Histone post-translational modifications in preimplantation mouse embryos and their role in nuclear architecture

In mammals, epigenetic markers are globally rearranged after fertilization: while gametes carry special epigenetic signatures and a unique nuclear organization, they attain embryo-specific patterns after fertilization. This “reprogramming” is promoted by the intimate contact between the parental inherited genomes and the oocyte cytoplasm over the first cell cycles of development. Interestingly, histone post-translational modifications (PTMs) are among the factors involved in this reprogramming.During the last few years, many studies focusing on epigenetic modifications have indeed shown that, immediately after fertilization, different histone PTM profiles create an asymmetry between the two parental genomes, although both parental genomes undergo global hyperacetylation and hypomethylation. Thereafter, histone PTMs reprogramming goes on (Beaujean et al., MRD 2014). It is hypothesized that this PTMs reprogramming is required for the embryonic genome activation (EGA). Recently, we for example put forward the importance of the PRC1 complex that binds H3K27me3, for proper EGA and development beyond the two-cell stage (Posfai et al., 2012). By the stage of implantation (blastocyst stage) two cell subpopulations forms: an outer layer of epithelial trophectoderm cells (TE) and the inner cell mass (ICM) located eccentrically within the blastocoelic cavity. Remarkably, some histone PTMs have been found to differ between the ICM vs. TE and to correlate with specific gene expression in each of these cell types (Dahl et al., 2010; Vermilyea et al., 2009).On the other hand, it is well known that diverse parts of the genome have different types of chromatin configuration depending on their function (centromeric and telomeric heterochromatin for instance). Interestingly, the mouse embryo presents a unique organization of the peri-centromeric heterochromatin that locates around the nucleoli. This configuration is rapidly acquired in the maternal pronucleus and more progressively in the paternal one (Martin et al., 2006; Aguirre-Lavin et al., 2012), probably due to the specific epigenetic marks present only in the paternal chromatin. During the 2-cell stage, dissociation of pericentromeric heterochromatin from nucleoli begins, concomitantly with the major phase of embryonic genome activation, although the importance of this remodeling is not yet well understood. Remarkably, it however seems that transcripts generated by pericentromeric satellite repeats are involved in this event and that interference with this phenomenon results in developmental arrest (Probst et al., 2010; Santenard et al., 2010; Fulka & Langerova 2014).Altogether, it suggests that histone PTMs may be closely correlated with the formation of a transcriptionally active or repressive state during early embryonic development and that they can modify chromatin organization and nuclear architecture during mouse embryonic development. It should also be mentioned that knock-outs of several histone modification enzymes have underlined the importance of PTMs during preimplantation development

Genome restructuring in mouse embryos during reprogramming and early development

AbstractAlthough a growing number of studies investigates functional genome organization in somatic cell nuclei, it is largely unknown how mammalian genome organization is established during embryogenesis. To address this question, we investigated chromocenter formation and the peculiar arrangements of chromosome domains in early mouse embryos. At the one-cell stage, we observed characteristic arrangements of chromosomes and chromocenter components. Subsequently, starting with the burst of zygotic genome transcription major rearrangements led to the establishment of somatic type chromocenters with a defined spatio-temporal organization. These processes appeared to be completed at the blastocyst stage with the onset of cell differentiation. During the same developmental period, a fraction of pericentric heterochromatin that was late replicating in the first cycle underwent switches in replication timing, spatial organization and epigenetic marks. Cloning experiments revealed that the genome organization typical for more advanced stages was quickly reverted into the one-cell stage-specific form after nuclear transfer, supporting the idea that reprogramming associated genome remodeling in normal and cloned embryos is determined by cytoplasmic factors. Together, the results suggest that distinct but characteristic forms of nuclear genome organization are required for genome reprogramming in early embryos and for proper regulation of differential gene expression patterns at later stages

Trichostatin A treatment of cloned mouse embryos improves constitutive heterochromatin remodeling as well as developmental potential to term

<p>Abstract</p> <p>Background</p> <p>Genome reprogramming in early mouse embryos is associated with nuclear reorganization and particular features such as the peculiar distribution of centromeric and pericentric heterochromatin during the first developmental stage. This zygote-specific heterochromatin organization could be observed both in maternal and paternal pronuclei after natural fertilization as well as in embryonic stem (ES) cell nuclei after nuclear transfer suggesting that this particular type of nuclear organization was essential for embryonic reprogramming and subsequent development.</p> <p>Results</p> <p>Here, we show that remodeling into a zygotic-like organization also occurs after somatic cell nuclear transfer (SCNT), supporting the hypothesis that reorganization of constitutive heterochromatin occurs regardless of the source and differentiation state of the starting material. However, abnormal nuclear remodeling was frequently observed after SCNT, in association with low developmental efficiency. When transient treatment with the histone deacetylase inhibitor trichostatin A (TSA) was tested, we observed improved nuclear remodeling in 1-cell SCNT embryos that correlated with improved rates of embryonic development at subsequent stages.</p> <p>Conclusion</p> <p>Together, the results suggest that proper organization of constitutive heterochromatin in early embryos is involved in the initial developmental steps and might have long term consequences, especially in cloning procedures.</p

