19 Improvement of the developmental competence of bovine somatic cell nuclear transfer embryos using latrunculin A during activation

2020 ◽  
Vol 32 (2) ◽  
pp. 135
Author(s):  
G. Vans Landschoot ◽  
V. Savy ◽  
L. D. Ratner ◽  
V. Alberio ◽  
D. F. Salamone
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
Developmental Competence ◽  
Donor Cells ◽  
Latrunculin A ◽  
D Cells ◽  
G1 Cells

Somatic cell nuclear transfer (SCNT) is an assisted reproductive technology with potential for its application in agriculture, biomedicine, and biotechnology. However, the SCNT efficiency is low. Failure in embryo production by SCNT could be associated mainly with chemical activation treatments or the donor cell type. In this context, we compare the use of latrunculin A (LatA), instead of cytochalasin B during the activation with roscovitine (Rosco), versus the treatment of donor cells with demecolcine (D-cells) followed by activation just with Rosco to compare cloning efficiency. The aim of this study was to evaluate the invitro developmental competence as well as the gene expression pattern of key genes (CDX2, OCT4, SOX2, and NANOG) in blastocysts obtained from the two treatments. To do this, cumulus-oocyte complexes were collected from cow ovaries obtained from slaughterhouses and were IVM for 21h. After cumulus-cell removal, enucleation was performed as described by Gambini et al. (2014 PLoS ONE 9, e110998; https://doi.org/10.1371/journal.pone.0110998). The G0/G1 cells or D-cells were fused to the oocytes. For activation, reconstructed zygotes were treated with 5μM ionomycin for 4min followed by 5-h incubation into different randomly activation groups: D-cells + 50μM Rosco (SCNT-Demec), G0/G1 cells + 50μM Rosco/10μM LatA (SCNT-LatA), and G0/G1 cells + 50μM Rosco/5μgmL−1 cytochalasin B (SCNT-Ctrol). Parthenogenetic controls were also included: Part-Demec, Part-LatA, and Part-Ctrol. Activated oocytes were cultured in synthetic oviductal fluid with amino acids medium until blastocyst stage. Rates of cleavage, morulae, and blastocysts were evaluated at Days 2, 5, and 7 of invitro culture, respectively. Relative abundance of mRNA coding for the four genes was compared between SCNT-Demec, SCNT-LatA, SCNT-Ctrol, and IVF groups by RTqPCR. Data was analysed by Fisher's exact test for invitro culture (P<0.05) or by one-way analysis of variance followed by Tukey post-hoc test (P=0.05). Cleavage rates from SCNT-Demec (n=247, 88%) and SCNT-LatA (n=112, 88%) were significantly higher than those from SCNT-Ctrol (n=123, 76%; Table 1). However, higher blastocyst rates were observed for the SCNT-LatA (n=112, 29%) group than for SCNT-Demec (n=247, 10%) and SCNT-Ctrol (n=123, 14%) (P<0.05). No differences were found for the relative abundance of mRNAs coding for SOX2 and CDX2 between all groups. The NANOG expression was significantly decreased in SCNT-Ctrol and SCNT-LatA compared with IVF embryos (P<0.05). The SCNT-Demec group did not differ from IVF embryos, and OCT4 expression analysis showed no difference among groups. In conclusion, LatA activation improved significantly blastocyst rates, whereas it did not affect gene expression when compared with IVF embryos. Our results suggest that this group could improve full-term developmental efficiency of SCNT embryos. Table 1.Cleavage, morulae, and blastocyst rates among the groups Group1 Treatment n Cleavage (%) Morulae (%) Blastocyst (%) SCNT-Demec Rosco 247 218 (88,26)b 41 (16,60)a 24 (9,72)a SCNT-Ctrol CB/Rosco 123 93 (75,61)a 25 (20,33)a 17 (13,82)a SCNT-LatA LatA 10μM/Rosco 112 99 (88,39)b 42 (37,50)b 33 (29,46)bc Part-Demec Rosco 141 133 (94,33)b 35 (24,82)a 20 (14,18)a Part-Ctrol CB/Rosco 84 76 (90,48)b 22 (26,19)ab 19 (22,62)ab Part-LatA LatA 10μM/Rosco 73 67 (91,78)b 30 (41,10)b 28 (38,36)c a-cStatistical differences between treatments (Fisher's test P<0.05). 1Treatments: donor cells with demecolcine (D-cells) + 50μM roscovitine (Rosco) (SCNT-Demec), G0/G1 cells + 50μM Rosco/10μM latrunculin A (LatA) (SCNT-LatA), and G0/G1 cells + 50μM Rosco/5μgmL−1 cytochalasin B (CB) (SCNT-Ctrol). Parthenogenetic controls were also included: Part-Demec, Part-LatA, and Part-Ctrol.

