Transcription of rRNA in early mouse embryos promotes chromatin reorganization and expression of major satellite repeats.

During the first cell cycles of early development, the chromatin of the embryo is highly reprogrammed while the embryonic genome starts its own transcription. The spatial organization of the genome is an important process that contributes to regulating gene transcription in time and space. It has, however, been poorly studied in the context of early embryos. To study the cause-and-effect link between transcription and spatial organization in embryos, we focused on ribosomal genes, which are silent initially but start to be transcribed in 2-cell mouse embryos. We demonstrated that ribosomal sequences and early unprocessed rRNAs are spatially organized in a very particular manner between 2-cell and 16-cell stage. By using drugs that interfere with ribosomal DNA transcription, we showed that this organization - which is totally different in somatic cells - depends on an active transcription of ribosomal genes and induces a unique chromatin environment that favors transcription of major satellite sequences once the 4-cell stage has been reached.

INRA96 Supplemented With Phospholipids Liposomes, A Promising Approach for Stallion Sperm Chilling.

Among biotechnologies of reproduction in the equine species, artificial insemination remains the most used technology especially for cooled transported sperm. Although the use of INRA96 extender has demonstrated its efficiency for long-term sperm storage at 4°C or 15°C, some stallions ("bad coolers") are excluded from such technology. Some years ago, we demonstrated that liposomes produced from egg yolk (EY) phospholipids could be an alternative to egg yolk plasma in stallion freezing extenders. To develop a new extender for sperm chilling, we evaluated the protective effect of liposomes produced from EY phospholipids on stallion sperm storage at 4°C. The sperm of stallions from two studs was diluted in INRA96 extender (as control) or an experimental extender (EE) composed of INRA96 supplemented with liposomes of EY phospholipids. After 24H (D1), 72H (D3), and 6 days (D6) or 7 days (D7), motility parameters were evaluated using Computer Assisted Semen Analyzer. Our results demonstrated that total and progressive motility decreased significantly after dilution and storage in INRA96 between D1 and D3 (P < .05) while no significant decrease was observed between D1 and D3 with EE. Regarding VAP parameter, no significant difference was observed between extenders except at D7 in stud 2. Moreover, total and progressive motility were maintained at a significantly higher level (D3, D6, D7) when sperm was stored in EE compared to INRA96. These promising results demonstrate that the supplementation of INRA96 extender with egg-yolk phospholipids liposomes allows a higher protection to stallion sperm cells.

Heat shock during in vitro maturation induces chromatin modifications in the bovine embryo.

Heat stress compromises bovine oocyte developmental competence, but the effects of high temperature during oocyte maturation on embryo chromatin organization is unknown. In this study bovine oocytes were exposed to heat shock (41°C) for 12 h during in vitro maturation and then submitted to in vitro fertilization. The heat shock did not affect (P > 0.05) the cleavage but reduced (P < 0.01) the blastocyst rate on Day 7 and Day 8. No effect (P > 0.05) on total cell number was found, but the heat shock increased (P < 0.05) the proportion of apoptotic cells in blastocysts at Day 8. Immunofluorescence analysis of H3K9me3 and HP1 was performed in embryos at 52 h post in vitro fertilization. An accumulation of H3K9me3 in the nuclei of embryos derived from heat-shocked oocytes at four-cell and eight-cell stages was found. Also, a non-expected higher proportion (P < 0.05) of four-cell stage embryos displaying nuclei with increased HP1 fluorescence was observed, suggesting an abnormal chromatin compaction in embryos from heat-shocked oocytes. Embryos at eight-cell stage derived from heat-shocked oocytes displayed lower (P < 0.05) relative amount of HSP40 transcripts than control ones. In conclusion, heat shock before fertilization has an effect on embryo chromatin, influencing the accumulation of H3K9me3 and HP1 in early embryos as well as further development.

Three-dimensional analysis of nuclear heterochromatin distribution during early development in the rabbit.

