Mouse Preimplantation Development Research Articles

Minor zygotic gene activation is essential for mouse preimplantation development

In mice, transcription initiates at the mid-one-cell stage and transcriptional activity dramatically increases during the two-cell stage, a process called zygotic gene activation (ZGA). Associated with ZGA is a marked change in the pattern of gene expression that occurs after the second round of DNA replication. To distinguish ZGA before and after the second-round DNA replication, the former and latter are called minor and major ZGA, respectively. Although major ZGA are required for development beyond the two-cell stage, the function of minor ZGA is not well understood. Transiently inhibiting minor ZGA with 5, 6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB) resulted in the majority of embryos arresting at the two-cell stage and retention of the H3K4me3 mark that normally decreases. After release from DRB, at which time major ZGA normally occurred, transcription initiated with characteristics of minor ZGA but not major ZGA, although degradation of maternal mRNA normally occurred. Thus, ZGA occurs sequentially starting with minor ZGA that is critical for the maternal-to-zygotic transition.

Novel key roles for structural maintenance of chromosome flexible domain containing 1 (Smchd1) during preimplantation mouse development.

Structural maintenance of chromosome flexible domain containing 1 (Smchd1) is a chromatin regulatory gene for which mutations are associated with facioscapulohumeral muscular dystrophy and arhinia. The contribution of oocyte- and zygote-expressed SMCHD1 to early development was examined in mice ( Mus musculus) using a small interfering RNA knockdown approach. Smchd1 knockdown compromised long-term embryo viability, with reduced embryo nuclear volumes at the morula stage, reduced blastocyst cell number, formation and hatching, and reduced viability to term. RNA sequencing analysis of Smchd1 knockdown morulae revealed aberrant increases in expression of a small number of trophectoderm (TE)-related genes and reduced expression of cell proliferation genes, including S-phase kinase-associated protein 2 ( Skp2). Smchd1 expression was elevated in embryos deficient for Caudal-type homeobox transcription factor 2 ( Cdx2, a key regulator of TE specification), indicating that Smchd1 is normally repressed by CDX2. These results indicate that Smchd1 plays an important role in the preimplantation embryo, regulating early gene expression and contributing to long-term embryo viability. These results extend the known functions of SMCHD1 to the preimplantation period and highlight important function for maternally expressed Smchd1 messenger RNA and protein.

The Role of Xist in X-Chromosome Dosage Compensation.

In each somatic cell of a female mammal one X chromosome is transcriptionally silenced via X-chromosome inactivation (XCI), initiating early in development. Although XCI events are conserved in mouse and human postimplantation development, regulation of X-chromosome dosage in preimplantation development occurs differently. In preimplantation development, mouse embryos undergo imprinted form of XCI, yet humans lack imprinted XCI and instead regulate gene expression of both X chromosomes by dampening transcription. The long non-coding RNA Xist/XIST is expressed in mouse and human preimplantation and postimplantation development to orchestrate XCI, but its role in dampening is unclear. In this review, we discuss recent advances in our understanding of the role of Xist in X chromosome dosage compensation in mouse and human.

Requirement of the 3'-UTR-dependent suppression of DAZL in oocytes for pre-implantation mouse development.

Functional oocytes are produced through complex molecular and cellular processes. In particular, the contribution of post-transcriptional gene regulation mediated by RNA-binding proteins (RBPs) is crucial for controlling proper gene expression during this process. DAZL (deleted in azoospermia-like) is one of the RBPs required for the sexual differentiation of primordial germ cells and for the progression of meiosis in ovulated oocytes. However, the involvement of DAZL in the development of follicular oocytes is still unknown. Here, we show that Dazl is translationally suppressed in a 3′-UTR-dependent manner in follicular oocytes, and this suppression is required for normal pre-implantation development. We found that suppression of DAZL occurred in postnatal oocytes concomitant with the formation of primordial follicles, whereas Dazl mRNA was continuously expressed throughout oocyte development, raising the possibility that DAZL is dispensable for the survival and growth of follicular oocytes. Indeed, follicular oocyte-specific knockout of Dazl resulted in the production of normal number of pups. On the other hand, genetically modified female mice that overexpress DAZL produced fewer numbers of pups than the control due to defective pre-implantation development. Our data suggest that post-transcriptional suppression of DAZL in oocytes is an important mechanism controlling gene expression in the development of functional oocytes.