Developmental competence and gene expression in preimplantation bovine embryos derived from somatic cell nuclear transfer using different donor cells

Zygote ◽  
2005 ◽  
Vol 13 (3) ◽  
pp. 187-195 ◽  
Author(s):  
Goo Jang ◽  
Hyun Yong Jeon ◽  
Kyung Hee Ko ◽  
Hee Jung Park ◽  
Sung Keun Kang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cumulus Cells ◽  
Cell Types ◽  
Developmental Competence ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Different Cell Types

This study compared the developmental competence of somatic cell nuclear transfer (SCNT) embryos reconstructed with different donor cells and analysed gene expression in the resulting embryos. Bovine fetal/adult ear fibroblasts and cumulus cells were used as donor cells and the developmental competence of the reconstructed embryos was monitored. The cell number and allocation in blastocysts were determined by differential staining. The Bax, E-cad, IF-tau, Hsp (heat shock protein) 70, Igf2r (insulin-like growth factor 2 receptor), DNMT (DNA methyltransferase) 1 and Mash (mammalian achaete-scute homologue) 2 genes were selected for gene expression analysis. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by semiquantitative reverse transcription-polymerase chain reaction. In experiment 1, development of SCNT preimplantation embryos and the cell numbers of inner cell masses and trophoblasts were not different among SCNT embryos derived from different cell types. In experiment 2, the relative expression of GAPDH and Hsp 70 transcripts was similar in all embryos. The expression of Bax, Igf2r and Mash2 transcripts was significantly increased in SCNT embryos reconstructed with adult fibroblasts. The E-cad transcript levels were reduced in SCNT embryos reconstructed with fetal fibroblasts. Relative abundance of DNMT1 in SCNT embryos derived from fetal fibroblasts was increased, and IF-tau expression in SCNT embryos derived from cumulus cells was increased. In conclusion, depending on the type of donor cells, preimplantation SCNT embryos displayed marked differences in gene expression. This may affect the developmental competence of SCNT embryos reconstructed with different cell types after implantation or during fetal growth in vivo.

35 USE OF METAPHASE DONOR CELLS AND ACTIVATION WITH ROSCOVITINE FOR SOMATIC CELL NUCLEAR TRANSFER IN BOVINE

2017 ◽  
Vol 29 (1) ◽  
pp. 125
Author(s):  
G. V. Landschoot ◽  
V. Savy ◽  
N. Canel ◽  
S. Ferraris ◽  
D. Salamone
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Cytochalasin B ◽  
M Cells ◽  
In Vitro Development ◽  
Donor Cells ◽  
G1 Cells ◽  
Vitro Development