Changes to the spatial organization of specific chromatin domains such as constitutive heterochromatin have been studied extensively in somatic cells. During early embryonic development, drastic epigenetic reprogramming of both the maternal and paternal genomes, followed by chromatin remodeling at the time of embryonic genome activation (EGA), have been observed in the mouse. Very few studies have been performed in other mammalian species (human, bovine, or rabbit) and the data are far from complete. During this work, we studied the three-dimensional organization of pericentromeric regions during the preimplantation period in the rabbit using specific techniques (3D-FISH) and tools (semi-automated image analysis). We observed that the pericentromeric regions (identified with specific probes for Rsat I and Rsat II genomic sequences) changed their shapes (from pearl necklaces to clusters), their nuclear localizations (from central to peripheral), as from the 4-cell stage. This reorganization goes along with histone modification changes and reduced amount of interactions with nucleolar precursor body surface. Altogether, our results suggest that the 4-cell stage may be a crucial window for events necessary before major EGA, which occurs during the 8-cell stage in the rabbit.

An Assessment of Fixed and Native Chromatin Preparation Methods to Study Histone Post-Translational Modifications at a Whole Genome Scale in Skeletal Muscle Tissue.

BACKGROUND: Genomic loci associated with histone marks are typically analyzed by immunoprecipitation of the chromatin followed by quantitative-PCR (ChIP-qPCR) or high throughput sequencing (ChIP-seq). Chromatin can be either cross-linked (X-ChIP) or used in the native state (N-ChIP). Cross-linking of DNA and proteins helps stabilizing their interactions before analysis. Despite X-ChIP is the most commonly used method, muscle tissue fixation is known to be relatively inefficient. Moreover, no protocol described a simple and reliable preparation of skeletal muscle chromatin of sufficient quality for subsequent high-throughput sequencing. Here we aimed to set-up and compare both chromatin preparation methods for a genome-wide analysis of H3K27me3, a broad-peak histone mark, using chicken P. major muscle tissue. RESULTS: Fixed and unfixed chromatin were prepared from chicken muscle tissues (Pectoralis major). Chromatin fixation, shearing by sonication or digestion and immunoprecipitation performed equivalently. High-quality Illumina reads were obtained (q30 > 93%). The bioinformatic analysis of the data was performed using epic, a tool based on SICER, and MACS2. Forty millions of reads were analyzed for both X-ChIP-seq and N-ChIP-seq experiments. Surprisingly, H3K27me3 X-ChIP-seq analysis led to the identification of only 2000 enriched regions compared to about 15,000 regions identified in the case of N-ChIP-seq. N-ChIP-seq peaks were more consistent between replicates compared to X-ChIP-seq. Higher N-ChIP-seq enrichments were confirmed by ChIP-qPCR at the PAX5 and SOX2 loci known to be enriched for H3K27me3 in myotubes and at the loci of common regions of enrichment identified in this study. CONCLUSIONS: Our findings suggest that the preparation of muscle chromatin for ChIP-seq in cross-linked conditions can compromise the systematic analysis of broad histone marks. Therefore, native chromatin preparation should be preferred to cross-linking when a ChIP experiment has to be performed on skeletal muscle tissue, particularly when a broad source signal is considered.

Sexual dimorphism of the feto-placental phenotype in response to a high fat and control maternal diets in a rabbit model.

