Fertilization triggers early proteomic symmetry breaking in mammalian embryos.

What is the relationship between the metabolism of preimplantation embryos and their developmental competence?

Human embryonic development: from peri-implantation to gastrulation.

Study question How multinucleation and fragmentation occur in the first mitosis in human embryos? Summary answer The first mitotic spindles had heterogeneity, and the shapes and movements of the spindles were strongly associated with multinucleation and fragmentation. What is known already Studies have shown that many problems occur during the first mitosis, such as fragmentation and multinucleation. However, the cell biologic mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, it is necessary to analysis the dynamics of DNA and cytoskeleton using live human embryos. Although microinjections of DNA or mRNA into mammalian embryos were widely used for live imaging during early mitosis, the method into human poses the risk of ethical issues. Therefore, we established a method used for real-time imaging in human embryos using chemical fluorescent labeling. Study design, size, duration Frozen human two-pronuclear stage embryos were donated for research by couples who had completed fertility treatment. We obtained written informed consent from patients who donated human embryos. The mean age of patients at embryo freezing was 32.00 ± 3.03 (SD) years. We imaged thirty-one living human embryos using chemical fluorescent labeling and confocal microscopy to visualize DNA and tubulin. Participants/materials, setting, methods Thirty-one human embryos were cultured in medium containing the live-cell stain probes SPY555-DNA and SPY650-Tubulin. The Embryos were imaged using an LSM980 laser scanning confocal microscope every 10–20 min for a period of eight hours. Three-dimensional (3D) visualizations of human embryos were performed using the Imaris software. The first spindle shapes were quantified by image analysis and calculated the aspect ratio of the spindles using ImageJ. Main results and the role of chance We discovered that over 81% of two-pronuclear zygotes became multinucleated 2-cell stage embryos. We demonstrated that there was variation in the first mitotic spindles. There was a significant difference between the aspect ratio (AR) of the spindles leading to the mononuclear and multinuclear types at the 2-cell stage (p < 0.005). In addition, the low-AR spindles were unstable and often had more defocused poles than the high-AR spindles, which lead to multinucleation at the 2-cell stage (55 % vs. 0%, p < 0.05). More embryo having high-aspect ratio spindles tend to develop blastocysts than those having low aspect-ratio spindles (p < 0.05). Moreover, the movement of the central spindles varied after chromosome segregation. The embryos with more fragments at two cell stage had a significantly longer period for the central spindles to transform into midbodies compared to those with fewer fragments. The central spindles of embryos with more fragments moved a longer distance from the beginning of furrow ingression to the completion of the first division (p < 0.05). These results suggest that the first mitotic spindle instability and heterogeneity cause many errors such as multinucleation, fragmentation, and embryonic development. Limitations, reasons for caution Imaged human embryos were donated from a fertility clinic, and detailed patient information, such as underlying disease and method of ovarian stimulation, was not available. It is possible that the human zygotes used in this study had a bias in patient characteristics. Wider implications of the findings Our study showed that there were instability and heterogeneity in the first mitotic spindle, which was strongly associated with multinucleation, fragmentation, and embryonic development. Revealing the mechanism underlying spindle formation in cleavage-stage embryos will contribute to improving the success rate of assisted reproduction technologies in humans. Trial registration number not applicable

