Preimplantation Mouse Embryos Research Articles

Donor template delivery by recombinant adeno-associated virus for the production of knock-in mice

BackgroundThe ability of recombinant adeno-associated virus to transduce preimplantation mouse embryos has led to the use of this delivery method for the production of genetically altered knock-in mice via CRISPR-Cas9. The potential exists for this method to simplify the production and extend the types of alleles that can be generated directly in the zygote, obviating the need for manipulations of the mouse genome via the embryonic stem cell route.ResultsWe present the production data from a total of 13 genetically altered knock-in mouse models generated using CRISPR-Cas9 electroporation of zygotes and delivery of donor repair templates via transduction with recombinant adeno-associated virus. We explore the efficiency of gene targeting at a total of 12 independent genetic loci and explore the effects of allele complexity and introduce strategies for efficient identification of founder animals. In addition, we investigate the reliability of germline transmission of the engineered allele from founder mice generated using this methodology. By comparing our production data against genetically altered knock-in mice generated via gene targeting in embryonic stem cells and their microinjection into blastocysts, we assess the animal cost of the two methods.ConclusionsOur results confirm that recombinant adeno-associated virus transduction of zygotes provides a robust and effective delivery route for donor templates for the production of knock-in mice, across a range of insertion sizes (0.9–4.7 kb). We find that the animal cost of this method is considerably less than generating knock-in models via embryonic stem cells and thus constitutes a considerable 3Rs reduction.

DNMT1 Y495C mutation interferes with maintenance methylation of imprinting control regions

Hereditary Sensory and Autonomic Neuropathy Type 1E (HSAN1E) is a rare autosomal dominant neurological disorder due to missense mutations in DNA methyltransferase 1 (DNMT1). To investigate the nature of the dominant effect, we compared methylomes of transgenic R1wtDnmt1 and R1Dnmt1Y495C mouse embryonic stem cells (mESCs) overexpressing WT and the mutant mouse proteins respectively, with the R1 (wild-type) cells. In case of R1Dnmt1Y495C, 15 out of the 20 imprinting control regions were hypomethylated with transcript level dysregulation of multiple imprinted genes in ESCs and neurons. Non-imprinted regions, minor satellites, major satellites, LINE1 and IAP repeats were unaffected. These data mirror the specific imprinting defects associated with transient removal of DNMT1 in mESCs, deletion of the maternal-effect DNMT1o variant in preimplantation mouse embryos, and in part, reprogramming to naïve human iPSCs. This is the first DNMT1 mutation demonstrated to specifically affect Imprinting Control Regions (ICRs), and reinforces the differences in maintenance methylation of ICRs over non-imprinted regions. Consistent with nervous system abnormalities in the HSAN1E disorder and involvement of imprinted genes in normal development and neurogenesis, R1Dnmt1Y495C cells showed dysregulated pluripotency and neuron marker genes, and yielded more slender, shorter, and extensively branched neurons. We speculate that R1Dnmt1Y495C cells produce predominantly dimers containing mutant proteins, leading to a gradual and specific loss of ICR methylation during early human development.

TEAD4 regulates KRT8 and YAP in preimplantation embryos in mice but not in cattle.

Lineage specification plays a vital role in preimplantation development. TEAD4 is an essential transcription factor for trophectoderm lineage specification in mice but not in cattle. Tead4, a critical transcription factor expressed during preimplantation development, is essential for the expression of trophectoderm-specific genes in mice. However, the functional mechanism of TEAD4 in mouse preimplantation development and its conservation across mammals remain unclear. Here, we report that Tead4 is a crucial transcription factor necessary for blastocyst formation in mice. Disruption of Tead4 through base editing results in developmental arrest at the morula stage. Additionally, RNA-seq analysis reveals dysregulation of 670 genes in Tead4 knockout embryos. As anticipated, Tead4 knockout led to a decrease in trophectoderm genes Cdx2 and Gata3. Intriguingly, we observed a reduction in Krt8, suggesting that Tead4 influences the integrity of the trophectoderm epithelium in mice. More importantly, we noted a dramatic decrease in nuclear Yap in outside cells for Tead4-deficient morula, indicating that Tead4 directly regulates Hippo signaling. In contrast, bovine embryos with TEAD4 depletion could still develop to blastocysts with normal expression of CDX2, GATA3, and SOX2, albeit with a decrease in total cell number and ICM cell number. In conclusion, we propose that Tead4 regulates mouse blastocyst formation via Krt8 and Yap, both of which are critical regulators of mouse preimplantation development.

