Ablation of ZC3H11A causes early embryonic lethality and dysregulation of metabolic processes.

ZC3H11A (zinc finger CCCH domain-containing protein 11A) is a stress-induced mRNA-binding protein required for efficient growth of nuclear-replicating viruses. The cellular functions of ZC3H11A during embryonic development are unknown. Here, we report the generation and phenotypic characterization of Zc3h11a knockout (KO) mice. Heterozygous null Zc3h11a mice were born at the expected frequency without distinguishable phenotypic differences compared with wild-type mice. In contrast, homozygous null Zc3h11a mice were missing, indicating that Zc3h11a is crucial for embryonic viability and survival. Zc3h11a -/- embryos were detected at the expected Mendelian ratios up to late preimplantation stage (E4.5). However, phenotypic characterization at E6.5 revealed degeneration of Zc3h11a -/- embryos, indicating developmental defects around the time of implantation. Transcriptomic analyses documented a dysregulation of glycolysis and fatty acid metabolic pathways in Zc3h11a-/- embryos at E4.5. Proteomic analysis indicated a tight interaction between ZC3H11A and mRNA-export proteins in embryonic stem cells. CLIP-seq analysis demonstrated that ZC3H11A binds a subset of mRNA transcripts that are critical for metabolic regulation of embryonic cells. Furthermore, embryonic stem cells with an induced deletion of Zc3h11a display an impaired differentiation toward epiblast-like cells and impaired mitochondrial membrane potential. Altogether, the results show that ZC3H11A is participating in export and posttranscriptional regulation of selected mRNA transcripts required to maintain metabolic processes in embryonic cells. While ZC3H11A is essential for the viability of the early mouse embryo, inactivation of Zc3h11a expression in adult tissues using a conditional KO did not lead to obvious phenotypic defects.

Pressurized intra-peritoneal aerosol chemotherapy (PIPAC): increased intraperitoneal pressure does not affect distribution patterns but leads to deeper penetration depth of doxorubicin in a sheep model.

BACKGROUND: Pressurized Intra-Peritoneal Aerosol Chemotherapy (PIPAC) is an innovative treatment against peritoneal carcinomatosis. Doxorubicin is a common intra-venous chemotherapy used for peritoneal carcinomatosis and for PIPAC. This study evaluated the impact of increased PIPAC intraperitoneal pressure on the distribution and cell penetration of doxorubicin in a sheep model. METHODS: Doxorubicin was aerosolized using PIPAC into the peritoneal cavity of 6 ewes (pre-alpes breed): N = 3 with 12 mmHg intraperitoneal pressure ("group 12") and N = 3 with 20 mmHg ("group 20"). Samples from peritoneum (N = 6), ovarian (N = 1), omentum (N = 1) and caecum (N = 1) were collected for each ewe. The number of doxorubicin positive cells was determined using the ratio between doxorubicine fluorescence-positive cell nuclei (DOXO+) over total number of DAPI positive cell nuclei (DAPI+). Penetration depth (µm) was defined as the distance between the luminal surface and the location of the deepest DOXO+ nuclei over the total number of cell nuclei that were stained with DAPI. Penetration depth (µm) was defined as the distance between the luminal surface and the location of the deepest DOXO+ nuclei. RESULTS: DOXO+ nuclei were identified in 87% of samples. All omental samples, directly localized in front of the nebulizer head, had 100% DOXO+ nuclei whereas very few nuclei were DOXO+ for caecum. Distribution patterns were not different between the two groups but penetration depth in ovary and caecum samples was significantly deeper in group 20. CONCLUSIONS: This study showed that applying a higher intra-peritoneal pressure during PIPAC treatment leads to a deeper penetration of doxorubicin in ovarian and caecum but does not affect distribution patterns.

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.

Turning off NADPH oxidase-2 by impeding p67phox activation in infected mouse macrophages reduced viral entry and inflammation.

