pellucida (ZP) porcine clones. Cumulus–oocyte complexes were recovered from slaughterhouse ovaries by follicular aspiration. Maturation was performed in TCM for 42 to 48 h at 39°C and 5% CO2. After denudation by treatment with hyaluronidase, mature oocytes were stripped of the ZP using a protease and then enucleated by micromanipulation; staining was performed with Hoechst 33342 to observe metaphase II. Ooplasms were placed in phytohemagglutinin to permit different membranes to adhere between each other; the ooplasm membrane was adhered to a porcine fetal fibroblast from an in vitro culture. Adhered membranes of the donor cell nucleus and enucleated oocyte cytoplasm were electrofused through the use of an electric pulse (80 V for 30 μs). All reconstituted embryos (RE) were electrically activated using an electroporator in activation medium (0.3 M mannitol, 1.0 mM CaCl2, 0.1 mM MgCl2, and 0.01% PVA) by a DC pulse of 1.2 kV cm–1 for 80 μs. Then, the oocytes were incubated in 2 mM 6-DMAP for 3 h. In vitro culture of free ZP embryos was achieved in a system of well of wells in 100 μL of medium, placing 3 activated oocytes per microwell (aggregation embryo), whereas the control group was cultivated with equal drops without microwells. Embryos were cultivated at 39°C in 5% O2, 5% CO2 for 7 days in SOF medium with a supplement of 10% fetal bovine serum on the fifth day. The RE were placed in microwells. Two experimental groups were used, control group (not added 1X) and 3 RE per microwell (3X). At Day 7, resulting blastocysts were classified according to their morphology and diameter to determine their quality and evaluate if the embryo aggregation improves it. Results demonstrated that aggregation improves in vitro embryo development rates until blastocyst stage and indicated that blastocysts rates calculated over total number of oocytes do not differ between groups (Table 1). Embryo aggregation improves cleavage per oocyte and cleavage per microwell rates, presenting statistical significant differences and increasing the probabilities of higher embryo development generation until the blastocyst stage with better quality and higher diameter. Table 1.Somatic cell nuclear transfer cloning and embryo aggregation

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