The oocyte-early embryo-specific subcortical maternal complex in mammals

Prior to zygotic genome activation, gene transcription is quiescent. The programs of the early embryonic development are mainly regulated by maternal-effect genes. The research of mammalian maternal-effect genes has long been hindered due to the limitation of research materials and techniques. Recently, a subcortical maternal complex (SCMC) was identified specifically in mammalian oocytes and preimplantation embryos. The SCMC is a protein complex consisting of numerous maternal effect proteins, and is required for mouse preimplantation development. Accumulating evidence suggests that SCMC can be used to identify new maternal-effect genes, to clarify the molecular mechanism of other maternal-effect genes, and to explore the regulation of early embryonic development in mammals. Additionally, the SCMC may be conserved in human oocytes and early embryos. Importantly, the mutations of human SCMC genes are associated with several human reproductive disorders. Collectively, these findings indicate that the SCMC may be an important module orchestrating the maternal-to-zygotic transition in mammalian early development. Further investigations of the SCMC will greatly promote the understanding of molecular regulation in mammalian early development, significantly contributing to human reproductive medicine.

Expression of Components of the Serotonergic System in Folliculogenesis and Preimplantation Development in Mice

The functions of serotonin include the growth and development regulation of female germ cells as well as early embryo development. RT-PCR analysis of mRNA expression of the genes of the enzymes for synthesis and degradation and transporters and receptors of serotonin during folliculogenesis and preimplantation development of mice was performed to discover the particular mechanisms of these functions. The mRNA of tryptophan hydroxylase tph1 and monoaminoxidase maoa; membrane transporter sert and vesicular transporter vmat2; and serotonin receptors htr1b, htr1d, htr2a, htr5b, and htr7 were revealed in granulosa cells. The expression of mRNA of the aromatic amino acid decarboxylase ddc and the htr2b receptor additionally appears in the yellow body. The expression of mRNA of the genes of the tph2, ddc, and maoa enzymes; the sert, vmat1, and vmat2 transporters; and quite a number of receptors is observed during the preimplantation development, and it is transitory in most of them. The expression of all components and its dynamics suggest that the serotonergic signaling system is functionally active in mouse folliculogenesis and preimplantation development.

Parental genetic material and oxygen concentration affect hatch dynamics of mouse embryo in vitro

BackgroundHatching is crucial for mammalian embryo implantation, since difficulties during this process can lead to implantation failure, ectopic pregnancy and consequent infertility. Despite years of intensive researches, how internal and external factors affecting embryo hatch are still largely unclear.MethodsThe effects of parental genetic material and oxygen concentration on hatch process were examined. Fertilized and parthenogenetic mouse preimplantation embryos were cultured in vitro under 5 and 20% oxygen for 120 h. Zona pellucida drilling by Peizo micromanipulation were performed to resemble the breach by sperm penetration.ResultsFirstly, parthenogenetic embryos had similarly high blastocyst developmental efficiency as fertilized embryos, but significantly higher hatch ratio than fertilized embryos in both O2 concentrations. 5% O2 reduced the hatch rate of fertilized embryos from 58.2 to 23.8%, but increased that of parthenogenetic embryos from 81.2 to 90.8% significantly. Analogously, 5% O2 decreased the ratio of Oct4-positive cells in fertilized blastocysts, whereas increased that in parthenogenetic blastocysts. Additionally, 5% O2 increased the total embryonic cell number in both fertilized and parthegenetic embryos, when compared to 20% O2, and the total cell number of fertilized embryos was also higher than that of parthegenetic embryos, despite O2 concentration. Real-time PCR revealed that the expression of key genes involving in MAPK pathway and superoxide dismutase family might contribute to preimplantation development and consequent blastocyst hatch in vitro. Finally, we showed that fertilized and parthenogenetic embryos have diverse hatch dynamics in vitro, although the zona pellucida integrity is not the main reason for their mechanistic differences.ConclusionBoth parental genetic material and O2 concentration, as the representative of intrinsic and extrinsic factors respectively, have significant impacts on mouse preimplantation development and subsequent hatch dynamics, probably by regulating the gene expression involving in MAPK pathway and superoxide dismutase family to control embryonic cell proliferation and allocation of ICM cells.