BACKGROUND: Targeting cells of the host immune system is a promising approach to fight against Influenza A virus (IAV) infection. Macrophage cells use the NADPH oxidase-2 (NOX2) enzymatic complex as a first line of defense against pathogens by generating superoxide ions O2- and releasing H2O2. Herein, we investigated whether targeting membrane -embedded NOX2 decreased IAV entry via raft domains and reduced inflammation in infected macrophages. METHODS: Confocal microscopy and western blots monitored levels of the viral nucleoprotein NP and p67phox, NOX2 activator subunit, Elisa assays quantified TNF-α levels in LPS or IAV-activated mouse or porcine alveolar macrophages pretreated with a fluorescent NOX inhibitor, called nanoshutter NS1. RESULTS: IAV infection in macrophages promoted p67phox translocation to the membrane, rafts clustering and activation of the NOX2 complex at early times. Disrupting rafts reduced intracellular viral NP. NS1 markedly reduced raft clustering and viral entry by binding to the C-terminal of NOX2 also characterized in vitro. NS1 decrease of TNF-α release depended on the cell type. CONCLUSION: NOX2 participated in IAV entry and raft-mediated endocytosis. NOX2 inhibition by NS1 reduced viral entry. NS1 competition with p67phox for NOX2 binding shown by in silico models and cell-free assays was in agreement with NS1 inhibiting p67phox translocation to membrane-embedded NOX2 in mouse and porcine macrophages. GENERAL SIGNIFICANCE: We introduce NS1 as a compound targeting NOX2, a critical enzyme controlling viral levels and inflammation in macrophages and discuss the therapeutic relevance of targeting the C-terminal of NADPH oxidases by probes like NS1 in viral infections.

Prosurvival effect of cumulus prostaglandin G/H synthase 2/prostaglandin2 signaling on bovine blastocyst: impact on in vivo posthatching development.

Apoptotic activity is a common physiological process which culminates at the blastocyst stage in the preimplantation embryo of many mammals. The degree of embryonic cell death can be influenced by the oocyte microenvironment. However, the prognostic significance of the incidence of apoptosis remains undefined. Prostaglandin E2 (PGE2) derived from prostaglandin G/H synthase-2 (PTGS2) activity is a well-known prosurvival factor that is mainly studied in oncology. PGE2 is the predominant PTGS2-derived prostaglandin present in the oocyte microenvironment during the periconceptional period. Using an in vitro model of bovine embryo production followed by transfer and collection procedures, we investigated the impact of periconceptional PGE2 on the occurrence of spontaneous apoptosis in embryos and on subsequent in vivo posthatching development. Different periconceptional PGE2 environments were obtained using NS-398, a specific inhibitor of PTGS2 activity, and exogenous PGE2. We assessed the level of embryonic cell death in blastocysts at day 8 postfertilization by counting total cell numbers, by the immunohistochemical staining of active caspase-3, and by quantifying terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling signals and apoptosis regulator (BCL-2/BAX) mRNA expression. Morphometric parameters were used to estimate the developmental stage of the embryonic disk and the extent of trophoblast elongation on day 15 conceptuses. Our findings indicate that periconceptional PGE2 signaling durably impacts oocytes, conferring increased resistance to spontaneous apoptosis in blastocysts and promoting embryonic disk development and the elongation process during preimplantation development.

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.

The perilipin-2 (adipophilin) coat of cytosolic lipid droplets is regulated by an Arf1-dependent mechanism in HC11 mouse mammary epithelial cells.

The cytosolic lipid droplets (cLDs) store excess intracellular lipids, and perilipin-2 is believed to protect cLDs from degradation. Here, we investigated the role of the small G-protein Arf1 and the proteasome in the fates of perilipin-2 and cLDs. In oleate-loaded cells, upon brefeldin A (BFA) treatment, perilipin-2 remained associated with cLDs for at least 30 min before significant release, and proteasomal degradation-mediated decrease was observed. Interestingly, the cLD population did not mimic the decline in perilipin-2. We tested several chemical modulators of regulators of Arf1 activity on the association of perilipin-2 with cLDs. QS11 and Exo2 accelerated the reduction in perilipin-2, although less than BFA. In contrast, Exo1 unexpectedly slowed down its degradation. Correlatively, BFA, QS11, and Exo2 enhanced the dissociation of perilipin-2 from cLDs, whereas Exo1 inhibited it. There was a synergistic effect of BFA with Exo2 and QS11, and of Exo2 with QS11, whereas Exo1 antagonized the effect of BFA without affecting that of Exo2 or QS11. We concluded that the Arf1 complex regulates the association of perilipin-2 with cLDs. Additionally, MG132 and BFA modified the number of cLDs over a relatively short period.

Heterochromatin reprogramming in rabbit embryos after fertilization, intra-, and inter-species SCNT correlates with preimplantation development.

