Chimerism in piglets developed from aggregated cloned embryos.
Ontology highlight
ABSTRACT:
SUBMITTER: Huang Y
PROVIDER: S-EPMC4821359 | biostudies-other | 2016 Apr
REPOSITORIES: biostudies-other
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Project description:This study was conducted to investigate whether the treatment of dog to pig interspecies somatic cell nuclear transfer (iSCNT) embryos with a histone deacetylase inhibitor, to improve nuclear reprogramming, can be applied to dog SCNT embryos. The dog to pig iSCNT embryos were cultured in fresh porcine zygote medium-5 (PZM-5) with 0, 1, or 10 µM suberoylanilide hydroxamic acid (SAHA) for 6 h, then transferred to PZM-5 without SAHA. Although there were no significant differences in cleavage rates, the rates of 5-8-cell stage embryo development were significantly higher in the 10 µM group (19.5 ± 0.8%) compared to the 0 µM groups (13.4 ± 0.8%). Acetylation of H3K9 was also significantly higher in embryos beyond the 4-cell stage in the 10 µM group compared to the 0 or 1 µM groups. Treatment with 10 µM SAHA for 6 h was chosen for application to dog SCNT. Dog cloned embryos with 0 or 10 µM SAHA were transferred to recipients. However, there were no significant differences in pregnancy and delivery rates between the two groups. Therefore, it can be concluded that although porcine oocytes support nuclear reprogramming of dog fibroblasts, treatment with a histone deacetylase inhibitor that supports nuclear reprogramming in dog to pig iSCNT embryos was not sufficient for reprogramming in dog SCNT embryos.
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Project description:The efficiency of cloning by somatic cell nuclear transfer (SCNT) has remained low. In most cloned embryos, epigenetic reprogramming is incomplete, and usually the genome is hypermethylated. The DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) could improve the developmental competence of cow, pig, cat and human SCNT embryos in previous studies. However, the parameters of 5-aza-dC treatment among species are different, and whether 5-aza-dC could enhance the developmental competence of porcine cloned embryos has still not been well studied. Therefore, in this study, we treated porcine fetal fibroblasts (PFF) that then were used as donor nuclei for nuclear transfer or fibroblast-derived reconstructed embryos with 5-aza-dC, and the concentration- and time-dependent effects of 5-aza-dC on porcine cloned embryos were investigated by assessing pseudo-pronucleus formation, developmental potential and pluripotent gene expression of these reconstructed embryos. Our results showed that 5-aza-dC significantly reduced the DNA methylation level in PFF (0 nM vs. 10 nM vs. 25 nM vs. 50 nM, 58.70% vs. 37.37% vs. 45.43% vs. 39.53%, P<0.05), but did not improve the blastocyst rate of cloned embryos derived from these cells. Treating cloned embryos with 25 nM 5-aza-dC for 24 h significantly enhanced the blastocyst rate compared with that of the untreated group. Furthermore, treating cloned embryos, but not donor cells, significantly promoted pseudo-pronucleus formation at 4 h post activation (51% for cloned embryos treated, 34% for donor cells treated and 36% for control, respectively, P<0.05) and enhanced the expression levels of pluripotent genes (Oct4, Nanog and Sox2) up to those of in vitro fertilized embryos during embryo development. In conclusion, treating cloned embryos, but not donor cells, with 5-aza-dC enhanced the developmental competence of porcine cloned embryos by promotion of pseudo-pronucleus formation and improvement of pluripotent gene expression.
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Project description:Most animals produced by somatic cell nuclear transfer (SCNT) are heteroplasmic for mitochondrial DNA (mtDNA). Oxidative phosphorylation (OXPHOS) in clones therefore requires the coordinated expression of genes encoded by the nuclear DNA and the two sources of mitochondria. Such interaction is rarely studied because most clones are generated using slaughterhouse oocytes of unrecorded origin. Here we traced the maternal lineages of seven diseased and five one-month-old live cloned piglets by sequencing their mtDNA. Additionally by using a 13K oligonucleotide microarray, we compared the expression profiles of nuclear and mtDNA-encoded genes that are involved in mitochondrial functions and regulation between the cloned groups and their age-matched controls (n=5 per group). We found that the oocytes used to generate the cloned piglets were of either the Large White or Duroc background, and oocyte genetic background was not related to the clones' survival. Expression profiles of mtDNA-encoded genes in clones and controls showed intermixed clustering patterns without treatment or maternal lineage-dependency. In contrast, clones and controls clustered separately for their global and nuclear DNA-encoded mitochondrial genes in the lungs for both the deceased and live groups. Functional annotation of differentially expressed genes encoded by both nuclear and mtDNA revealed abnormal gene expression in the mitochondrial OXPHOS pathway in deceased clones. Among the nine differentially expressed genes of the OXPHOS pathway, seven were down-regulated in deceased clones compared to controls, suggesting deficiencies in mitochondrial functions. Together, these data demonstrate that the coordination of expression of mitochondrial genes encoded by nuclear and mtDNA is disrupted in the lung of diseased clones.
