scholarly journals Single-Cell Sequencing of Primate Preimplantation Embryos Reveals Chromosome Elimination Via Cellular Fragmentation and Blastomere Exclusion

2018 ◽  
Author(s):  
Brittany L. Daughtry ◽  
Jimi L. Rosenkrantz ◽  
Nathan H. Lazar ◽  
Suzanne S. Fei ◽  
Nash Redmayne ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Cell ◽  
Chromosome Instability ◽  
Complex Mixture ◽  
Chromosome Elimination ◽  
Early Embryo ◽  
Preimplantation Embryos ◽  
Cleavage Stage ◽  
Primate Model ◽  
Cell Stage

ABSTRACTAneuploidy that arises during meiosis and/or mitosis is a major contributor to early embryo loss. We previously demonstrated that human preimplantation embryos encapsulate mis-segregated chromosomes into micronuclei while undergoing cellular fragmentation and that fragments can contain chromosomal material, but the source of this DNA was unknown. Here, we leveraged the use of a non-human primate model and single-cell DNA-sequencing (scDNA-seq) to examine the chromosomal content of 471 individual samples comprising 254 blastomeres, 42 polar bodies, and 175 cellular fragments from a large number (N=50) of disassembled rhesus cleavage-stage embryos. Our analysis revealed that the frequency of aneuploidy and micronucleation is conserved between humans and macaques and that cellular fragments encapsulate whole and/or partial chromosomes lost from blastomeres. Single-cell/fragment genotyping demonstrated that these chromosome-containing cellular fragments (CCFs) can be either maternal or paternal in origin and display DNA damage via double-stranded breaks. Chromosome breakage and abnormal cytokinesis resulted in reciprocal losses/gains at the terminal ends of chromosome arms, uniparental genome segregation, and mixoploidy between blastomeres. Combining time-lapse imaging with scDNA-seq, we also determined that multipolar divisions at the zygote or 2-cell stage generated chaotic aneuploidy encompassing a complex mixture of maternal and paternal chromosomes. Despite frequent chromosomal mis-segregation at the cleavage-stage, we show that CCFs and non-dividing aneuploid blastomeres exhibiting extensive DNA damage are prevented from incorporation at the blastocyst stage. These findings suggest that embryos respond to chromosomal errors by encapsulation into micronuclei, elimination by cellular fragmentation, and selection against highly aneuploid blastomeres to overcome chromosome instability during preimplantation development.

Regulation of desmocollin transcription in mouse preimplantation embryos

Development ◽  
1995 ◽  
Vol 121 (3) ◽  
pp. 743-753 ◽  
Author(s):  
J.E. Collins ◽  
J.E. Lorimer ◽  
D.R. Garrod ◽  
S.C. Pidsley ◽  
R.S. Buxton ◽  
...  
Keyword(s):  
Protein Expression ◽  
Molecular Mechanisms ◽  
Inner Cell Mass ◽  
Splice Variants ◽  
Cell Mass ◽  
Cumulus Cells ◽  
Early Embryo ◽  
Preimplantation Embryos ◽  
Cell Stage ◽  
Cleavage Stages

The molecular mechanisms regulating the biogenesis of the first desmosomes to form during mouse embryogenesis have been studied. A sensitive modification of a reverse transcriptase-cDNA amplification procedure has been used to detect transcripts of the desmosomal adhesive cadherin, desmocollin. Sequencing of cDNA amplification products confirmed that two splice variants, a and b, of the DSC2 gene are transcribed coordinately. Transcripts were identified in unfertilized eggs and cumulus cells and in cleavage stages up to the early 8-cell stage, were never detected in compact 8-cell embryos, but were evident again either from the 16-cell morula or very early blastocyst (approx 32-cells) stages onwards. These two phases of transcript detection indicate DSC2 is encoded by maternal and embryonic genomes. Previously, we have shown that desmocollin protein synthesis is undetectable in eggs and cleavage stages but initiates at the early blastocyst stage when desmocollin localises at, and appears to regulate assembly of, nascent desmosomes that form in the trophectoderm but not in the inner cell mass (Fleming, T. P., Garrod, D. R. and Elsmore, A. J. (1991), Development 112, 527–539). Maternal DSC2 mRNA is therefore not translated and presumably is inherited by blastomeres before complete degradation. Our results suggest, however, that initiation of embryonic DSC2 transcription regulates desmocollin protein expression and thereby desmosome formation. Moreover, data from blastocyst single cell analyses suggest that embryonic DSC2 transcription is specific to the trophectoderm lineage. Inhibition of E-cadherin-mediated cell-cell adhesion did not influence the timing of DSC2 embryonic transcription and protein expression. However, isolation and culture of inner cell masses induced an increase in the amount of DSC2 mRNA and protein detected. Taken together, these results suggest that the presence of a contact-free cell surface activates DSC2 transcription in the mouse early embryo.

