Single-cell RNA-seq of goat early embyos and GFFs cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Capra hircus
PROVIDER: GSE199933 | GEO | 2022/04/04
REPOSITORIES: GEO
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Project description:Somatic cell nuclear transfer (SCNT) into an enucleated oocyte can reprogram a differentiated nucleus to a totipotent state. However, the time course of transcriptional reprogramming has not been determined. To monitor the inactivation of somatic genes after SCNT in the bovine model system, we determined transcript levels of 159 genes highly expressed in fetal fibroblast nuclear donor cells, but not in metaphase II recipient oocytes. For 18 of these genes significantly higher transcript levels were found in four-cell SCNT embryos compared with four-cell embryos derived by in vitro fertilization (IVF), indicating incomplete silencing of somatic genes at this stage. RNA sequencing revealed that nearly 600 genes with no transcripts in oocytes were activated in eight-cell IVF embryos, in agreement with major embryonic genome activation (EGA). De novo transcription in SCNT embryos was delayed (16 genes before the 16-cell stage, 300 and >800 genes at the 16-cell and blastocyst stages). Intron-containing primary transcripts as another option to detect embryonic gene transcription revealed activation of >2,200 genes in IVF embryos at the eight-cell stage, while only 828 genes were activated in SCNT embryos. At the 16-cell stage, a higher number of activated genes was found in SCNT (1,917) than in IVF embryos (738). Self-organizing tree algorithm clustering confirmed that in SCNT embryos transcription of genes that are normally activated during major EGA is delayed. Our study provides detailed insight into the timing of genome activation in cloned embryos that is substantially different from IVF embryos in spite of similar developmental kinetics.
2021-05-31 | GSE125828 | GEO
Project description:Somatic cell nuclear transfer (SCNT) into an enucleated oocyte can reprogram a differentiated nucleus to a totipotent state. However, the time course of transcriptional reprogramming has not been determined. To monitor the inactivation of somatic genes after SCNT in the bovine model system, we determined transcript levels of 159 genes highly expressed in fetal fibroblast nuclear donor cells, but not in metaphase II recipient oocytes. For 18 of these genes significantly higher transcript levels were found in four-cell SCNT embryos compared with four-cell embryos derived by in vitro fertilization (IVF), indicating incomplete silencing of somatic genes at this stage. RNA sequencing revealed that nearly 600 genes with no transcripts in oocytes were activated in eight-cell IVF embryos, in agreement with major embryonic genome activation (EGA). De novo transcription in SCNT embryos was delayed (16 genes before the 16-cell stage, 300 and >800 genes at the 16-cell and blastocyst stages). Intron-containing primary transcripts as another option to detect embryonic gene transcription revealed activation of >2,200 genes in IVF embryos at the eight-cell stage, while only 828 genes were activated in SCNT embryos. At the 16-cell stage, a higher number of activated genes was found in SCNT (1,917) than in IVF embryos (738). Self-organizing tree algorithm clustering confirmed that in SCNT embryos transcription of genes that are normally activated during major EGA is delayed. Our study provides detailed insight into the timing of genome activation in cloned embryos that is substantially different from IVF embryos in spite of similar developmental kinetics.
2021-05-31 | GSE125724 | GEO
Project description:Global hypermethylations of histone H3 lysine 9 (H3K9) tri- and di-methylation (H3K9me3/2) have been identified in bovine cloned embryos at the embryonic genome activation (EGA) stage (eight-cell stage), but the intrinsic reason for these anomalies remains elusive. To ascertain key factors responsible for aberrant H3K9 methylation, we performed RNA sequencing of transcripts in eight-cell bovine in vitro fertilized (IVF) and SCNT embryo. From the differentially expressed genes (DEGs) between IVF and SCNT embryos, we identified that the unsuccessful reactivation of two histone demethylases, KDM4D and KDM4E, is responsible for the incomplete H3K9me3/2 demethylation in SCNT embryos at the EGA stage. By mRNA injection, ectopic expression of either KDM4D or KDM4E could erase H3K9me3/2 barriers, improve blastocyst formation, and elevate cloning efficiency of bovine SCNT. To examine the detailed genes responsive to KDM4E overexpression, we also performed RNA sequencing of bovine eight-cell SCNT embryos with KDM4E compensation and found an obvious restoration of global transcriptional patterns in SCNT embryos. Our study first provides the transcriptome data set of bovine IVF and SCNT embryos during EGA with or without KDM4E overexpression, which advance the understanding of incomplete nuclear reprogramming, and contribute to the practical implications for genetically modified livestock breeding using SCNT.
