H3K27me3 at Pericentromeric Heterochromatin is a Defining Feature of the Early Mouse Blastocyst

Early mouse development is characterized by structural and epigenetic changes at the chromatin level while cells progress towards differentiation. At blastocyst stage, the segregation of the three primordial lineages is accompanied by establishment of differential patterns of DNA methylation and post-translational modifications of histones, such as H3K27me3. In this study, we have analysed the dynamics of H3K27me3 at pericentromeric heterochromatin (PCH) during development of the mouse blastocyst, in comparison with cultured embryonic cells. We show that this histone modification is first enriched at PCH in the whole embryo and evolves into a diffuse distribution in epiblast during its specification and maturation. Concomitantly, the level of transcription from major satellite decreases. Stem cells derived from blastocyst (naïve ESCs and TSCs) do not fully maintain the H3K27me3 enrichment at PCH. Moreover, the dynamic of H3K27me3 at PCH during in vitro conversion from naïve to primed pluripotent state and during ESCs derivation suggests that the mechanisms underlying the control of this histone mark at PCH are different in embryo and in vitro. We also conclude that the non-canonical presence of H3K27me3 at PCH is a defining feature of embryonic cells in the young blastocyst before epiblast segregation.

p38-MAPK-mediated translation regulation during early blastocyst development is required for primitive endoderm differentiation in mice

Successful specification of the two mouse blastocyst inner cell mass (ICM) lineages (the primitive endoderm (PrE) and epiblast) is a prerequisite for continued development and requires active fibroblast growth factor 4 (FGF4) signaling. Previously, we identified a role for p38 mitogen-activated protein kinases (p38-MAPKs) during PrE differentiation, but the underlying mechanisms have remained unresolved. Here, we report an early blastocyst window of p38-MAPK activity that is required to regulate ribosome-related gene expression, rRNA precursor processing, polysome formation and protein translation. We show that p38-MAPK inhibition-induced PrE phenotypes can be partially rescued by activating the translational regulator mTOR. However, similar PrE phenotypes associated with extracellular signal-regulated kinase (ERK) pathway inhibition targeting active FGF4 signaling are not affected by mTOR activation. These data indicate a specific role for p38-MAPKs in providing a permissive translational environment during mouse blastocyst PrE differentiation that is distinct from classically reported FGF4-based mechanisms.

Dynamic erasure of X chromosome upregulation during iPSC reprogramming and in the inner cell mass

SummaryRecent studies have provided substantial evidence supporting Ohno’s hypothesis that upregulation of active X chromosome genes balances the dosage relative to autosomal genes. Here, we found the evidence for erasure of active X-chromosome upregulation upon reactivation of inactive X in female cells during iPSC reprogramming and in the inner cell mass of mouse blastocyst. Moreover, our analysis revealed that the kinetics of erasure of X upregulation is tightly linked with the dynamics of X reactivation.Abstract Figure

MiRNA Profiling: Mouse Blastocyst Extracellular Microvesicles Inhibit Melanoma Cell Invasion

Background: Microvesicles (MVs) mediate the transmission of information between cells through the miRNAs, proteins, lipids and mRNAs carried by MVs, which inhibit/promote the function of receptor cells. This study confirmed that blastocyst MVs inhibited the invasion of melanoma cells and showed a complex miRNA pedigree. Functional miRNAs were found, and the mechanism of inhibiting invasiveness was analyzed based on the miRNA lineage characteristics of mouse blastocyst MVs, combined with the miRNA bioinformatics information of miRBase, microRNA.org, NCBI and other databases.Methods: MVs derived from D3.5 mouse blastocysts were isolated and the miRNA gene expression of MV was detected by fluorescence quantitative polymerase chain reaction (qPCR). The dynamic changes in tumor cell invasiveness in the co-culture system of blastocyst MVs and melanoma B16-F10 cells were monitored in real time. The MV miRNAs expression profile of blastocyst was identified using the qPCR array. Bioinformatics tools were used to analyze the characteristics of miRNA pedigree in blastocyst MV to screen functional miRNAs, which reduce tumor cell invasiveness and to predict functional miRNA target genes. The target genes were analyzed by KEGG and GO pathway enrichment analyses using David and FunRich software.Results: The average diameter of round MVs extracted from mouse blastocysts was 196.4 ±7.8 nm. When B16-F10 melanoma cells were co-cultured with mouse blastocyst MVs, no significant difference in invasive ability was found between the two groups before 72 h, but increased significantly after 72 h (P < 0.05, P < 0.01). A total of 336 miRNAs were analyzed in mouse blastocyst MVs. Further analysis showed that 224 miRNAs were associated with invasion. Also, 32 invasion-related miRNAs with the strongest differential expression were verified by real-time quantitative polymerase chain reaction (qPCR). The GO analysis of the target genes showed that the 32 functional miRNAs were mainly related to 8 biological processes, including DNA binding, sequence-specific DNA binding, metal ion binding, chromatin binding, nucleotide binding, sequence specificity, DNA binding transcription factor activity and ubiquitin-protein ligase activity. The KEGG pathway analysis showed that the 32 functional miRNAs were mainly related to the PI3K-Akt signaling pathway.Conclusion: Blastocyst MVs carrying miRNAs regulated the decrease in the invasive ability of melanoma cells through the PI3K-Akt signaling pathway.

Expression of IL-1β and implantation serine proteases is required for mouse blastocyst hatching

Mammalian blastocyst hatching is critically an indispensable process for successful implantation. One of the major challenges in IVF clinics is to achieve superior embryonic development with intrinsically potent hatching-competent blastocyst. However, the molecular regulation of hatching phenomenon is poorly understood. In this study, we examined the expression and function of one of the cytokines, IL-1β during blastocyst hatching in the mouse. In particular, the expression of IL-1β (Interleukin-1β), IL-1ra (Interleukin-1 receptor antagonist) and their functional receptor IL-1rt1 (Interleukin 1 receptor type-1) in morulae, zona intact- and hatched- blastocysts was studied. Supplementation of IL-1β to cultured embryos accelerated blastocyst development with improved hatching (treated: 89.6 ± 3.6% vs untreated: 65.4 ± 4.1%). When embryos were treated with IL-1ra, blastocyst hatching was decreased (treated: 28.8 ± 3.1% vs untreated: 67.5 ± 3.8%). Moreover, IL-1β and IL-1ra influenced the expression of hatching enzymes viz., implantation serine proteases (ISP 1 and ISP 2). While IL-1β increased the embryonic mRNA expression of ISPs (ISP1: 2-4; ISP2: 9-11 fold), IL-1ra decreased expression. The protein localization studies revealed increased nuclear presence predominantly of ISP 2 in IL-1β treated blastocysts. This is the first report to show the functional significance of embryonic IL-1β in regulating hatching-associated proteases, particularly ISP2. These findings have implications in our understanding of molecular regulation of blastocyst hatching and implantation failure in other species including humans.