Early epigenetic reprogramming in fertilized, cloned, and parthenogenetic embryos

Despite ongoing research in a number of species, the efficiency of embryo production by nuclear transfer remains low. Incomplete epigenetic reprogramming of the nucleus introduced in the recipient oocyte is one factor proposed to limit the success of this technique. Nonetheless, knowledge of reprogramming factors has increased—thanks to comparative studies on reprogramming of the paternal genome brought by sperm on fertilization—and will be reviewed here. Another valuable model of reprogramming is the one obtained in the absence of sperm fertilization through artificial activation—the parthenote—and will also be introduced. Altogether the objective of this review is to have a better understanding on the mechanisms responsible for the resistance to reprogramming, not only because it could improve embryonic development but also as it could benefit therapeutic reprogramming research

Random allocation of blastomere descendants to the trophectoderm and ICM of the bovine blastocyst

The first lineage specification during mammalian embryo development can be visually distinguished at the blastocyst stage. Two cell lineages are observed on the embryonic-abembryonic axis of the blastocyst: the inner cell mass and the trophectoderm. The timing and mechanisms driving this process are still not fully understood. In mouse embryos, cells seem prepatterned to become certain cell lineage because the first cleavage plane has been related with further embryonic-abembryonic axis at the blastocyst stage. Nevertheless, this possibility has been very debatable. Our objective was to determine whether this would be the case in another mammalian species, the bovine. To achieve this, cells of in vitro produced bovine embryos were traced from the 2-cell stage to the blastocyst stage. Blastocysts were then classified according to the allocation of the labeled cells in the embryonic and/or abembryonic part of the blastocyst. Surprisingly, we found that there is a significant percentage of the embryos (∼60%) with labeled and nonlabeled cells randomly distributed and intermingled. Using time-lapse microscopy, we have identified the emergence of this random pattern at the third to fourth cell cycle, when cells started to intermingle. Even though no differences were found on morphokinetics among different embryos, these random blastocysts and those with labeled cells separated by the embryonic-abembryonic axis (deviant pattern) are significantly bigger; moreover deviant embryos have a significantly higher number of cells. Interestingly, we observed that daughter cells allocation at the blastocyst stage is not affected by biopsies performed at an earlier stage

Early patterning of cloned mouse embryos contributes to post-implantation development

AbstractSeveral research groups have suggested that the embryonic–abembryonic (Em–Ab) axis in the mouse can be predicted by the first cleavage plane of the early embryo. Currently, it is not known whether this early patterning occurs in cloned embryos produced by nuclear transfer and whether it affects development to term. In this work, the relationship between the first cleavage plane and the Em–Ab axis was determined by the labeling of one blastomere in cloned mouse embryos at the 2-cell stage, followed by ex-vivo tracking until the blastocyst stage. The results demonstrate that approximately half of the cloned blastocysts had an Em–Ab axis perpendicular to the initial cleavage plane of the 2-cell stage. These embryos were classified as “orthogonal” and the remainder as “deviant”. Additionally, we report here that cloned embryos were significantly more often orthogonal than their naturally fertilized counterparts and overexpressed Sox2. Orthogonal cloned embryos demonstrated a higher rate of post-implantation embryonic development than deviant embryos, but cloned pups did not all survive. These results reveal that the angular relationship between the Em–Ab axis and the first cleavage plane can influence later development and they support the hypothesis that proper early patterning of mammalian embryos is required after nuclear transfer

5-Methylcytosine and 5-Hydroxymethylcytosine Spatiotemporal Profiles in the Mouse Zygote

Background: In the mouse zygote, DNA methylation patterns are heavily modified, and differ between the maternal and paternal pronucleus. Demethylation of the paternal genome has been described as an active and replication-independent process, although the mechanisms responsible for it remain elusive. Recently, 5-hydroxymethylcytosine has been suggested as an intermediate in this demethylation. Methodology/principal findings: In this study, we quantified DNA methylation and hydroxymethylation in both pronuclei of the mouse zygote during the replication period and we examined their patterns on the pericentric heterochromatin using 3D immuno-FISH. Our results demonstrate that 5-methylcytosine and 5-hydroxymethylcytosine localizations on the pericentric sequences are not complementary; indeed we observe no enrichment of either marks on some regions and an enrichment of both on others. In addition, we show that DNA demethylation continues during DNA replication, and is inhibited by aphidicolin. Finally, we observe notable differences in the kinetics of demethylation and hydroxymethylation; in particular, a peak of 5-hydroxymethylcytosine, unrelated to any change in 5-methylcytosine level, is observed after completion of replication. Conclusion/significance: Together our results support the already proposed hypothesis that 5-hydroxymethylcytosine is not a simple intermediate in an active demethylation process and could play a role of its own during early development

Ring-Like Distribution of Constitutive Heterochromatin in Bovine Senescent Cells

Background: Cells that reach ‘‘Hayflick limit’ ’ of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining. Methods: We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH). Results: Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli

Deciphering pluripotency in rabbit embryos by single-cell RNA sequencing

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