Cloning of domestic species by somatic cell nuclear transfer (SCNT) continues to be inefficient, probably due to an incomplete reprogramming of the reconstituted embryo. The ability of the embryonic cytoplasm to support reprogramming fluctuates within the cell cycle (Egli et al. 2007 Nature 447, 679–85). In this context, we compared the development capability and second polar body (2PB) extrusion of embryos produced by metaphase (M) cells, in comparison with G0/G1 cells, which are commonly used as nuclear donors. Because M cells have 2 sets of chromosomes (in contrast with G0/G1 cells, which have only 1 set), an activation protocol that impedes 2PB extrusion is required to produce reconstituted embryos with the correct ploidy. Therefore, we performed SCNT with M or G0/G1 cells, followed by different activation protocols, and evaluated in vitro development and 2PB extrusion of the reconstituted embryos. Cow oocytes were in vitro matured and enucleated as described by Gambini et al. (2014 PLoS One 14, 9). A group of cells at 70 to 80% confluence was synchronized in M stage using 0.05 μg mL−1 demecolcine for 3 to 4 h and used as nuclear donors for SCNT (M group). Another group of cells was induced into quiescence by serum starvation for 3 to 4 days before SCNT (G0/G1 group). For activation, reconstituted embryos were treated with 5 µM ionomycin (Io) for 4 min followed by 5-h incubation in 50 μM roscovitine for M group, or in 50 μM roscovitine and 5 μg mL−1 cytochalasin B for G0/G1 group. Parthenogenetic controls were activated with Io followed by 50 μM roscovitine alone (ROSCO) or with 5 μg mL−1 cytochalasin B (ROSCO/CB). Hoescht 33342 staining was performed 16 h post-Io to evaluate 2PB extrusion. Other activated oocytes were cultured in SOFaa medium and rates of cleavage, morulas, and blastocysts were evaluated at Days 2, 5 and 7 of in vitro development, respectively. Data were analysed by Fisher’s exact test (P < 0.05). Rates of 2PB extrusion were 72.72 (n = 33), 65.63 (n = 32), 80 (n = 15), and 42.86 (n = 14) for M, G0/G1, ROSCO, and ROSCO/CB, respectively. Results of in vitro development are shown in Table 1. In conclusion, somatic M cells can be used as donors to produce cloned embryos. The M and G0/G1 groups were able to induce cloned blastocysts, even though rates did not differed statistically from controls groups (ROSCO and ROSCO/CB). The M group was as effective as G0/G1. Although further analysis is required to establish the quality of the embryos, our results are encouraging for use in SCNT. Table 1.In vitro development of NT embryos produced with M and G0/G1 donor cells

49 TRANSCRIPT LEVEL OF mRNA IN BOVINE CLONED OR RE-CLONED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 142
Author(s):  
G. Jang ◽  
H. Y. Jeon ◽  
K. H. Ko ◽  
H. J. Oh ◽  
H. J. Park ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Developmental Competence ◽  
Gene Transcript ◽  
Embryo Viability ◽  
Guanine Nucleotide Binding Protein ◽  
Glut 1 ◽  
Small Nuclear Ribonucleoprotein ◽  
Donor Cells ◽  
Fetal Fibroblasts

Gene expression in embryos played important roles during preimplantation development and had the potential to be used as an indicator for embryo viability. The purpose of this study was (1) to compare the developmental competence of cloned, or re-cloned embryos (Experiment 1); (2) to analyze the transcripts level of the related implantation, metabolic, and imprinting genes in IVF, cloned and re-cloned embryos (Experiment 2). The SCNT was performed according to the established system in our laboratory (Theriogenology 2006 65, 1800–1812). For producing cloned embryos, fetal fibroblasts as donor cells were used and a viable cloned calf was born. Recloned embryos derived from ear fibroblasts of the cloned calf, genetically same with donor fetal donor cells, were produced. The couplets were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) for up to 7 days. The developmental competence up to blastocysts was observed under a microscope. The implantation (Bax, E-cad, If-tau, Hsp 70, Igf2r, and DNMT1), metabolic [LDHA (Lactate Dehydrogenase A), G6PD (Glucose-6 Phosphate Dehydrogenase), PGK (Phosphogycerate Kinase), Na/K ATPase, and Glut-1], and imprinting [GNAS (guanine nucleotide binding protein, alpha stimulating), UBE3a (ubiquitin protein ligase E3A), Mest (mesoderm specific transcript), SNRPN (small nuclear ribonucleoprotein polypeptide N), and Ndn (necdin)] genes were selected. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by conventional semi-quantitive RT-PCR. In Experiment 1, development competence of SCNT pre-implantation embryo was not different between cloned or re-cloned embryos (26% vs. 22%). In Experiment 2, the relative expression of Bax, Hsp70, If-tau, and Igf2r transcript was not different in IVF, cloned, and re-cloned embryos. Expression of E-cad and DNMT1 was higher in re-cloned embryos than any other group. Transcripts levels of LDHA, Na/K ATPase, and Glut-1 showed the similar relative abundance in IVF, cloned and re-cloned embryos. Expression of G6PD and PGF was increased in re-cloned and cloned embryos, respectively. Compared to IVF and cloned embryos, in re-cloned embryos, relative abundance of related imprinting genes (Ube3a, Mest, SNRPN, and Ndn) was increased. In conclusion, this study demonstrated that, whereas developmental competence of cloned or re-cloned embryos was not different, gene transcript levels were observed differently. It was suggested that alteration of gene expression in the re-cloned embryos derived from the cloned calf, genetically same with initial donor cells, might have affected the fetal development and births of re-cloned offspring.