124. DNA METHYLATION IN THE 2-CELL MOUSE EMBRYO IS THE RESULT OF DNMT ACTIVITY DURING DEVELOPMENT FROM THE ZYGOTE TO THE 2-CELL STAGE

2009 ◽  
Vol 21 (9) ◽  
pp. 43
Author(s):  
Y. Li ◽  
H. D. Morgan ◽  
L. Ganeshan ◽  
C. O'Neill
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Cytosine Methylation ◽  
De Novo ◽  
Culture Conditions ◽  
Early Embryo ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Stage Of Development ◽  
First Time

In an accompanying abstract we show for the first time that global demethylation of both paternally- and maternally-derived genomes occurs prior to syngamy. It is commonly considered that new methylation of the genome does not commence until late in the preimplantation stage. Yet embryos during cleavage stage are known to show DNA methylation. This creates a paradox, if global demethylation occurs by the time of syngamy yet remethylation does not occur until the blastocysts stage, how can cleavage stage embryos possess methylated DNA. We examined this paradox. We examined DNA methylation in 2-cell embryos by confocal microscopy of anti-methylcytosine immunofluorescence and propidium iodide co-staining of whole mounts. We confirmed that DNA in late zygotes was substantially demethylated in both the male and female pronuclei. By the 2-cell stage, embryos collected direct from the oviduct showed high levels of cytosine methylation. We assessed whether this accumulation of cytosine methylation during the early 2-cell stage was a consequence of DNA methyltransferase (DNMT) activity. This was achieved by treating late stage zygotes with the DNMT inhibitor RG108 (5 μM) for the period of development spanning pronuclear stage 5 to early 2-cell stage. The embryos that developed in the presence of the DNA methyltransferase inhibitor showed significantly less methylcytosine staining than the embryos in the untreated culture conditions (P<0.001). Treatment of embryos during this period with RG108 significantly reduced their capacity to develop to normal blastocysts, indicating that this early DNA re-methylation reaction was important for the normal development of the embryo. Our results show for the first time that de novo methylation of the genome occurs as early as the 2-cell stage of development and that this is mediated by a RG108-sensitive DNMT activity. The results substantially change our understanding of epigenetic reprogramming in the early embryo.

scholarly journals Single-cell sequencing of primate preimplantation embryos reveals chromosome elimination via cellular fragmentation and blastomere exclusion

2019 ◽  
Vol 29 (3) ◽  
pp. 367-382 ◽  
Author(s):  
Brittany L. Daughtry ◽  
Jimi L. Rosenkrantz ◽  
Nathan H. Lazar ◽  
Suzanne S. Fei ◽  
Nash Redmayne ◽  
...  
Keyword(s):  
Single Cell ◽  
Chromosome Elimination ◽  
Preimplantation Embryos ◽  
Single Cell Sequencing

Ago2 and GW182 expression in mouse preimplantation embryos: a link between microRNA biogenesis and GW182 protein synthesis

2010 ◽  
Vol 22 (4) ◽  
pp. 634 ◽  
Author(s):  
Xing-Hui Shen ◽  
Young-Joon Han ◽  
Xiang-Shun Cui ◽  
Nam-Hyung Kim
Keyword(s):  
Protein Synthesis ◽  
Internal Standard ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Internal Reference ◽  
Cell Stage ◽  
Transcript Levels ◽  
Microrna Biogenesis

MicroRNA-mediated RNA interference appears to play a role in early development and differentiation processes in preimplantation embryos. However, the expression of its key effectors, including Ago2, a key component of the RNA-induced silencing complex, and GW182, a critical component of GW bodies (GWBs), has not been assessed in preimplantation embryos. To characterise the roles of Ago2 and GW182 in early embryo development, we determined their transcription and protein synthesis in mouse embryos. Transcript levels of Ago2 and GW182 increased steadily from the one-cell stage through to the blastocyst stage when data were not normalised against an internal reference. However, when normalised against the internal standard, transcript levels for both genes were highest in four-cell stage embryos and decreased steadily through to the blastocyst stage. Indirect immunocytochemistry showed that both AGO2 and GW182 proteins were expressed in each stage in the early embryo and were observed to colocalise in the morula and blastocyst stages. Specific silencing of mRNA expression by short interference (si) RNA against Ago2 or Dicer1 decreased the expression of selected apoptosis- and development-related microRNAs, but did not inhibit development up to the blastocyst stage. However, transcription levels of Oct3/4, Nanog and Sox2 were decreased in both Ago2- and Dicer1-knockdown embryos at the blastocyst stage. Furthermore, although knockdown of these genes did not change transcript levels of GW182, GW182 protein synthesis was decreased in blastocyst stage embryos. These results suggest that Ago2 and Dicer1 regulate GW182 protein expression in mouse embryos, which is linked to microRNA biogenesis and likely to be important for differentiation in the blastocyst stage.