Chimeric PRMT6 protein produced by an endogenous retrovirus promoter regulates cell fate decision in mouse preimplantation embryos†.

Murine endogenous retrovirus with leucine tRNA primer, also known as MERVL, is expressed during zygotic genome activation in mammalian embryos. Here we show that protein arginine N-methyltransferase 6 (Prmt6) forms a chimeric transcript with MT2B2, one of the long terminal repeat sequences of murine endogenous retrovirus with leucine tRNA primer, and is translated into an elongated chimeric protein (PRMT6MT2B2) whose function differs from that of the canonical PRMT6 protein (PRMT6CAN) in mouse preimplantation embryos. Overexpression of PRMT6CAN in fibroblast cells increased asymmetric dimethylation of the third arginine residue of both histone H2A (H2AR3me2a) and histone H4 (H4R3me2a), while overexpression of PRMT6MT2B2 increased only H2AR3me2a. In addition, overexpression of PRMT6MT2B2 in one blastomere of mouse two-cell embryos promoted cell proliferation and differentiation of the blastomere into epiblast cells at the blastocyst stage, while overexpression of PRMT6CAN repressed cell proliferation. This is the first report of the translation of a chimeric protein (PRMT6MT2B2) in mouse preimplantation embryos. Our results suggest that analyzing chimeric transcripts with murine endogenous retrovirus with leucine tRNA primer will provide insight into the relationship between zygotic genome activation and subsequent intra- and extra-cellular lineage determination.

Does TFAP2C govern conflicting cell fates in mouse preimplantation embryos?

Transcription factor AP2 gamma (TFAP2C) is a well-established regulator of the trophoblast lineage in mice and humans, but a handful of studies indicate that TFAP2C may play an important role in pluripotency. Here, we hypothesize and provide new evidence that TFAP2C functions as an activator of trophoblast and pluripotency genes during preimplantation embryo development.

PFOS and F–53B disrupted inner cell mass development in mouse preimplantation embryo

Perfluorooctane sulfonic acid (PFOS) is a perfluoroalkyl and polyfluoroalkyl substance (PFAS) widely used in daily life. As its toxicity was confirmed, it has been gradually substituted by F–53B (chlorinated polyfluoroalkyl sulfonates, Cl-PFESAs) in China. PFOS exposure during prenatal development may hinder the development of preimplantation embryos, as indicated by recent epidemiological research and in vivo assays. However, the embryotoxicity data for F–53B are scarce. Furthermore, knowledge about the toxicity of F–53B and PFOS exposure to internal follicular fluid concentrations on early preimplantation embryo development remains limited. In this study, internal exposure concentrations of PFOS (10 nM) and F–53B (2 nM) in human follicular fluid were chosen to study the effects of PFAS on early mouse preimplantation embryo development. We found that both PFOS and F–53B treated zygotes exhibited higher ROS activity in 8-cell embryos but not in 2-cell stage embryos. PFOS and F–53B significantly affected the proportion and aggregation of the inner cell mass (ICM) in the blastocyst, but not the total cell number. Mouse embryonic stem cells (mESCs, isolated from the ICM) and embryoid body (EB) assays were employed to assess the toxicity of PFOS and F–53B on the development and differentiation of embryonic pluripotent cells. These results suggested that mESCs exhibited more DNA damage and abnormal germ layer differentiation after brief exposure to PFOS or F–53B. Finally, RNA-sequencing revealed that PFOS and F–53B exposure affected mESCs biosynthetic processes and chromatin-nucleosome assembly. Our results indicate that F–53B has potential risks as an alternative to PFOS, which disrupts ICM development and differentiation.