To investigate the embryonic genome organization upon fertilization and somatic cell nuclear transfer (SCNT), we tracked HP1ß and CENP, two well-characterized protein markers of pericentric and centromeric compartments respectively, in four types of embryos produced by rabbit in vivo fertilization, rabbit parthenogenesis, rabbit-to-rabbit, and bovine-to-rabbit SCNT. In the interphase nuclei of rabbit cultured fibroblasts, centromeres and associated pericentric heterochromatin are usually isolated. Clustering into higher-order chromatin structures, such as the chromocenters seen in mouse and bovine somatic cells, could not be observed in rabbit fibroblasts. After fertilization, centromeres and associated pericentric heterochromatin are quite dispersed in rabbit embryos. The somatic-like organization is progressively established and completed only by the 8/16-cell stage, a stage that corresponds to major embryonic genome activation in this species. In SCNT embryos, pericentric heterochromatin distribution typical for rabbit and bovine somatic cells was incompletely reverted into the 1-cell embryonic form with remnants of heterochromatin clusters in 100% of bovine-to-rabbit embryos. Subsequently, the donor cell nuclear organization was rapidly re-established by the 4-cell stage. Remarkably, the incomplete remodeling of bovine-to-rabbit 1-cell embryos was associated with delayed transcriptional activation compared with rabbit-to-rabbit embryos. Together, the results confirm that pericentric heterochromatin spatio-temporal reorganization is an important step of embryonic genome reprogramming. It also appears that genome reorganization in SCNT embryos is mainly dependent on the nuclear characteristics of the donor cells, not on the recipient cytoplasm.

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.

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.

Reliability of Hoechst 33342 staining under wide-field microscopy for evaluation of the nuclear status of living dog oocytes.

Due to the marked cytoplasmic opacity of canine oocytes, the diagnosis of their nuclear status is difficult. The objective of the present study was to evaluate the accuracy of Hoechst staining observed under epifluorescence wide-field microscopy [living oocyte observation (LivOO)] by comparison to a reference technique [DNA staining with ethidium homodimer-2 under confocal microscopy; fixed oocyte observation (FixOO)]. Four Hoechst 33342 concentrations (200 ng, 500 ng, 1 µg, 2 µg/mL) were tested and 1 µg/mL was the lowest one with the lowest proportion of oocytes in which DNA was missed. At this concentration, LivOO procedure did not affect the degeneration rate. On 379 oocytes observed individually with the two techniques successively, diagnosis of meiosis resumption by LivOO was exact in 87.3% of the cases, but the meiosis resumption rate was underestimated (23.5% versus 34.3% with FixOO; p < 0.001). Diagnosis for metaphase II was exact in 80% of the cases, but LivOO detected only 72.7% of the metaphase II oocytes present. Metaphase rates did not differ between LivOO and FixOO. This study contributes to a better interpretation of in vitro maturation results. The developmental potential of metaphase II canine oocytes sorted after Hoechst staining is to be evaluated.

PTGS2-related PGE2 affects oocyte MAPK phosphorylation and meiosis progression in cattle: late effects on early embryonic development.

During the periovulatory period, the induction of prostaglandin G/H synthase-2 (PTGS2) expression in cumulus cells and associated prostaglandin E2 (PGE2) production are implicated in the terminal differentiation of the cumulus-oocyte complex. During the present study, the effects of the PTGS2/PGE2 pathway on the developmental competence of bovine oocytes were investigated using an in vitro model of maturation, fertilization, and early embryonic development. The specific inhibition of PTGS2 activity with NS-398 during in vitro maturation (IVM) significantly restricted mitogen-activated protein kinase (MAPK) activation in oocytes at the germinal vesicle breakdown stage and reduced both cumulus expansion and the maturation rate after 22 h of culture. In addition, significantly higher rates of abnormal meiotic spindle organization were observed after 26 h of culture. Periconceptional PTGS2 inhibition did not affect fertilization but significantly reduced the speed of embryo development. Embryo output rates were significantly decreased on Day 6 postfertilization but not on Day 7. However, total blastomere number was significantly lower in embryos obtained after PTGS2 inhibition. The addition of PGE2 to IVM and in vitro fertilization cultures containing NS-398 overrode oocyte maturation and early embryonic developmental defects. Protein and mRNA expression for the prostaglandin E receptor PTGER2 were found in oocytes, whereas the PTGER2, PTGER3, and PTGER4 subtypes were expressed in cumulus cells. This study is the first to report the involvement of PGE2 in oocyte MAPK activation during the maturation process. Taken together, these results indicate that PGE2-mediated interactions between somatic and germ cells during the periconceptional period promote both in vitro oocyte maturation and preimplantation embryonic development in cattle.