scholarly journals Rainbow-seq: combining cell lineage tracking with single-cell RNA sequencing in preimplantation embryos

2018 ◽  
Author(s):  
Fernando Biase ◽  
Qiuyang Wu ◽  
Riccardo Calandrelli ◽  
Marcelo Rivas-Astroza ◽  
Shuigeng Zhou ◽  
...  
Keyword(s):  
Cell Division ◽  
Single Cell ◽  
Cell Fate ◽  
Fluorescent Protein ◽  
Lineage Tracing ◽  
Preimplantation Embryos ◽  
Marker Genes ◽  
Regulation Of Transcription ◽  
Rna Seq ◽  
Cell Stage

SUMMARYSingle-cell RNA-seq experiments cannot record cell division history and therefore cannot directly connect intercellular differences at a later developmental stage to their progenitor cells. We developed Rainbow-seq to combine cell division lineage tracing with single-cell RNA-seq. With distinct fluorescent protein genes as lineage markers, Rainbow-seq enables each single-cell RNA-seq experiment to simultaneously read single-cell transcriptomes and decode the lineage marker genes. We traced the lineages deriving from each blastomere in two-cell mouse embryos and observed inequivalent contributions to the embryonic and abembryonic poles in 72% of the blastocysts evaluated. Rainbow-seq on four- and eight-cell embryos with lineage tracing triggered at two-cell stage exhibited remarkable transcriptome-wide differences between the two cell lineages at both stages, including genes involved in negative regulation of transcription and signaling. These data provide critical insights on cell fate choices in cleavage embryos. Rainbow-seq bridged a critical gap between cellular division history and single-cell RNA-seq assays.

scholarly journals Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maya Sen ◽  
Dylan Mooijman ◽  
Alex Chialastri ◽  
Jean-Charles Boisset ◽  
Mina Popovic ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cost Effective ◽  
Dna Demethylation ◽  
Mouse Embryos ◽  
Preimplantation Embryos ◽  
Mammalian Development ◽  
Cell Stage ◽  
Stage Of Development

AbstractDNA methylation (5mC) is central to cellular identity. The global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of gametes to the cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to 5mC erasure remains unclear. Here, we report a single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, allowing us to systematically probe the dynamics of global demethylation. When applied to mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells.

Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 485-493 ◽  
Author(s):  
A.F. Pereira ◽  
L.M. Melo ◽  
V.J.F. Freitas ◽  
D.F. Salamone
Keyword(s):  
Dna Damage ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Dna Breaks ◽  
Embryo Production ◽  
Cell Stage ◽  
Γh2ax Foci

SummaryIn vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P < 0.05), no differences in the number of γH2AX foci or area were detected among the treatments. γH2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.

scholarly journals Whole-Genome DNA Methylation Dynamics of Sheep Preimplantation Embryo Investigated by Single-Cell DNA Methylome Sequencing

2021 ◽  
Vol 12 ◽  
Author(s):  
Zijing Zhang ◽  
Jiawei Xu ◽  
Shijie Lyu ◽  
Xiaoling Xin ◽  
Qiaoting Shi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Single Cell ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Preimplantation Embryo ◽  
Preimplantation Embryos ◽  
Whole Genome ◽  
Cell Stage ◽  
Preimplantation Embryo Development ◽  
Developmental Block