Small noncoding RNA are known to play a regulatory role in various biological processes including development. Here we aimed to investigate the spatiotemporal expression pattern of the miR-130 microRNA family (miR-130a, miR-130b, and miR-301b) throughout the bovine pre-implantation- stage embryos and to experimentally validate the target of miR-130b. For this, triplicate pools of in vitro-produced immature or mature oocytes (n = 100), zygote (n = 100), 2-cell (n = 50), 4-cell (n = 30), 8-cell (n = 30), morula (n = 20), and blastocyst (n = 20) stage embryos were used to profile the 3 miRNA and one of the predicted target gene MSK1 using SYBR green-based real-time PCR system. Furthermore, based on its expression profile result, miR-130b was selected for localization in all stages of embryos using 3′digoxigenin labeled, LNA-modified oligonucleotide probes. Reporter assay was conducted in cultured bovine cumulus cells to validate the target of miR-130b. The predicted MSK1 gene 3′ untranslated region- containing dual-luciferase miRNA target expression vector (pmirGLO; 600 ng), having renilla luciferase as a control reporter (Promega, Madison, WI, USA), was cotransfected with miR-130b precursor (20 pmol) and/or miR-130b inhibitor (20 pmol) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in Opti-MEM1 Medium with 4 independent transfections. Cells were lysed 48 h post-transfection, and luciferase assay was performed in luminometer. Normalization of firefly luciferase activity was based on renilla luciferase activity, and data were presented as mean ± SD. The expression profiling result shows that miR-130b was abundant (>8 to 12-fold) at morula and blastocyst stages. This was further validated by in situ localization, where high fluorescent intensity was seen in the same stages. Greater abundance of miR-301b was evident immediately after fertilization at zygote stage, whereas miR-130a was abundant in equal amount from oocyte until 8-cell stage, after which expression reduced at morula and blastocyst stages. Verification of MSK1 as target of miR-130b using MSK1-pmirGLO-vector showed a significant reduction (>45%) in expression of luciferase when cells were cotransfected with miR-130b precursor compared with cotransfection with miR-130b inhibitor (95%) or only MSK1 construct transfection (100%). MSK1, which was validated as target of miR-130b in the current study for the first time, is known to regulate the phosphorylation of CREB and ATF1 and is required for inhibits of Wnt-fi-catenin pathway and cell proliferation in colon cancer cells (Morán OP et al. 2008 J. Cell Biol. 183(4), 697-710). The results from this study evidenced the spatiotemporal expression of members of the miR130 family during bovine embryo development and their potential regulatory mechanism in the expression of developmentally important gene. The overexpression or inhibition of these miRNA in bovine oocytes and embryos might help in fully understanding their specific role in bovine embryogenesis.

DNA methylation is a crucial element in the epigenetic regulation of mammalian embryonic development. However, its dynamic patterns have not been analysed at the genome scale in human pre-implantation embryos due to technical difficulties and the scarcity of required materials. Here we systematically profile the methylome of human early embryos from the zygotic stage through to post-implantation by reduced representation bisulphite sequencing and whole-genome bisulphite sequencing. We show that the major wave of genome-wide demethylation is complete at the 2-cell stage, contrary to previous observations in mice. Moreover, the demethylation of the paternal genome is much faster than that of the maternal genome, and by the end of the zygotic stage the genome-wide methylation level in male pronuclei is already lower than that in female pronuclei. The inverse correlation between promoter methylation and gene expression gradually strengthens during early embryonic development, reaching its peak at the post-implantation stage. Furthermore, we show that active genes, with the trimethylation of histone H3 at lysine 4 (H3K4me3) mark at the promoter regions in pluripotent human embryonic stem cells, are essentially devoid of DNA methylation in both mature gametes and throughout pre-implantation development. Finally, we also show that long interspersed nuclear elements or short interspersed nuclear elements that are evolutionarily young are demethylated to a milder extent compared to older elements in the same family and have higher abundance of transcripts, indicating that early embryos tend to retain higher residual methylation at the evolutionarily younger and more active transposable elements. Our work provides insights into the critical features of the methylome of human early embryos, as well as its functional relation to the regulation of gene expression and the repression of transposable elements.

ABSTRACTLIMKs (LIMK1 and LIMK2) are serine/threonine protein kinases that involve in various cellular activities such as cell migration, morphogenesis and cytokinesis. However, its roles during mammalian early embryo development are still unclear. In the present study, we disrupted LIMK1/2 activity to explore the functions of LIMK1/2 during mouse early embryo development. We found that p-LIMK1/2 mainly located at the cortex of each blastomeres from 2-cell to 8-cell stage, and p-LIMK1/2 also expressed at morula and blastocyst stage in mouse embryos. Inhibition of LIMK1/2 activity by LIMKi 3 (BMS-5) at the zygote stage caused the failure of embryo early cleavage, and the disruption of LIMK1/2 activity at 8-cell stage caused the defects of embryo compaction and blastocyst formation. Fluorescence staining and intensity analysis results demonstrated that the inhibition of LIMK1/2 activity caused aberrant cortex actin expression and the decrease of phosphorylated cofilin in mouse embryos. Taken together, we identified LIMK1/2 as an important regulator for cofilin phosphorylation and actin assembly during mouse early embryo development.