Sperm toxicity testing: UK best practice guideline from the Association of Biomedical Andrologists

In order to ensure the quality and integrity of diagnostic semen analysis results, materials used should be tested to ensure that they do not interfere with sperm function. As a toxicity test, complex sperm function testing may be considered controversial, since the fertilizing capacity of single sperm can never be assured. In preference, sperm motility offers a unique means of assessing the toxicity of reagents and materials before they are used in routine practice. Motility is the semen parameter most likely to be influenced by the external environment. Indeed, it is the main reason that laboratories insist on supplying their own approved specimen containers and ensuring that patients, as far as possible, adhere to strict conditions for sample collection and transport prior to testing. This differs to other indirect tests of toxicity such as the mouse embryo assay, whereby the rate of mouse pre-implantation embryo development to the blastocyst stage is compared. This guideline is aimed at health care scientists who deal with andrology in both general pathology and specialised fertility laboratories, and provides a model approach to sperm toxicity testing. For assisted reproduction clinics, the same methodology can be used to test any consumables that are used for sperm processing, and as an indirect guide for any consumables that come into direct contact with oocytes and pre-implantation embryos.

Cell Polarity-Dependent Regulation of Cell Allocation and the First Lineage Specification in the Preimplantation Mouse Embryo.

During the first few days in the mouse preimplantation embryo, two types of cells, polar and apolar cells are generated from spherical totipotent blastomeres. Sequential morphogenetic events, polarization, compaction, and asymmetric division, are essential for the generation of the first distinct cell populations, polar and apolar cells, which establish the outer/inner configuration within the embryo. This leads to position-dependent activation of the Hippo signaling pathway and lineage-specific gene expression to form the trophectoderm and inner cell mass in a blastocyst. It is still unknown how each morphogenetic event is initiated and how it influences subsequent events. In this chapter, we briefly review the two classic models of mouse preimplantation development and discuss recent studies providing novel insights into the self-organizing ability of the preimplantation mouse embryo. Advances in live cell imaging and mathematical modeling contribute a greater understanding to lineage specification and cell fate commitment at the single cell level. Differential molecular and mechanistic characteristics created by the presence and absence of the apical domain in polar and apolar cells, respectively, dictate cell allocation, divisional orientation, and differential activation of the Hippo signaling pathway.

Asymmetric distribution of CRUMBS polarity complex proteins from compacted 8-cell to blastocyst stage during mouse preimplantation development

During mouse preimplantation development, blastomeres are equipotent until polarity establishment at compacted 8-cell stage. The intrinsic nature of polarity is the asymmetric distribution of polarity proteins between inside and outside blastomeres along the direction of apical-basal axis. This study investigated the early developmental temporal and spatial expression of the main CRUMBS polarity complex proteins in the mouse preimplantation embryo. We observed that Crb3, Pals1, Patj and Mpdz are transcribed in the mouse preimplantation embryo. However, the asymmetric distribution of these polarity proteins is not established until the compacted 8-cell stage. From compaction and thereafter, CRB3 and PALS1 are progressively enriched in the apical membrane, while PATJ and MPDZ are discretely localized at both tight junctions and the apical membrane adjacent to tight junctions. These temporal and spatial distribution patterns suggest that CRUMBS polarity complex might be involved in the cell polarity establishment in the early mouse embryo and reinforce the viewpoint that developmentally spatial asymmetries are first set up at the compaction stage. The present study provides a foundation for further investigation on the functions of CRUMBS polarity complex in trophectoderm specification and blastocyst morphogenesis.