Maternal environment during early developmental stages plays a seminal role in the establishment of adult phenotype. Using a rabbit model, we previously showed that feeding dams with a diet supplemented with 8% fat and 0.2% cholesterol (HH diet) from the prepubertal period and throughout gestation induced metabolic syndrome in adult offspring. Here, we examined the effects of the HH diet on feto-placental phenotype at 28 days post-coïtum (term = 31 days) in relation to earlier effects in the blastocyst (Day 6). At 28 days, both male and female HH fetuses were intrauterine growth retarded and dyslipidemic, with males more affected than females. Lipid droplets accumulated in the HH placentas' trophoblast, consistent with the increased concentrations in cholesteryl esters (3.2-fold), triacylglycerol (2.5-fold) and stored FA (2.12-fold). Stored FA concentrations were significantly higher in female compared to male HH placentas (2.18-fold, p<0.01), whereas triacylglycerol was increased only in HH males. Trophoblastic lipid droplet accumulation was also observed at the blastocyst stage. The expression of numerous genes involved in lipid pathways differed significantly according to diet both in term placenta and at the blastocyst stage. Among them, the expression of LXR-α in HH placentas was reduced in HH males but not females. These data demonstrate that maternal HH diet affects the blastocyst and induces sex-dependent metabolic adaptations in the placenta, which appears to protect female fetuses from developing severe dyslipidemia.

3D-FISH analysis of embryonic nuclei in mouse highlights several abrupt changes of nuclear organization during preimplantation development.

BACKGROUND: Embryonic development proceeds through finely tuned reprogramming of the parental genomes to form a totipotent embryo. Cells within this embryo will then differentiate and give rise to all the tissues of a new individual. Early embryonic development thus offers a particularly interesting system in which to analyze functional nuclear organization. When the organization of higher-order chromatin structures, such as pericentromeric heterochromatin, was first analyzed in mouse embryos, specific nuclear rearrangements were observed that correlated with embryonic genome activation at the 2-cell stage. However, most existing analyses have been conducted by visual observation of fluorescent images, in two dimensions or on z-stack sections/projections, but only rarely in three dimensions (3D). RESULTS: In the present study, we used DNA fluorescent in situ hybridization (FISH) to localize centromeric (minor satellites), pericentromeric (major satellites), and telomeric genomic sequences throughout the preimplantation period in naturally fertilized mouse embryos (from the 1-cell to blastocyst stage). Their distribution was then analyzed in 3D on confocal image stacks, focusing on the nucleolar precursor bodies and nucleoli known to evolve rapidly throughout the first developmental stages. We used computational imaging to quantify various nuclear parameters in the 3D-FISH images, to analyze the organization of compartments of interest, and to measure physical distances between these compartments. CONCLUSIONS: The results highlight differences in nuclear organization between the two parental inherited genomes at the 1-cell stage, i.e. just after fertilization. We also found that the reprogramming of the embryonic genome, which starts at the 2-cell stage, undergoes other remarkable changes during preimplantation development, particularly at the 4-cell stage.

Chromatin and epigenetic modifications during early mammalian development.

In mammals, the embryonic genome is transcriptionally inactive after fertilization and embryonic gene expression is initiated during the preimplantation developmental period, during so-called "embryonic genome activation (EGA)". EGA is dependent on the presence of the basal transcriptional machinery components but also on the parental genome reorganization after fertilization. Indeed, during the first cell cycles, the embryonic nuclei undergo intense remodelling that participates in the regulation of embryonic development. Among the mechanisms of this remodeling, it appears that modifications of epigenetic marks are essential especially at the time of embryonic genome activation. This review will focus on DNA methylation and histone modifications such as acetylation or methylation which are important to produce healthy embryos. We will also consider nuclear higher-order structures, such as chromosomes territories and pericentric heterochromatin clusters. The relevance of these chromatin epigenetic modifications has been sustained by the work performed on cloned embryos produced through nuclear transfer of somatic donor cells. It is indeed believed that incomplete reprogramming of the somatic nucleus, in other words, the incomplete re-establishment of the embryonic epigenetic patterns and peculiar nuclear organization may be among the causes of development failure of cloned animals. This will also be discussed in this review.

Nuclear dynamics of histone H3 trimethylated on lysine 9 and/or phosphorylated on serine 10 in mouse cloned embryos as new markers of reprogramming?