The early stages of mammalian embryonic development involve the participation and cooperation of numerous complex processes, including nutritional, genetic, and epigenetic mechanisms. However, in embryos cultured in vitro, a developmental block occurs that affects embryo development and the efficiency of culture. Although the block period is reported to involve the transcriptional repression of maternal genes and transcriptional activation of zygotic genes, how epigenetic factors regulate developmental block is still unclear. In this study, we systematically analyzed whole-genome methylation levels during five stages of sheep oocyte and preimplantation embryo development using single-cell level whole genome bisulphite sequencing (SC-WGBS) technology. Then, we examined several million CpG sites in individual cells at each evaluated developmental stage to identify the methylation changes that take place during the development of sheep preimplantation embryos. Our results showed that two strong waves of methylation changes occurred, namely, demethylation at the 8-cell to 16-cell stage and methylation at the 16-cell to 32-cell stage. Analysis of DNA methylation patterns in different functional regions revealed a stable hypermethylation status in 3′UTRs and gene bodies; however, significant differences were observed in intergenic and promoter regions at different developmental stages. Changes in methylation at different stages of preimplantation embryo development were also compared to investigate the molecular mechanisms involved in sheep embryo development at the methylation level. In conclusion, we report a detailed analysis of the DNA methylation dynamics during the development of sheep preimplantation embryos. Our results provide an explanation for the complex regulatory mechanisms underlying the embryo developmental block based on changes in DNA methylation levels.

Cell interactions in preimplantation embryos: evidence for involvement of saccharides of the poly- N-acetyllactosamine series

Development ◽  
1985 ◽  
Vol 87 (1) ◽  
pp. 115-128
Author(s):  
S. Rastan ◽  
S. J. Thorpe ◽  
P. Scudder ◽  
S. Brown ◽  
H. C. Gooi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Culture Medium ◽  
De Novo ◽  
Embryonic Cell ◽  
Preimplantation Embryos ◽  
Carbohydrate Binding ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Antigen Activity ◽  
Surface Carbohydrates

Roles of cell surface carbohydrates containing the 3-fucosyl-N-acetyllactosamine and poly-Nacetyllactosamine sequences (SSEA-1 and I antigens, respectively) in the compaction of mouse embryos have been investigated using the endo-β-galactosidase of Bacteroides fragilis to modify the surface of cleavage-stage embryos. Treatment with this enzyme abolished SSEA-1 activity and diminished I antigen activity on the embryonic cell surface. Embryos cultured in the presence of endo-β-galactosidase from the 2- to 4-cell stage onwards, or treated with the enzyme at the compacting 8-cell stage, continued to compact and proceeded to form blastocysts at the normal rate. However, when compacted 8- to 16-cell embryos were experimentally decompacted in calcium-free medium, treated for 1 h with endo-β-galactosidase and returned to normal culture medium, the time taken for 50 % of the embryos to recompact was prolonged five-fold. There was an even greater delay if these embryos were maintained in culture medium containing the enzyme. Blastocysts were eventually formed under both conditions. Thus, endo-β-galactosidase did not affect compaction unless the embryos were first decompacted. On the assumption that recompaction and de novo compaction occur by similar mechanisms, we propose that carbohydrate-binding molecules are involved which have high affinities for poly-Nacetyllactosamine structures and protect them from digestion by endo-β-galactosidase.

scholarly journals Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development

2019 ◽  
Author(s):  
Maya Sen ◽  
Dylan Mooijman ◽  
Jean-Charles Boisset ◽  
Alex Chialastri ◽  
Mina Popovic ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cost Effective ◽  
Dna Demethylation ◽  
Preimplantation Embryos ◽  
Mammalian Development ◽  
Cell Stage ◽  
Stage Of Development ◽  
Genome Wide

AbstractDNA methylation (5mC) is central to cellular identity and the global erasure of 5mC from the parental genomes during preimplantation mammalian development is critical to reset the methylome of terminally differentiated gametes to the pluripotent cells in the blastocyst. While active and passive modes of demethylation have both been suggested to play a role in this process, the relative contribution of these two mechanisms to genome-wide 5mC erasure remains unclear. Here, we report a new high-throughput single-cell method (scMspJI-seq) that enables strand-specific quantification of 5mC, thereby allowing us to systematically probe the dynamics of global demethylation. First, when applied to hybrid mouse embryonic stem cells, we identified substantial cell-to-cell strand-specific 5mC heterogeneity, with a small group of cells displaying asymmetric levels of 5mCpG between the two DNA strands of a chromosome suggesting loss of maintenance methylation. Next, using scMspJI-seq in preimplantation mouse embryos, we discovered that methylation maintenance is active till the 16-cell stage followed by passive demethylation in a fraction of cells within the early blastocyst at the 32-cell stage of development. Finally, we found that human preimplantation embryos qualitatively show temporally delayed yet similar demethylation dynamics as mouse preimplantation embryos. Collectively, these results demonstrate that scMspJI-seq is a sensitive and cost-effective method to map the strand-specific genome-wide patterns of 5mC in single cells, thereby enabling quantitative investigation of methylation dynamics in developmental systems.

Sign in / Sign up

Export Citation Format

Share Document