Genomic imprinting of Xist by maternal H3K27me3.

Maternal imprinting at the Xist gene is essential to achieve paternal allele-specific imprinted X-chromosome inactivation (XCI) in female mammals. However, the mechanism underlying Xist imprinting is unclear. Here we show that the Xist locus is coated with a broad H3K27me3 domain that is established during oocyte growth and persists through preimplantation development in mice. Loss of maternal H3K27me3 induces maternal Xist expression and maternal XCI in preimplantation embryos. Our study thus identifies maternal H3K27me3 as the imprinting mark of Xist.

Maintenance of Mest imprinted methylation in blastocyst-stage mouse embryos is less stable than other imprinted loci following superovulation or embryo culture.

Assisted reproductive technologies are fertility treatments used by subfertile couples to conceive their biological child. Although generally considered safe, these pregnancies have been linked to genomic imprinting disorders, including Beckwith–Wiedemann and Silver–Russell Syndromes. Silver–Russell Syndrome is a growth disorder characterized by pre- and post-natal growth retardation. The Mest imprinted domain is one candidate region on chromosome 7 implicated in Silver–Russell Syndrome. We have previously shown that maintenance of imprinted methylation was disrupted by superovulation or embryo culture during pre-implantation mouse development. For superovulation, this disruption did not originate in oogenesis as a methylation acquisition defect. However, in comparison to other genes, Mest exhibits late methylation acquisition kinetics, possibly making Mest more vulnerable to perturbation by environmental insult. In this study, we present a comprehensive evaluation of the effects of superovulation and in vitro culture on genomic imprinting at the Mest gene. Superovulation resulted in disruption of imprinted methylation at the maternal Mest allele in blastocysts with an equal frequency of embryos having methylation errors following low or high hormone treatment. This disruption was not due to a failure of imprinted methylation acquisition at Mest in oocytes. For cultured embryos, both the Fast and Slow culture groups experienced a significant loss of maternal Mest methylation compared to in vivo-derived controls. This loss of methylation was independent of development rates in culture. These results indicate that Mest is more susceptible to imprinted methylation maintenance errors compared to other imprinted genes.

Dynamic composition of the nuclear envelope during mouse pre-implantation development

Dynamic composition of the nuclear envelope during mouse pre-implantation development

Single-Cell 5-Formylcytosine Landscapes of Mammalian Early Embryos and ESCs at Single-Base Resolution

Active DNA demethylation in mammals involves ten-eleven translocation (TET) family protein-mediated oxidation of 5-methylcytosine (5mC). However, base-resolution landscapes of 5-formylcytosine (5fC) (an oxidized derivative of 5mC) at the single-cell level remain unexplored. Here, we present "CLEVER-seq"(chemical-labeling-enabled C-to-T conversion sequencing), which is a single-cell, single-base resolution 5fC-sequencing technology, based on biocompatible, selective chemical labeling of 5fC and subsequent C-to-T conversion during amplification and sequencing. CLEVER-seq shows intrinsic 5fC heterogeneity in mouse early embryos, Epi stem cells (EpiSCs), and embryonic stem cells (ESCs). CLEVER-seq of mouse early embryos also reveals the highly patterned genomic distribution and parental-specific dynamics of 5fC during mouse early pre-implantation development. Integrated analysis demonstrates that promoter 5fC production precedes the expression upregulation of a clear set of developmentally and metabolically critical genes. Collectively, our work reveals the dynamics of active DNA demethylation during mouse pre-implantation development and provides an important resource for further functional studies of epigenetic reprogramming in single cells.