2018-01-16 | GSE99294 | GEO
Project description:Production of embryos with high developmental competence by somatic cell nuclear transfer (scNT) is far less efficient than for in vitro fertilized (IVF) embryos, likely due to an accumulation of errors in genome reprogramming that results in aberrant expression of RNA transcripts, including messenger RNAs (mRNA) and, possibly, microRNAs (miRNA). Thus, our objectives were to use RNAseq to determine the dynamics of mRNA expression in early developing scNT and IVF embryos in the context of the maternal-to-embryonic transition (MET) and to correlate apparent transcriptional dysregulation in cloned embryos with miRNA expression profiles. Comparisons between scNT and IVF embryos indicated large scale transcriptome differences, which were most evident at the 8-cell and morula stages for genes associated with biological functions critical for the MET. For two miRNAs previously identified as differentially expressed in scNT morulae, miR-34a and miR-345, negative correlations with some predicted mRNA targets were apparent, though not widespread among the majority of predicted targets. Moreover, although large-scale aberrations in expression of mRNAs were evident during the MET in cattle scNT embryos, these changes were not consistently correlated with aberrations in miRNA expression at the same developmental stage, suggesting that other mechanisms controlling gene expression may be involved.
2021-06-18 | GSE178436 | GEO
Project description:Although somatic cell nuclear transfer (SCNT) cloning is more efficient in bovine than in all 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 pre-implantation period. Therefore, we evaluated the transcriptome response of the endometrium to SCNT embryos (produced from five different donor cell cultures) as compared to embryos derived from in vitro fertilization (IVF). SCNT embryos and IVF embryos were cultured under identical conditions to the blastocyst stage (Day 8) and transferred to recipients. The recipients were slaughtered at day 18 of pregnancy and the uterus was recovered. Pregnancy was verified by the presence of at least one normally developed embryo. Transcriptome profiling of endometrium samples using a custom cDNA microarray covering transcripts expressed in the endometrium and/or oviduct epithelium revealed 58 transcripts that were differently abundant between endometrium samples from SCNT vs. IVF pregnancies. Prominent examples are NR2F2 (encoding the orphan nuclear receptor COUP-TFII) and GJA1 (encoding connexin 43). Both transcripts are known to play important roles in placentation and were significantly less abundant in endometrium from SCNT vs. IVF pregnancies. These findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo-maternal communication already in the pre- or peri-implantation period. Endometrium transcriptome profiles may serve as a novel readout to evaluate SCNT embryos for their ability to induce pregnancy with a functional placenta. Keywords: response to different embryos Nineteen German Fleckvieh (Simmental) heifers were slaughtered at day 18 of pregnancy. Cycle-synchronized recipient heifers received either IVP or SCNT embryos at day 7 of the estrous cycle. Animals were slaughtered at day 18. Endometrial (intercaruncular) tissue samples were obtained from 10 pregnant animals after transfer of IVP embryos and from 9 pregnant animals after transfer of SCNT embryos.
2009-03-12 | E-GEOD-13663 | biostudies-arrayexpress
Project description:Although somatic cell nuclear transfer (SCNT) cloning is more efficient in bovine than in all 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 pre-implantation period. Therefore, we evaluated the transcriptome response of the endometrium to SCNT embryos (produced from five different donor cell cultures) as compared to embryos derived from in vitro fertilization (IVF). SCNT embryos and IVF embryos were cultured under identical conditions to the blastocyst stage (Day 8) and transferred to recipients. The recipients were slaughtered at day 18 of pregnancy and the uterus was recovered. Pregnancy was verified by the presence of at least one normally developed embryo. Transcriptome profiling of endometrium samples using a custom cDNA microarray covering transcripts expressed in the endometrium and/or oviduct epithelium revealed 58 transcripts that were differently abundant between endometrium samples from SCNT vs. IVF pregnancies. Prominent examples are NR2F2 (encoding the orphan nuclear receptor COUP-TFII) and GJA1 (encoding connexin 43). Both transcripts are known to play important roles in placentation and were significantly less abundant in endometrium from SCNT vs. IVF pregnancies. These findings suggest that placental failure in bovine clone pregnancies may originate from abnormal embryo-maternal communication already in the pre- or peri-implantation period. Endometrium transcriptome profiles may serve as a novel readout to evaluate SCNT embryos for their ability to induce pregnancy with a functional placenta. Keywords: response to different embryos
2009-03-13 | GSE13663 | GEO
Project description:The efficiency of somatic cell nuclear transfer (scNT) for production of viable offspring is relatively low as compared to in vitro fertilization (IVF), presumably due to deficiencies in epigenetic reprogramming of the donor cell genome. Such defects may also involve the population of small non-coding RNAs (sncRNAs), which are important during early embryonic developmeNT.The objective of this study was to examine dynamic changes in relative abundance of sncRNAs during the maternal-to embryonic transition (MET) in bovine embryos produced by scNT as compared to IVF by using RNA sequencing. When comparing populations of miRNA in scNT versus IVF embryos, only miR-2340, miR-345, and miR34a were differentially expressed in morulae, though many more miRNAs were differentially expressed when comparing across developmental stages. Also of interest, distinct populations of piwi-interacting like RNAs (pilRNAs) were identified in bovine embryos prior to and during embryonic genome activation (EGA) as compared bovine embryos post EGA and differentiated cells. Overall, sncRNA sequencing analysis of preimplantation embryos revealed largely similar profiles of sncRNAs for IVF and scNT embryos at the 2-cell, 8-cell, morula and blastocyst stages of developmeNT.However, these sncRNA profiles, including miRNA, piRNA and tRNA fragments, were notably distinct prior to and after completion of the MET.