87 Determining the impact of CO2 concentration and pH on mouse pre-implantation embryo development

87 Determining the impact of CO2 concentration and pH on mouse pre-implantation embryo development

#124 : Microtubule Stability is Associated with the Functional Activity of Mitochondria for the Mouse Preimplantation Embryo Development

Background and Aims: Mitochondria have a pivotal role in the quality of embryos and good development to a healthy embryo pregnancy. Also, mitochondrial dysfunction increases during the aging of the embryos. But there is still a debate about the main cause of mitochondrial dysfunction in the aged embryos. We investigate that effect of microtubule stabilizer (MTS) as Taxol, EpD and microtubule disturber (MTD) as VNB in the preimplantation embryo of the mouse. Method: We performed preimplantation embryo culture with MTS and MTD. Then we assess development ratio and mitochondria functional activity between control and MTS and MTD. We performed comparable analysis of mitochondrial motility between MTS treated and MTD treated embryos by confocal live imaging. Results: In the MTS treated embryos, well-developed embryos with microtubules formation were present in the ooplasm. However, in the MTD treated embryos group, less development and poor microtubule polymer formation was observed in the ooplasm. Microtubules was also demonstrated by depolymerization and the clustering form in the ooplasm of MTD treated embryos. The high dynamic movement of the cytoskeleton with a mitochondrion compared to the MTD treated embryos. Conclusion: Our data suggest that the new function of MTS as Taxol, EpD shows promoting competence of embryo development with enhanced activity of a mitochondrial function. Therefore, MTS is useful as a supplement in vitro culture media of embryos.

MTOR hypoactivity leads to trophectoderm cell failure by enhancing lysosomal activation and disrupting the cytoskeleton in preimplantation embryo

BackgroundMetabolic homeostasis is closely related to early impairment of cell fate determination and embryo development. The protein kinase mechanistic target of rapamycin (mTOR) is a key regulator of cellular metabolism in the body. Inhibition of mTOR signaling in early embryo causes postimplantation development failure, yet the mechanisms are still poorly understood.MethodsPregnancy mice and preimplantation mouse embryo were treated with mTOR inhibitor in vivo and in vitro respectively, and subsequently examined the blastocyst formation, implantation, and post-implantation development. We used immunofluorescence staining, RNA-Seq smart2, and genome-wide bisulfite sequencing technologies to investigate the impact of mTOR inhibitors on the quality, cell fate determination, and molecular alterations in developing embryos.ResultsWe showed mTOR suppression during preimplantation decreases the rate of blastocyst formation and the competency of implantation, impairs the post implantation embryonic development. We discovered that blocking mTOR signaling negatively affected the transformation of 8-cell embryos into blastocysts and caused various deficiencies in blastocyst quality. These included problems with compromised trophectoderm cell differentiation, as well as disruptions in cell fate specification. mTOR suppression significantly affected the transcription and DNA methylation of embryos. Treatment with mTOR inhibitors increase lysosomal activation and disrupts the organization and dynamics of the actin cytoskeleton in blastocysts.ConclusionsThese results demonstrate that mTOR plays a crucial role in 8-cell to blastocyst transition and safeguards embryo quality during early embryo development.

Enhancement of preimplantation mouse embryo development with optimized in vitro culture dish via stabilization of medium osmolarity.

We evaluated the efficacy of the newly developed optimized in vitro culture (OIVC) dish for cultivating preimplantation mouse embryos. This dish minimizes the need for mineral oil and incorporates microwells, providing a stable culture environment and enabling independent monitoring of individual embryos. Mouse pronuclear (PN) zygotes and two-cell-stage embryos were collected at 18 and 46 hours after human chorionic gonadotropin injection, respectively. These were cultured for 120 hours using potassium simplex optimized medium (KSOM) to reach the blastocyst stage. The embryos were randomly allocated into three groups, each cultured in one of three dishes: a 60-mm culture dish, a microdrop dish, and an OIVC dish that we developed. The OIVC dish effectively maintained the osmolarity of the KSOM culture medium over a 5-day period using only 2 mL of mineral oil. This contrasts with the significant osmolarity increase observed in the 60-mm culture dish. Additionally, the OIVC dish exhibited higher blastulation rates from two-cell embryos (100%) relative to the other dish types. Moreover, blastocysts derived from both PN zygotes and two-cell embryos in the OIVC dish group demonstrated significantly elevated mean cell numbers. Use of the OIVC dish markedly increased the number of cells in blastocysts derived from the in vitro culture of preimplantation mouse embryos. The capacity of this dish to maintain medium osmolarity with minimal mineral oil usage represents a breakthrough that may advance embryo culture techniques for various mammals, including human in vitro fertilization and embryo transfer programs.