Exploratory bioinformatics investigation reveals importance of \u201cjunk\u201d DNA in early embryo development

BackgroundInstead of testing predefined hypotheses, the goal of exploratory data analysis (EDA) is to find what data can tell us. Following this strategy, we re-analyzed a large body of genomic data to study the complex gene regulation in mouse pre-implantation development (PD).ResultsStarting with a single-cell RNA-seq dataset consisting of 259 mouse embryonic cells derived from zygote to blastocyst stages, we reconstructed the temporal and spatial gene expression pattern during PD. The dynamics of gene expression can be partially explained by the enrichment of transposable elements in gene promoters and the similarity of expression profiles with those of corresponding transposons. Long Terminal Repeats (LTRs) are associated with transient, strong induction of many nearby genes at the 2-4 cell stages, probably by providing binding sites for Obox and other homeobox factors. B1 and B2 SINEs (Short Interspersed Nuclear Elements) are correlated with the upregulation of thousands of nearby genes during zygotic genome activation. Such enhancer-like effects are also found for human Alu and bovine tRNA SINEs. SINEs also seem to be predictive of gene expression in embryonic stem cells (ESCs), raising the possibility that they may also be involved in regulating pluripotency. We also identified many potential transcription factors underlying PD and discussed the evolutionary necessity of transposons in enhancing genetic diversity, especially for species with longer generation time. ConclusionsTogether with other recent studies, our results provide further evidence that many transposable elements may play a role in establishing the expression landscape in early embryos. It also demonstrates that exploratory bioinformatics investigation can pinpoint developmental pathways for further study, and serve as a strategy to generate novel insights from big genomic data.

PDGFRα+ Cells in Embryonic Stem Cell Cultures Represent the InVitro Equivalent of the Pre-implantation Primitive Endoderm Precursors.

SummaryIn early mouse pre-implantation development, primitive endoderm (PrE) precursors are platelet-derived growth factor receptor alpha (PDGFRα) positive. Here, we demonstrated that cultured mouse embryonic stem cells (mESCs) express PDGFRα heterogeneously, fluctuating between a PDGFRα+ (PrE-primed) and a platelet endothelial cell adhesion molecule 1 (PECAM1)-positive state (epiblast-primed). The two surface markers can be co-detected on a third subpopulation, expressing epiblast and PrE determinants (double-positive). In vitro, these subpopulations differ in their self-renewal and differentiation capability, transcriptional and epigenetic states. In vivo, double-positive cells contributed to epiblast and PrE, while PrE-primed cells exclusively contributed to PrE derivatives. The transcriptome of PDGFRα+ subpopulations differs from previously described subpopulations and shows similarities with early/mid blastocyst cells. The heterogeneity did not depend on PDGFRα but on leukemia inhibitory factor and fibroblast growth factor signaling and DNA methylation. Thus, PDGFRα+ cells represent the in vitro counterpart of in vivo PrE precursors, and their selection from cultured mESCs yields pure PrE precursors.

Detection of Bidirectional Promoter-Derived lncRNAs from Small-Scale Samples Using Pre-Amplification-Free Directional RNA-seq Method.

Development of high-throughput sequencing technologies has uncovered the immensity of the long noncoding RNA (lncRNA) world. Divergently transcribed lncRNAs from bidirectional gene promoters, called promoter-associated noncoding RNAs (pancRNAs), account for ~20% of the total number of lncRNAs, and this major fraction is involved in many biological processes, such as development and cancer formation. Recently, we have found that the pancRNAs activate their partner genes, as represented by the fact that pancIl17d, a pancRNA that is transcribed from the antisense strand of the promoter region of Interleukin 17d (Il17d) at the onset of zygotic gene activation (ZGA), is essential for mouse preimplantation development through Il17d upregulation. The discovery of the expression of a specific set of pancRNAs during ZGA was achieved by using a method that generates directional RNA-seq libraries from small-scale samples. Although there are several methods available for small-scale samples, most of them require a pre-amplification procedure that frequently generates some amplification biases toward a subset of transcripts. We provide here a highly sensitive and reproducible method based on the preparation of directional RNA-seq libraries from as little as 100 mouse oocytes or embryos without pre-amplification for the quantification of lncRNAs as well as mRNAs.