2021-06-18 | GSE178437 | GEO
Project description:Mammalian oocytes can reprogram somatic cells into totipotent state, which allows animal cloning through somatic cell nuclear transfer (SCNT). However, the great majority of SCNT embryos fail to develop to term due to poorly defined reprogramming defects. Here we demonstrate that histone H3 lysine 9 trimethylation (H3K9me3) in donor nuclei is a major epigenetic barrier that prevents efficient nuclear reprogramming in mouse oocytes. Comparative transcriptome analysis of early embryos revealed reprogramming resistant regions (RRRs) where transcriptional activation at 2-cell embryos is inhibited by SCNT compared to in vitro fertilization (IVF). RRRs significantly overlap with H3K9me3 enrichment in donor somatic cells. Importantly, removal of the H3K9me3 by ectopic expression of an H3K9me3 demethylase Kdm4d in recipient oocytes not only reactivates most RRRs, but also greatly improves development of SCNT embryos. Furthermore, the use of Suv39h1/2-depleted somatic nuclei as donors also greatly improves the development of SCNT embryos. Our study thus reveals H3K9me3 as an epigenetic barrier in SCNT-mediated reprogramming and provides a feasible method for improving mammalian cloning efficiency.
2014-11-07 | GSE59073 | GEO
Project description:In this study, we explored x-inactivation in monkey embryos (ICM and TE separately) and pluripotent stem cells (IVF derived ES, SCNT-derived ES and monkey iPS) To elucidate x-inactivation in experimentally reprogrammed pluripotent cells, we derived pluripotent stem cells by both SCNT and iPS approaches from same parental skin fibroblasts. We also compared gene patterns of those cells to IVF-derived counterpart. The transcriptomes of rhesus monkey embryonic stem cell lines derived by both SCNT (CRES) and iPS (RiPS) from same monkey skin fibroblasts were compared each other. Both experimentally reprogrammed cells were also compared with IVF-derived counterpart (ORMES23). Finally, the adult somatic skin fibroblasts were analyzed. Three biological replicates of each cell line (A, B, C) were analyzed.
2012-08-31 | E-GEOD-36252 | biostudies-arrayexpress
Project description:Zygotic genome activation (ZGA) marks the beginning of the embryonic program for a totipotent embryo, which gives rise to inner cell mass (ICM), where pluripotent epiblast arises, and extraembryonic trophectoderm. While much has been learned about pluripotency regulation, how ZGA is connected to the first lineage segregation in early embryos remains elusive. Here, we investigated the role of nuclear receptor transcription factors (NR TFs), whose motifs are highly enriched in accessible chromatin at the 2-cell (2C) to 8-cell (8C) stages after ZGA. We found NR5A2, an NR TF highly induced upon ZGA, is required for early development. Genome-wide chromatin binding in 2C and 8C embryos showed NR5A2 bound cis-regulatory elements which strongly enrich B1 elements where its motif is embedded. RNA-seq showed while the zygotic genome was largely activated at the 2C stage, 4-8C-specific gene activation was substantially impaired in Nr5a2 KD embryos. At the 8C stage, NR5A2 preferentially regulated its targets including a subset of early ICM genes. Consistently, NR5A2 was required for opening 8C-specific binding sites. Interestingly, while NR5A2’s 2C-binding sites are largely closed in blastocyst, 8C-specific binding sites tend to stay open and are bound by master pluripotency TFs (NANOG, SOX2, and OCT4) in blastocyst or ESC. Further analyses showed NR5A2 regulates early ICM genes at the 8C stage, master pluripotency TFs regulate both early and late ICM genes at later stages. Taken together, these data identify NR5A2 as a key regulator in totipotent embryos that connects ZGA to the first lineage segregation in early mouse development.
2023-10-12 | GSE229740 | GEO