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Project description:Although somatic cell nuclear transfer (SCNT) cloning is more efficient in cattle than in any other species tested so far, there is a high rate of pregnancy failure that has been linked to structural and functional abnormalities of the placenta. We tested the hypothesis that these changes may originate from disturbed embryo-maternal interactions in the peri-implantation period. Therefore, we evaluated the response of the endometrium to SCNT embryos (produced from 7 different fetal fibroblast cell lines) as compared with embryos derived from in vitro fertilization (IVF). SCNT embryos and IVF embryos were cultured under identical conditions to the blastocyst stage (day 7) and were transferred to corresponding recipients, which were slaughtered at day 18 of pregnancy. The mRNA profiles of endometrium samples were obtained using a custom cDNA microarray enriched for transcripts differentially expressed in the endometrium and/or oviduct epithelium during the estrous cycle and/or early pregnancy. Overall, the variation in mRNA profiles was greater in the SCNT group than in the IVF group. Furthermore, 58 transcripts were differentially abundant in endometria from SCNT and IVF pregnancies. Prominent examples are orphan nuclear receptor COUP-TFII and connexin 43, both known to play important roles in uterine receptivity and conceptus placentation. These findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo-maternal communication that develops during the peri-implantation period. Endometrium transcriptome profiles may serve as a tool to evaluate SCNT embryos for their ability to establish pregnancy and develop a functional placenta.
| S-EPMC2666995 | biostudies-literature
Project description:Epigenetic reprogramming is necessary in somatic cell nuclear transfer (SCNT) embryos in order to erase the differentiation-associated epigenetic marks of donor cells. However, such epigenetic memories often persist throughout the course of clonal development, thus decreasing cloning efficiency. Here, we explored reprogramming-refractory regions in bovine SCNT blastocyst transcriptomes. We observed that histone genes residing in the 1.5 Mb spanning the cow HIST1 cluster were coordinately downregulated in SCNT blastocysts. In contrast, both the nonhistone genes of this cluster, and histone genes elsewhere remained unaffected. This indicated that the downregulation was specific to HIST1 histone genes. We found that, after trichostatin A treatment, HIST1 histone genes were derepressed, and DNA methylation at their promoters was decreased to the level of in vitro fertilization embryos. Therefore, our results indicate that the reduced expression of HIST1 histone genes is a consequence of poor epigenetic reprogramming in SCNT blastocysts.
| S-EPMC4856087 | biostudies-literature
Project description:In general, pig embryos established by somatic cell nuclear transfer (SCNT) are transferred at the one-cell stage because of suboptimal embryo culture conditions. Improvements in embryo culture can increase the practical application of late embryo transfer. The goal of this study was to evaluate embryos cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro, and to track the in vivo developmental competency of SCNT-derived blastocysts from these GM-CSF embryos. The receptor for GM-CSF was up-regulated in in vitro-produced embryos when compared to in vivo-produced cohorts, but the level decreased when GM-CSF was present. In vitro fertilized (IVF) embryos, supplemented with GM-CSF (2 or 10 ng/ml), showed a higher frequency of development to the blastocyst stage compared to controls. The total cell numbers of the blastocysts also increased with supplementation of GM-CSF. Molecular analysis demonstrates that IVF-derived blastocysts cultured with GM-CSF exhibit less apoptotic activity. Similarly, an increase in development to the blastocyst stage and an increase in the average total-cell number in the blastocysts were observed when SCNT-derived embryos were cultured with either concentration of GM-CSF (2 or 10 ng/ml). When SCNT-derived embryos, cultured with 10 ng/ml GM-CSF, were transferred into six surrogates at Day 6, five of the surrogates became pregnant and delivered healthy piglets. Our findings suggest that supplementation of GM-CSF can provide better culture conditions for IVF- and SCNT-derived embryos, and pig SCNT-derived embryos cultured with GM-CSF in vitro can successfully produce piglets when transferred into surrogates at the blastocyst stage. Thus, it may be practical to begin performing SCNT-derived embryo transfer at the blastocyst stage.