Towards a CRISPR view of early human development: applications, limitations and ethical concerns of genome editing in human embryos.

Developmental biologists have become increasingly aware that the wealth of knowledge generated through genetic studies of pre-implantation mouse development might not easily be translated to the human embryo. Comparative studies have been fueled by recent technological advances in single-cell analysis, allowing in-depth analysis of the human embryo. This field could shortly gain more momentum as novel genome editing technologies might, for the first time, also allow functional genetic studies in the human embryo. In this Spotlight article, we summarize the CRISPR-Cas9 genome editing system and discuss its potential applications and limitations in human pre-implantation embryos, and the ethical considerations thereof.

Roles and regulations of Hippo signaling during preimplantation mouse development.

During preimplantation development, mouse embryos form two types of cells, the trophoectoderm (TE) and inner cell mass (ICM), by the early blastocyst stage. This process does not require maternal factors localized in the zygotes, and embryos self-organize at the blastocyst stage through intercellular communications. In terms of the mechanisms of cell fate specification, three historical models have been proposed: the positional model, and the original and newer versions of the polarity model. Recent studies have revealed that the intercellular Hippo signaling pathway plays a central role in the specification of the first cell fates. Hippo signaling is active in the inner cells but inactive in the outer cells. The Hippo-active inner and Hippo-inactive outer cells take the fates of the ICM and the TE, respectively. At the 32-cell stage, E-cadherin-mediated cell-cell adhesion and cell polarization by the Par-aPKC system activates and inactivates the Hippo pathway, respectively. Both mechanisms involve regulation of angiomotin, and cooperation of these mechanisms establishes cell position-dependent activation of Hippo signaling. At the 16-cell stage, however, asymmetric cell division produces the initial differences in Hippo signaling. At this stage, cell polarity is controlled by both Par-aPKC-dependent and -independent mechanisms. All three historical models are explained by the different regulations and roles of Hippo signaling. Based on these findings, I would like to propose the model by which the differences in Hippo signaling among blastomeres is first produced by asymmetric cell division and then enhanced and stabilized by cell position-dependent mechanisms until their fates are fixed.

The effect of poly(ADP-ribosyl)ation inhibition on the porcine cumulus-oocyte complex during in vitro maturation

Poly(ADP-ribosyl)ation (PARylation) plays important roles in DNA repair, apoptosis, transcriptional regulation, and cell death, and occurs via the activity of poly(ADP-ribose) polymerases (PARPs). Previous studies have shown that PARylation affects mouse and porcine pre-implantation development and participates in mechanisms of autophagy. However, there have not yet been reported the role of PARylation during in vitro maturation (IVM) of porcine oocytes. Thus, we investigated the effect of PARylation inhibition on this process; cumulus-oocyte complexes (COCs) were cultured with 3-aminobenzamide (3-ABA, PARP inhibitor) during porcine IVM. Full cumulus expansion was significantly reduced (10.34 ± 1.23 [3-ABA] vs. 48.17 ± 2.03% [control]), but nuclear maturation rates were not changed in the 3-ABA treatment group. Especially, we observed that cumulus cells were little expanded after 22 h in 3-ABA treated COCs. The mRNA expression levels of oocyte maturation- and cumulus expansion-related genes were evaluated at 22 and 44 h. GDF9, BMP15, COX-2, and PTX3 expression were upregulated at 44 h, whereas the levels of HAS2 and TNFAIP6 were downregulated in the 3-ABA treated group. Furthermore, 3-ABA treatment significantly decreased the developmental rate (28.24 ± 1.06 vs. 40.24 ± 3.03%) and total cell number (41.12 ± 2.10 vs. 50.38 ± 2.27), but increased the total apoptotic index (6.44 ± 0.81 vs. 3.08 ± 0.51) in parthenogenetically activated embryos. In conclusion, these results showed that PARylation regulates cumulus expansion through the regulation of gene expression and affects developmental competence and quality in parthenogenetic embryos.