Lactate regulates major zygotic genome activation by H3K18 lactylation in mammals.

Lactate is present at a high level in the microenvironment of mammalian preimplantation embryos in vivo and in vitro. However, its role in preimplantation development is unclear. Here, we report that lactate is highly enriched in the nuclei of early embryos when major zygotic genome activation (ZGA) occurs in humans and mice. The inhibition of its production and uptake results in developmental arrest at the 2-cell stage, major ZGA failure, and loss of lactate-derived H3K18lac, which could be rescued by the addition of Lac-CoA and recapitulated by overexpression of H3K18R mutation. By profiling the landscape of H3K18lac during mouse preimplantation development, we show that H3K18lac is enriched on the promoter regions of most major ZGA genes and correlates with their expressions. In humans, H3K18lac is also enriched in ZGA markers and temporally concomitant with their expressions. Taken together, we profile the landscapes of H3K18lac in mouse and human preimplantation embryos, and demonstrate the important role for H3K18lac in major ZGA, showing that a conserved metabolic mechanism underlies preimplantation development of mammalian embryos.

Proline and Proline Analogues Improve Development of Mouse Preimplantation Embryos by Protecting Them against Oxidative Stress.

The culture of embryos in the non-essential amino acid L-proline (Pro) or its analogues pipecolic acid (PA) and L-4-thiazolidine carboxylic acid (L4T) improves embryo development, increasing the percentage that develop to the blastocyst stage and hatch. Staining of 2-cell and 4-cell embryos with tetramethylrhodamine methyl ester and 2',7'-dichlorofluorescein diacetate showed that the culture of embryos in the presence of Pro, or either of these analogues, reduced mitochondrial activity and reactive oxygen species (ROS), respectively, indicating potential mechanisms by which embryo development is improved. Inhibition of the Pro metabolism enzyme, proline oxidase, by tetrahydro-2-furoic-acid prevented these reductions and concomitantly prevented the improved development. The ways in which Pro, PA and L4T reduce mitochondrial activity and ROS appear to differ, despite their structural similarity. Specifically, the results are consistent with Pro reducing ROS by reducing mitochondrial activity while PA and L4T may be acting as ROS scavengers. All three may work to reduce ROS by contributing to the GSH pool. Overall, our results indicate that reduction in mitochondrial activity and oxidative stress are potential mechanisms by which Pro and its analogues act to improve pre-implantation embryo development.

Time-lapse observation of mouse preimplantation embryos using a simple closed glass capillary method

Time-lapse observation is a popular method for analyzing mammalian preimplantation embryos, but it often requires expensive equipment and skilled techniques. We previously developed a simply and costly embryo-culture system in a sealed tube that does not require a CO2 incubator. In the present study, we developed a new time-lapse observation system using our previous culture method and a glass capillary. Zygotes were placed in a glass capillary and sunk in oil for observation under a stereomicroscope. Warming the capillary using a thermoplate enabled most of the zygotes to develop into blastocysts and produce healthy offspring. This time-lapse observation system captured images every 30 min for up to 5 days, which confirmed that the developmental speed and quality of the embryos were not affected, even with fluorescence. Overall, this new system is a simple time-lapse observation method for preimplantation embryos that does not require dedicated machines and advanced techniques.