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.

Donor Cells at the G1 Phase Enhance Homogeneous Gene Expression Among Blastomeres in Bovine Somatic Cell Nuclear Transfer Embryos

2012 ◽  
Vol 14 (1) ◽  
pp. 20-28 ◽  
Author(s):  
Daisaku Iwamoto ◽  
Aya Kasamatsu ◽  
Atsushi Ideta ◽  
Manami Urakawa ◽  
Kazuya Matsumoto ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
G1 Phase ◽  
Donor Cells

22 OXAMFLATIN TREATMENT ENHANCES NUCLEAR REPROGRAMMING BY INHIBITING XIST EXPRESSION AND REDUCING DNA METHYLATION IN PORCINE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 125
Author(s):  
J. Mao ◽  
M. T. Zhao ◽  
K. M. Whitworth ◽  
L. D. Spate ◽  
K. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Somatic Cell ◽  
Real Time ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Xist Gene ◽  
Nuclear Reprogramming ◽  
Xist Expression ◽  
Donor Cells

Treatment of cloned embryos with histone deacetylase inhibitors (HDACi) enhances developmental potential by alteration of epigenetic status. Oxamflatin is one of the potent HDACi. In our previous study, development to Day 7 blastocysts was enhanced when the porcine somatic cell nuclear transfer (SCNT) embryos were treated with oxamflatin for 16 h. The objective of the present study was to investigate the effect of oxamflatin treatment on XIST gene expression and DNA methylation of XIST gene and centromeric repeat element in Day 7 SCNT blastocysts. Somatic cell nuclear transfer was performed on enucleated metaphase II oocytes using a transgene female cell line. Cloned embryos were electrically fused and activated, treated with 150 nM oxamflatin for 16 h and cultured in PZM3 under 5% CO2, 5% oxygen, and 90% N2 for 7 days. Clones without Oxamflatin treatment were used as controls. For XIST methylation, IVF blastocysts at Day 7 were used as controls. Blastocysts at Day 7 were pooled from each treatment group and processed for methylation analysis by bisulfite sequencing and gene expression by quantitative real-time PCR. This experiment was replicated 4 times. The percent of CpG methylation in donor cells before SCNT was also determined. Data were analysed by using SAS version 9.3 (SAS Institute Inc., Cary, NC, USA). In donor cells, 45.3 ± 5.8% of CpGs in a centromeric repeat element (9 CpGs in GenBank Z75640) were methylated. In the SCNT embryos, oxamflatin treatment reduced methylation from 27.3 ± 3.1% in the control to 18.2 ± 3.2% (P < 0.05). The average methylation in XIST (11 CpGs in GenBank KC149530.1) in donor cells was 42.4 ± 6.4%. This CpG island had 2 sites that were not methylated in any of the samples. However, the remaining 9 CpGs were methylated in 8 of 15 samples; for example, showing a parental imprint of ~50%. This implied that the CpG island studied represented the real-time status of the XIST locus in the cell and provides a good marker for reprogramming studies. XIST methylation level in Day 7 blastocysts was not different between oxamflatin (11.8 ± 3.2%) and control (11.8 ± 3.2%). However, XIST methylation in SCNT embryos was higher than in the same age IVF blastocysts (11.7 ± 1.7 v. 0.6 ± 2.4%; P < 0.01). Oxamflatin treatment tended to decrease XIST expression in Day 7 blastocysts compared with controls (18.8 ± 0.8 v. 21.7 ± 0.8; P < 0.1) as measured by real-time PCR. Interestingly, XIST gene expression was positively correlated with its methylation (P < 0.05). In conclusion, these results indicate that during nuclear reprogramming there was a dramatic decrease in DNA methylation from donor cells to Day 7 SCNT embryos. The higher methylation of XIST in SCNT embryos compared with IVF embryos suggests that the reprogramming of donor cells was not completed, which may be a contributor to low cloning efficiency. Oxamflatin treatment of SCNT embryos may enhance nuclear reprogramming by inhibiting XIST expression and reducing DNA methylation, resulting in better embryo development.

