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

Three-dimensional immunofluorescence in situ hybridization in preimplantation mouse embryos

It is now well established that three-dimensional organization of chromatin in the nucleus plays a vital role in regulating the genome. In this context, three-dimensional fluorescence in situ hybridization (3D FISH) has become a major technique used to visualize the location of DNA sequences in the nuclei and several variations of the technique have been published. In this article we describe a protocol which has been optimized for embryos to suit this peculiar experimental model without altering the 3D shape. Moreover, we will describe how to carry immuno-3D FISH to simultaneously check the localization of proteins and DNA sequences in embryos. The protocol is applicable to all preimplantation stages with several probes, thereby allowing multicolor FISH. With this method it is therefore possible to investigate nuclear localization of several genomic sequences together and apply automated imaging analyses

Carte épigénétique de l’embryon de poulet

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Three-Dimensional Fluorescence In Situ Hybridization in Mouse Embryos Using Repetitive Probe Sequences

Chapitre 31International audienceA 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

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

International audienceAmong 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

H3S10 phosphorylation marks constitutive heterochromatin during interphase in early mouse embryos until the 4-cell stage : H3S10P marks constitutive heterochromatin in early mouse embryos

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, HP1β 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 HP1β. 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

Status of replication according to the PN stage and time of fixation.

<p>Embryos were first sorted according to their PN stage and the timing of their fixation. Then, for each category, embryos were sorted again according to their replication staining. Early PN3 embryos are exclusively in pre-replication, PN3 are in majority in early replication, early PN4 are mostly in mid/late replication and most of the late PN4 have completed replication.</p

Relationship between DNA demethylation and replication.

<p>A) Quantification of the 5MeC paternal/maternal ratio in control (methanol) and aphidicolin treated embryos with representative images (z-projections of 3D-stacks). Images were rotated if necessary to have the maternal PN on the left and the paternal PN on the right. Scale Bar: 10 µm. B) Separate quantification of 5MeC for both pronuclei in control and aphidicolin treated embryos. 4 experiments were performed and a total of 59 (5+17+25+12) control embryos versus 61 (7+18+22+14) aphidicolin treated ones were quantified.</p