Statistical analysis of 3D images detects regular spatial distributions of centromeres and chromocenters in animal and plant nuclei.

In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear "compartments". Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types.

PGE2 Supplementation of Oocyte Culture Media Improves the Developmental and Cryotolerance Performance of Bovine Blastocysts Derived From a Serum-Free in vitro Production System, Mirroring the Inner Cell Mass Transcriptome.

The culture media used throughout the in vitro production (IVP) of bovine embryos remain complex. The serum added to culture media in order to improve embryo development negatively impacts the cryotolerance of blastocysts. Periconceptional prostaglandin E2 (PGE2) signaling is known to exert prosurvival effects on in vitro-generated blastocysts. The purpose of the present study was to evaluate the effects on developmental and cryotolerance performance of a serum-free (SF) IVP system that included defined oocyte culture media supplemented or not with PGE2, versus serum-containing (SC) IVP. RNA-sequencing analysis was used to examine the gene expression of ICM derived under the different IVP conditions. We assessed the degree of cryotolerance of grade-I blastocysts during a three-day post-thaw culture by measuring survival and hatching rates, counting trophectoderm and inner cell mass (ICM) blastomere numbers. We also determined the proportion of ICM cells expressing octamer-binding transcription factor 4 protein (OCT4/POU5F1). We showed that grade-I blastocyst development rates under SF + PGE2 conditions were similar to those obtained under SC conditions, although the cleavage rate remained significantly lower. SC IVP conditions induced changes to ICM gene expression relative to several metabolic processes, catabolic activities, cell death and apoptosis. These alterations were associated with significantly higher levels of ICM cell death at day 7 post-fertilization, and lower survival and hatching rates after thawing. SF IVP conditions supplemented or not with PGE2 induced changes to ICM gene expression related to DNA replication, metabolism and double-strand break repair processes, and were associated with significantly larger ICM cell populations after thawing. SF + PGE2 IVP induced changes to ICM gene expression related to epigenetic regulation and were associated with a significantly higher proportion of ICM cells expressing OCT4. For the first time, our study thus offers a comprehensive analysis of the ICM transcriptome regulated by IVP culture conditions in terms of the cellular changes revealed during culture for three days after thawing.

Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos.

Efficient reprograming of the donor cell genome in nuclear transfer (NT) embryos is linked to the ability of the embryos to sustain full-term development. As the nuclear architecture has recently emerged as a key factor in the regulation of gene expression, we questioned whether early bovine embryos obtained from transfer of cultured fibroblasts into enucleated oocytes would adopt an embryo-like nuclear organization. We studied the dynamics of constitutive heterochromatin in the stages prior to embryonic genome activation by distribution analysis of heterochromatin protein CBX1 (HP1), centromeric proteins CENPA and CENPB, and histone H3 three-methylated at lysine 9. Then we applied descriptive, quantitative, and co-localization analyses. A dramatic reorganization of heterochromatic blocks of somatic donor cells was first observed in the late one-cell stage NT embryos. Then at two- and four-cell stages, we found two types of NT embryos: one displaying noncondensed heterochromatin patches similar to IVF embryos, whereas the second type displayed condensed heterochromatin blocks, normally observed in IVF embryos only after the eight-cell stage. These analyses discriminate for the first time two contrasted types of nuclear organization in NT embryos, which may correspond to different functional states of the nuclei. The relationship with the somatic nucleus reprograming efficiency is discussed.

The nuclear localization of WAP and CSN genes is modified by lactogenic hormones in HC11 cells.