293 EFFECT OF MEDIUM TYPE FOR CULTURE OF ADIPOSE-DERIVED MESENCHYMAL STEM CELLS ON PRE-IMPLANTATION DEVELOPMENT OF CLONED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 294
Author(s):  
G. A. Kim ◽  
H. J. Oh ◽  
J. Kim ◽  
T. H. Lee ◽  
J. H. Lee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Culture Medium ◽  
Culture Media ◽  
Formation Rate ◽  
Donor Cells

Mesenchymal stem cells (MSC) have been known as useful donor cells for somatic cell nuclear transfer (SCNT). It has been suggested that the culture condition of donor cells causes different results on preimplantation development of SCNT embryos. In this study, we investigated the patterns of gene expression of adipose-derived mesenchymal stem cells (ad-MSC) in different culture media (DMEM and RKME), and examined the effect of ad-MSC, with the gene expression changed, used as donor cells on the preimplantation development of cloned embryos. Canine ad-MSC were isolated from fat tissue of 3-year-old female beagle and were cultured in DMEM supplemented with 10% fetal bovine serum (MSC-DMEM) and RKME (MSC-MSC) provided from RNL Bio Corp. (Seoul, Korea). Total RNA was extracted from ad-MSC cultured in each culture medium. After synthesising cDNA of each sample, quantitative RT-PCR was done according to the Takara Bio Inc. guidelines and using the 7300 Real Time PCR Cycler System (Applied Biosystems, Carlsbad, CA, USA). The level of all tested gene transcription was normalized to β-actin expression levels. The relative quantification of gene expression was analysed by the 2–ΔΔCt method. The data from all experiments were analysed by Student’s t-test using a statistical analysis GraphPad Prism 4.02 (GraphPad Software Inc., San Diego, CA, USA). Significance was determined at P < 0.05. The stemness, the reprogramming-related gene expression level of donor cells of MSC-DMEM and MSC-MSC were compared. In order to confirm the effect of MSC cultured in 2 different culture media on somatic cell nuclear transfer, we performed interspecies somatic cell nuclear transfer (iSCNT). The enucleated bovine oocytes were injected, respectively, with donor cells of MSC-DMEM and MSC-MSC, and were fused by electrofusion. The iSCNT embryos were cultured in modified SOF at 38.5°C for 7 days in an atmosphere of 5% CO2 and 5% O2, and the developmental ability of iSCNT embryos was observed under the microscope. The MSC-MSC contained a significantly higher amount of Sox2, Nanog, Oct4, Stella, HDAC1, DNMT1, and MeCP2 than the MSC-DMEM, whereas the amount of Rex1 was not different in either MSC-MSC or MSC-DMEM. In the development ability of iSCNT embryos, MSC-DMEM embryos resulted in a 16-cell embryo formation rate that was higher than that of MSC-MSC embryos (9.09 and 5.30%, respectively; P < 0.05). However, the blastocyst formation rate was not different between MSC-DMEM embryos and MSC-MSC embryos (4.5 and 3.2%, respectively; P > 0.05). These results demonstrate that the gene expression of ad-MSC can be modified, by culture media, into a state where reprogramming is easily done. Even so, ad-MSC with gene expression changed by culture medium did not influence the developmental ability of blastocysts. In conclusion, the alteration of gene-related stemness and reprogramming in canine ad-MSC would not be able to effectively control reprogramming in SCNT. This study was supported by RDA (#PJ0089752012), RNL Bio (#550-20120006), IPET (#311062-04-1-SB010), Research Institute for Veterinary Science, and Nestlé Purina Korea.

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.

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