The oxidation and incorporation of glucose and glutamine by embryos derived from cultured zygotes was compared with the utilization of these substrates by embryos recovered directly from the reproductive tract of pregnant females. The oxidation of glutamine was greater at the blastocyst stage than at the 2-cell stage. Embryos derived from outbred females (Qs) were less active in the oxidation of glutamine than those from hybrid (B10D2F1) females and development in culture was detrimental to this oxidation, especially in blastocysts from the outbred stock. The oxidation of glutamine was stimulated by the presence of glucose at the 2-cell stage but reduced by its presence at the blastocyst stage. Maternal genotype had no effect on the oxidation of glucose at either the 2-cell or blastocyst stage, and only at the blastocyst stage was there evidence of a detrimental effect of culture. The oxidation of glucose was stimulated by the presence of glutamine at the 2-cell stage but depressed by its addition at the blastocyst stage. Incorporation of glutamine increased with development, but this was reduced at the blastocyst stage by development in culture, especially if the blastocysts were derived from outbred females. Incorporation of glucose also increased with development. At the 2-cell stage, culture reduced incorporation of this substrate, especially into the acid-soluble fraction of embryos from outbred females. In blastocysts, incorporation of glucose into the acid-insoluble fraction was depressed by culture and in embryos from outbred females. In contrast to glucose oxidation, incorporation of glucose into the acid-soluble fraction was reduced by the addition of glutamine at the 2-cell stage but increased by its addition at the blastocyst stage.

Quantification of transcription activities in mammalian preimplantation embryos is challenging due to a huge amount of maternally stored transcripts and paucity of research materials. Here, we investigate genome-wide transcription activities of mouse and human preimplantation embryos by quantifying elongating RNA polymerase II. Two transcriptional waves are identified in early mouse embryos, with summits at the 2-cell and 8-cell stages. Gene collections with different expression patterns are obtained, with genes mainly transcribed at the mouse early/late 2-cell stage designated as zygotic genome activation-early/late 2-cell (ZGA-E2C/L2C). ZGA-E2C genes are short and have low promoter CpG density. Protein translation/degradation not only regulates transcription activity through stepwise orchestration of histone modifications, transcriptional initiation, and elongation in early mouse embryos but also controls on/off switching of ZGA-E2C/L2C genes in maternal aged mouse embryos. Genes mainly transcribed at the mouse 2-cell stage can also be transcribed as early as the human 2-cell stage.

Chapter 1 Ontogeny of Erythropoiesis in the Mammalian Embryo

Abstract: DNA methylation is essential for normal mammalian development and plays critical roles in various biological processes, including genomic imprinting, X-chromosome inactivation and repression of transposable elements. Although DNA methylation patterns are relatively stable in somatic cells, global reprogramming of DNA methylation occurs during mammalian preimplantation development. Advances in DNA methylation profiling techniques have been revealing the DNA methylation dynamics in mammalian embryos. Recently, we and other groups reported genome-scale DNA methylation analyses of human oocytes and preimplantation embryos, highlighting both the similarities and differences in the DNA methylation dynamics between humans and mice. In this review, we introduce the current knowledge of DNA methylation dynamics during early mammalian development. We also discuss the possibility of the application of genome-scale DNA methylation analysis techniques to human gametes and embryos for diagnostic purposes.