| S-EPMC4392757 | biostudies-literature
Project description:Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components. Moreover, autophagy is essential for preimplantation development in mammals. Here we show that autophagy is also important for reprogramming in somatic cell nuclear transfer (SCNT). Our data indicate that unlike fertilized oocytes, autophagy is not triggered in SCNT embryos during 6 hours of activation. Mechanistically, the inhibited autophagic induction during SCNT activation is due to the cytochalasin B (CB) caused depolymerization of actin filaments. In this study, we induced autophagy during SCNT activation by rapamycin and pp242, which could restore the expected level of autophagy and significantly enhance the development of SCNT embryos to the blastocyst stage when compared with the control (68.5% and 68.7% vs. 41.5%, P < 0.05). Furthermore, the treatment of rapamycin and pp242 accelerates active DNA demethylation indicated by the conversion of 5 mC to 5 hmC, and treatment of rapamycin improves degradation of maternal mRNA as well. Thus, our findings reveal that autophagy is important for development of SCNT embryos and inhibited autophagic induction during SCNT activation might be one of the serious causes of low efficiency of SCNT.
| S-EPMC4672298 | biostudies-literature
Project description:Incomplete reprogramming of pluripotent genes in cloned embryos is associated with low cloning efficiency. Epigenetic modification agents have been shown to enhance the developmental competence of cloned embryos; however, the effect of the epigenetic modification agents on pluripotent gene reprogramming remains unclear. Here, we investigated Nanog reprogramming and the expression patterns of pluripotent transcription factors during early embryo development in pigs. We found that compared with fertilized embryos, cloned embryos displayed higher methylation in the promoter and 5'-untranslated region and lower methylation in the first exon of Nanog. When 5-aza-2'-deoxycytidine (5-aza-dC) or trichostatin A (TSA) enhanced the development of porcine cloned embryos, Nanog methylation reprogramming was also improved, similar to that detected in fertilized counterparts. Furthermore, our results showed that the epigenetic modification agents improved the expression levels of Oct4 and Sox2 and effectively promoted Nanog transcription in cloned embryos. In conclusion, our results demonstrated that the epigenetic modification agent 5-aza-dC or TSA improved Nanog methylation reprogramming and the expression patterns of pluripotent transcription factors, thereby resulting in the enhanced expression of Nanog and high development of porcine cloned embryos. This work has important implications in the improvement of cloning efficiency.
| S-EPMC4487246 | biostudies-literature
Project description:Most animals produced by somatic cell nuclear transfer (SCNT) are heteroplasmic for mitochondrial DNA (mtDNA). Oxidative phosphorylation (OXPHOS) in clones therefore requires the coordinated expression of genes encoded by the nuclear DNA and the two sources of mitochondria. Such interaction is rarely studied because most clones are generated using slaughterhouse oocytes of unrecorded origin. Here we traced the maternal lineages of seven diseased and five one-month-old live cloned piglets by sequencing their mtDNA (short SNP information in single mitochondrial gene). Additionally by using a 13K oligonucleotide microarray, we compared the expression profiles of nuclear and mtDNA-encoded genes that are involved in mitochondrial functions and regulation between the cloned groups and their age-matched controls (n=5 per group). We found that the oocytes used to generate the cloned piglets were of either the Large White or Duroc background, and oocyte genetic background was not related to the clones’ survival. Expression profiles of mtDNA-encoded genes in clones and controls showed intermixed clustering patterns without treatment or maternal lineage-dependency. In contract, clones and controls clustered separately for their global and nuclear DNA-encoded mitochondrial genes in the lungs for both the deceased and live groups. Functional annotation of differentially expressed genes encoded by both nuclear and mtDNA revealed abnormal gene expression in the mitochondrial OXPHOS pathway in deceased clones. Among the nine differentially expressed genes of the OXPHOS pathway, seven were down-regulated in deceased clones compared to controls, suggesting deficiencies in mitochondrial functions. Together, these data demonstrate that the coordination of expression of mitochondrial genes encoded by nuclear and mtDNA is disrupted in the lung of diseased clones.
2015-06-25 | GSE68877 | GEO
Project description:Incomplete DNA methylation reprogramming in cloned embryos leads to low cloning efficiency. Our previous studies showed that the epigenetic modification agents 5-aza-2'-deoxycytidine (5-aza-dC) or trichostatin A (TSA) could enhance the developmental competence of porcine cloned embryos. Here, we investigated genomic methylation dynamics and specific gene expression levels during early embryonic development in pigs. In this study, our results showed that there was a typical wave of DNA demethylation and remethylation of centromeric satellite repeat (CenRep) in fertilized embryos, whereas in cloned embryos, delayed demethylation and a lack of remethylation were observed. When cloned embryos were treated with 5-aza-dC or TSA, CenRep methylation reprogramming was improved, and this was similar to that detected in fertilized counterparts. Furthermore, we found that the epigenetic modification agents, especially TSA, effectively promoted silencing of tissue specific genes and transcription of early embryo development-related genes in porcine cloned embryos. In conclusion, our results showed that the epigenetic modification agent 5-aza-dC or TSA could improve genomic methylation reprogramming in porcine cloned embryos and regulate the appropriate expression levels of genes related to early embryonic development, thereby resulting in high developmental competence.
| S-EPMC4219995 | biostudies-literature