Characterization of H3K9me3 and DNA methylation co-marked CpG-rich regions during mouse development

BackgroundH3K9me3 and DNA methylation co-marked CpG-rich regions (CHMs) are functionally important in mouse pre-implantation embryos, but their characteristics in other biological processes are still largely unknown.ResultsIn this study, we performed a comprehensive analysis to characterize CHMs during 6 mouse developmental processes, identifying over 2,600 CHMs exhibiting stable co-mark of H3K9me3 and DNA methylation patterns at CpG-rich regions. We revealed the distinctive features of CHMs, including elevated H3K9me3 signals and a significant presence in euchromatin and the potential role in silencing younger long terminal repeats (LTRs), especially in some ERVK subfamilies. The results highlight the distinct nature of universal CHMs compared to CpG-rich nonCHMs in terms of location, LTR enrichment, and DNA sequence features, enhancing our understanding of CpG-rich regions' regulatory roles.ConclusionsThis study characterizes the features of CHMs in multiple developmental processes and broadens our understanding of the regulatory roles of CpG-rich regions.

GATA transcription factors drive initial Xist upregulation after fertilization through direct activation of long-range enhancers.

X-chromosome inactivation (XCI) balances gene expression between the sexes in female mammals. Shortly after fertilization, upregulation of Xist RNA from one X chromosome initiates XCI, leading to chromosome-wide gene silencing. XCI is maintained in all cell types, except the germ line and the pluripotent state where XCI is reversed. The mechanisms triggering Xist upregulation have remained elusive. Here we identify GATA transcription factors as potent activators of Xist. Through a pooled CRISPR activation screen in murine embryonic stem cells, we demonstrate that GATA1, as well as other GATA transcription factors can drive ectopic Xist expression. Moreover, we describe GATA-responsive regulatory elements in the Xist locus bound by different GATA factors. Finally, we show that GATA factors are essential for XCI induction in mouse preimplantation embryos. Deletion of GATA1/4/6 or GATA-responsive Xist enhancers in mouse zygotes effectively prevents Xist upregulation. We propose that the activity or complete absence of various GATA family members controls initial Xist upregulation, XCI maintenance in extra-embryonic lineages and XCI reversal in the epiblast.

Oocyte-Specific Deletion of Slc6a9 Encoding the GLYT1 Glycine Transporter Eliminates Glycine Transport in Mouse Preimplantation Embryos and Their Ability to Counter Hypertonic Stress.

Early preimplantation mouse embryos are sensitive to increased osmolarity, which can block their development. To overcome this, they accumulate organic osmolytes to maintain cell volume. The main organic osmolyte used by early mouse embryos is glycine. Glycine is transported during the mature egg and 1-cell to 4-cell embryo stages by a transporter identified as GLYT1, encoded by the Slc6a9 gene. Here, we have produced an oocyte-specific knockout of Slc6a9 by crossing mice that have a segment of the gene flanked by LoxP elements with transgenic mice expressing iCre driven by the oocyte-specific Gdf9 promoter. Slc6a9 null oocytes failed to develop glycine transport activity during meiotic maturation. However, females with these oocytes were fertile. When enclosed in their cumulus-oocyte complex, Slc6a9 null oocytes could accumulate glycine via GLYT1 transport in their coupled cumulus cells, which may support female fertility in vivo. In vitro, embryos derived from Slc6a9 null oocytes displayed a clear phenotype. While glycine rescued complete preimplantation development of wild type embryos from increased osmolarity, embryos derived from null oocytes failed to develop past the 2-cell stage even with glycine. Thus, Slc6a9 is required for glycine transport and protection against increased osmolarity in mouse eggs and early embryos.

Blastomeres of 8-cell mouse embryos differ in their ability to generate embryonic stem cells and produce lines with different transcriptional signatures.

Embryonic stem cell (ESC) derivation from single blastomeres of 8-cell mouse embryos results in lower derivation rates than that from whole blastocysts, raising a biological question about the developmental potential of sister blastomeres. We aimed to assess the ability of 8-cell blastomeres to produce epiblast cells and ESC lines after isolation, and the properties of the resulting lines. Our results revealed unequal competence among sister blastomeres to produce ESC lines. At least half of the blastomeres possess a lower potential to generate ESCs, although culture conditions and blastomeres plasticity can redirect their non-pluripotent fate towards the epiblast lineage, allowing us to generate up to seven lines from the same embryo. Lines originated from the same embryo segregated into two groups according to their transcriptional signatures. While the expression of genes related to pluripotency and development was higher in one group, no differences were found in their trilineage differentiation ability. These results may help to improve our understanding of the ESC derivation process from single blastomeres and cell fate determination in the preimplantation mouse embryos.