Somatic cell nuclear transfer (SCNT) is the injection of a donor nucleus into an enucleated egg. Despite the use of this technology for many years in research, it is still quite inefficient. One of the causes for this is thought to be incorrect or incomplete genome reprogramming. Embryos produced by nuclear transfer (cloned embryos) very often present abnormal epigenetic signatures and irregular chromatin reorganization. Of these two issues, the issue of chromatin rearrangements within the nuclei after transfer is the least studied. It is known that cloned embryos often present pericentromeric heterochromatin clumps very similar to the chromocenters structures present in the donor nuclei. Therefore, it is believed that the somatic nuclear configuration of donor nuclei, especially that of the chromocenters, is not completely lost after nuclear transfer, in other words, not well reprogrammed. To further investigate pericentromeric heterochromatin reorganization after nuclear transfer, we decided to study its rearrangements in cumulus-derived clones using several related epigenetic markers such as H3S10P, H3K9me3, and the double marker H3K9me3S10P. We observed that two of these markers, H3S10P and H3K9me3S10P, are the ones found on the part of the pericentromeric heterochromatin that is remodeled correctly, resembling exactly the embryonic heterochromatin configuration of naturally fertilized embryos. Conversely, H3K9me3 and heterochromatin protein 1 beta (HP1ß)-associated protein were also detected in the perinuclear clumps of heterochromatin, making obvious the maintenance of the somatic epigenetic signature within these nuclear regions. Our results demonstrate that H3S10P and H3K9me3S10P could be good candidates for evaluating heterochromatin reorganization following nuclear reprogramming.

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. CONCLUSIONS/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.

H3S10 phosphorylation marks constitutive heterochromatin during interphase in early mouse embryos until the 4-cell stage.

Phosphorylation of histone H3 at Ser10 (H3S10P) has been linked to a variety of cellular processes, such as chromosome condensation and gene activation/silencing. Remarkably, in mammalian somatic cells, H3S10P initiates in the pericentromeric heterochromatin during the late G2 phase, and phosphorylation spreads throughout the chromosomes arms in prophase, being maintained until the onset of anaphase when it gets dephosphorylated. Considerable studies have been carried out about H3S10P in different organisms; however, there is little information about this histone modification in mammalian embryos. We hypothesized that this epigenetic modification could also be a marker of pericentromeric heterochromatin in preimplantation embryos. We therefore followed the H3S10P distribution pattern in the G1/S and G2 phases through the entire preimplantation development in in vivo mouse embryos. We paid special attention to its localization relative to another pericentromeric heterochromatin marker, HP1ß and performed immunoFISH using specific pericentromeric heterochromatin probes. Our results indicate that H3S10P presents a remarkable distribution pattern in preimplantation mouse embryos until the 4-cell stage and is a better marker of pericentromeric heterochromatin than HP1ß. After the 8-cell stage, H3S10P kinetic is more similar to the somatic one, initiating during G2 in chromocenters and disappearing upon telophase. Based on these findings, we believe that H3S10P is a good marker of pericentromeric heterochromatin, especially in the late 1- and 2-cell stages as it labels both parental genomes and that it can be used to further investigate epigenetic regulation and heterochromatin mechanisms in early preimplantation embryos.

Three-dimensional fluorescence in situ hybridization in mouse embryos using repetitive probe sequences.

A common problem in research laboratories that study the mammalian embryo is the limited supply of live material. For this reason, new methods are constantly being developed and existing methods for in vitro models using cells in culture are being adapted to represent embryogenesis. Three-dimensional fluorescence in situ hybridization (3D-FISH) is an important tool to study where genomic sequences are positioned within nuclei without interfering with this 3D organization. When used in the embryo, this technique provides vital information about the distribution of specific sequences in relation to embryonic nuclear substructures such as nucleolar precursor bodies and chromocenters. In this chapter, we will present a detailed description of FISH in order to perform 3D-FISH in the early preimplantation murine embryos.