Whey acidic protein (WAP) and casein (CSN) genes are among the most highly expressed milk protein genes in the mammary gland of the lactating mouse. Their tissue-specific regulation depends on the activation and recruitment of transcription factors, and chromatin modifications in response to hormonal stimulation. We have investigated if another mechanism, such as specific positioning of the genes in the nucleus, could be involved in their functional regulation. Fluorescent in situ hybridization was used to study the nuclear localization of WAP and CSN genes in mouse mammary epithelial cells (HC11) cultured in the absence and presence of lactogenic hormones. Automatic 3D image processing and analysis tools were developed to score gene positions. In the absence of lactogenic hormones, both genes are distributed non-uniformly within the nucleus: the CSN locus was located close to the nuclear periphery and the WAP gene tended to be central. Stimulation by lactogenic hormones induced a statistically significant change to their distance from the periphery, which has been described as a repressive compartment. The detection of genes in combination with the corresponding chromosome-specific probe revealed that the CSN locus is relocated outside its chromosome territory following hormonal stimulation, whereas the WAP gene, which is already sited more frequently outside its chromosome territory in the absence of hormones, is not affected. We conclude that milk protein genes are subject to nuclear repositioning when activated, in agreement with a role for nuclear architecture in gene regulation, but that they behave differently as a function of their chromosomal context.

Contrasting epigenetic states of heterochromatin in the different types of mouse pluripotent stem cells.

Mouse embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) represent naive and primed pluripotency states, respectively, and are maintained in vitro by specific signalling pathways. Furthermore, ESCs cultured in serum-free medium with two kinase inhibitors (2i-ESCs) are thought to be the ground naïve pluripotent state. Here, we present a comparative study of the epigenetic and transcriptional states of pericentromeric heterochromatin satellite sequences found in these pluripotent states. We show that 2i-ESCs are distinguished from other pluripotent cells by a prominent enrichment in H3K27me3 and low levels of DNA methylation at pericentromeric heterochromatin. In contrast, serum-containing ESCs exhibit higher levels of major satellite repeat transcription, which is lower in 2i-ESCs and even more repressed in primed EpiSCs. Removal of either DNA methylation or H3K9me3 at PCH in 2i-ESCs leads to enhanced deposition of H3K27me3 with few changes in satellite transcript levels. In contrast, their removal in EpiSCs does not lead to deposition of H3K27me3 but rather removes transcriptional repression. Altogether, our data show that the epigenetic state of PCH is modified during transition from naive to primed pluripotency states towards a more repressive state, which tightly represses the transcription of satellite repeats.

Cloned rabbits produced by nuclear transfer from adult somatic cells.

We have developed a method to produce live somatic clones in the rabbit, one of the mammalian species considered up to now as difficult to clone. To do so, we have modified current cloning protocols proven successful in other species by taking into account both the rapid kinetics of the cell cycle of rabbit embryos and the narrow window of time for their implantation after transfer into foster recipients. Although our method still has a low level of efficiency, it has produced several clones now proven to be fertile. Our work indicates that cloning can probably be carried out successfully in any mammalian species by taking into account physiological features of their oocytes and embryos. Our results will contribute to extending the use of rabbit models for biomedical research.

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.

Dynamics of DNA methylation levels in maternal and paternal rabbit genomes after fertilization.

The reprogramming of DNA methylation in early embryos has been considered to be essential for the reprogramming of differentiated parental genomes to totipotency, the transcription of embryonic genome activation (EGA) and subsequent development. However, its degree appears to differ as a function of species and it may be altered by the in vitro environment. While the rabbit is a pertinent model for species with a delayed EGA because both in vivo and in vitro developed embryos are easily available, the status of DNA methylation levels in both parental genomes after fertilization remains controversial. In order to generate precise data on the DNA methylation status in rabbit zygotes, we first of all defined five pronuclear (PN) stages during the first cell cycle and then classified in vivo and in vitro developed rabbit zygotes according to these PN stages. Using this classification we precisely quantified both methylated DNA and the total DNA content during the one cell stage. The quantification of methylated DNA, normalized for the total DNA content, showed that both pronuclei displayed distinct patterns of DNA methylation reprogramming. In the maternal pronucleus (MP) the methylation level remained constant throughout the one cell stage, thanks to maintenance methylation during the S phase. Conversely, in the paternal pronucleus (PP) partial demethylation occurred before replication, probably as a result of active DNA demethylation, while maintenance methylation subsequently occurred during the S phase. Interestingly, we showed that PP DNA methylation reprogramming was partially altered by the in vitro environment. Taken together, our approach evidenced that rabbit is one of the species displaying partial DNA demethylation in the PP, and for the first time demonstrated maintenance methylation activity in both pronuclei during the first S phase.