Essential role of Mg2+ in mouse preimplantation embryo development revealed by TRPM7 chanzyme-deficient gametes

TRPM7 (transient receptor potential cation channel subfamily M member 7) is a chanzyme with channel and kinase domains essential for embryo development. Using gamete-specific Trpm7-null lines, we report that TRPM7-mediated Mg2+ influx is indispensable for reaching the blastocyst stage. TRPM7 is expressed dynamically from gametes to blastocysts; displays stage-specific localization on the plasma membrane, cytoplasm, and nucleus; and undergoes cleavage that produces C-terminal kinase fragments. TRPM7 underpins Mg2+ homeostasis, and excess Mg2+ but not Zn2+ or Ca2+ overcomes the arrest of Trpm7-null embryos; expressing Trpm7 mRNA restores development, but mutant versions fail or are partially rescued. Transcriptomic analyses of Trpm7-null embryos reveal an abundance of oxidative stress-pathway genes, confirmed by mitochondrial dysfunction, and a reduction in transcription factor networks essential for proliferation; Mg2+ supplementation corrects these defects. Hence, TRPM7 underpins Mg2+ homeostasis in preimplantation embryos, prevents oxidative stress, and promotes gene expression patterns necessary for developmental progression and cell-lineage specification.

The salt-and-pepper pattern in mouse blastocysts is compatible with signaling beyond the nearest neighbors

Embryos develop in a concerted sequence of spatiotemporal arrangements of cells. In the preimplantation mouse embryo, the distribution of the cells in the inner cell mass evolves from a salt-and-pepper pattern to spatial segregation of two distinct cell types. The exact properties of the salt-and-pepper pattern have not been analyzed so far. We investigate the spatiotemporal distribution of NANOG- and GATA6-expressing cells in the ICM of the mouse blastocysts with quantitative three-dimensional single-cell-based neighborhood analyses. A combination of spatial statistics and agent-based modeling reveals that the cell fate distribution follows a local clustering pattern. Using ordinary differential equations modeling, we show that this pattern can be established by a distance-based signaling mechanism enabling cells to integrate information from the whole inner cell mass into their cell fate decision. Our work highlights the importance of longer-range signaling to ensure coordinated decisions in groups of cells to successfully build embryos.

Daxx knockdown promoted rDNA transcription without impairing H3.3 expression in mouse preimplantation embryos.

During fertilization, DAXX (death domain-associated protein) mediates histone variant H3.3 incorporation into heterochromatin, which plays an important role in the maintenance of genomic integrity. rDNA, the ribosomal gene, is included in the first wave of gene activation after fertilization. Our and other studies indicated that loss of Daxx disturbs rDNA heterochromatinization and promotes rDNA transcription without change in protein expression of H3.3. However, maternal and zygotic deletion of Daxx impairs blastocyst development. Whether Daxx knockdown affects H3.3 expression and improves the rDNA transcription in preimplantation development has not been reported. In the present study, we injected HA-labelled H3.3 (H3.3-HA) into oocytes during ICSI procedure, and detected H3.3 and DAXX by immunofluorescent staining. Then, we knockdowned Daxx and detected the gene expression levels of Daxx, H3.3, 18s and 47s rRNA. We also performed immunofluorescent staining of B23, γH2A and EdU incorporation to demonstrate nuclear structure, DNA damage and replication. We found injection of H3.3-HA did not impair preimplantation development. Daxx siRNA did not change expression of H3.3 mRNA, and the development of two-cell embryos and blastocysts, but the overall replication and expression levels of rRNA were increased compared with that in the control group. Finally, knockdown of DAXX did not aggravate the DNA damage but loosened the nucleolus. We concluded that Daxx knockdown promoted DNA replication and rDNA transcription, but did not affect H3.3 expression and subsequent preimplantation development.