Stanniocalcins (STC) are a small family of secreted homodimeric glycoprotein hormones consisting of STC1 and STC2. A previous study in Drosophila (Tolias and Stroumbakis 1998 Dev. Genes Evol. 208, 274–282) indicated that maternally derived STC is required during embryogenesis. However, little information is available for mammalian embryos. The aim of this study was to examine the expression of STC and assess their roles during the preimplantation stage of bovine embryo development. Immature cumulus–oocyte complexes were aspirated from follicles of bovine ovaries collected at a local abattoir and matured in vitro for 24 h at 39°C under an atmosphere of 5% CO2 in air with maximum humidity in TCM-199 supplemented with 10% (vol/vol) fetal calf serum and 10 ng mL–1 of epidermal growth factor. Matured cumulus–oocyte complexes were inseminated with fertile bull semen (Day 0). Embryos were cultured in vitro, and subsequently, 4 pools of 10 embryos each at the zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, and blastocyst stages were collected from 4 different replicate cultures and stored at –80°C until analysis. Total RNA was isolated using an RNeasy Micro Kit and a random primer was used during cDNA synthesis. The expression of STC1, STC2, and reference genes (YWHAZ, PPIA, SDHA) was examined. Quantitative real-time PCR was used to compare transcript abundance, and data were normalized to the geometric averages of the reference genes. The expression levels were analysed using the relative standard curve method, and means were compared using Student’s t-test. Despite being members of the same family and having large sequence similarity, the expression of each gene was unique and stage dependent during embryo development. Expression of STC1 was detected in all the stages examined. Expression was transiently reduced at the 2-cell stage, with no significant change until the 8-cell stage but with a slight increase at the 16-cell stage. In contrast, STC2 was barely detectable before the 8-cell stage. Expression at the 8- and 16-cell stages was significantly (P < 0.0001) higher compared with all other stages, with a peak at the 16-cell stage. This significantly higher expression pattern of STC2 during the critical stages of maternal to zygotic control of development may suggest an important role during this critical period of embryo development. Supported by Science Foundation Ireland (07/SRC/B1156).

Study question What is the effect of oxygen (O2) levels on the transcriptomic profile and embryonic genome activation of bovine embryos during the in vitro culture? Summary answer Normoxia (20% O2) delayed transcriptomic reprogramming and embryonic genome activation (EGA), and induced changes in energy metabolism gene expression. What is known already Mammalian preimplantation embryo development is a complex sequence of events where, within a week, terminally differentiated need to be reprogrammed to totipotency and subsequently diverge to embryonic and extraembryonic cell lineages for post-implantation embryo development. This period of development is highly sensitive to environmental changes such as oxygen levels (O2), pH, and temperature. Many IVF clinics still use normoxia for the embryo culture instead of hypoxia. In addition, a new hypothesis of sequential O2 hypoxia followed by ultrahypoxia has emerged. The molecular evidence about the O2 effect mostly comes from mice which are not developmentally similar to humans. Study design, size, duration In this study, we used triplicate bovine embryos as a model for human embryogenesis to compare the influence of O2 levels on preimplantation embryonic development by culturing embryos either in normoxic (20% O2) or physiological hypoxic (6% O2) conditions, or sequential hypoxia until 16-cell stage and then switching to ultrahypoxic culture (2% O2). Participants/materials, setting, methods As the readout for varied O2 effects, we performed RNA sequencing using the 5’ targeted STRT-N method on single embryos. We compared zygotes, 4-, 8-, 16-cell, and blastocyst stage embryos grown in either normoxic or hypoxic conditions, adding ultrahypoxia for blastocyst stage embryos as the third condition. Main results and the role of chance The effect of O2 was not observed at the cleavage rate, however the clear difference was seen at the blastocyst formation rate. The highest number of embryos that reached the blastocyst stage was in hypoxia condition (36%), in normoxia it was 13% and in ultrahypoxia, this number was only 4.6%. Transcriptomic profiling showed that normoxic conditions slowed down maternal transcript degradation and EGA. At the blastocyst stage, normoxia embryos did not have key energy metabolism genes for glycolysis upregulated, but rather only depended on oxidative phosphorylation metabolism. When we compared hypoxia and ultrahypoxia blastocysts, they had similar transcription profiles. Both conditions induced proper upregulation of the energy metabolism genes involved in glycolysis and lipid metabolism which are typical for in vivo embryos. We found constant hypoxia culture system provides the best blastocyst formation rate and induces appropriate energy metabolism needed for later stages of development. Normoxia alters the EGA, and energy metabolism and decreases blastocyst formation rate. Despite having a similar transcription profile as hypoxia, ultrahypoxia led to the lowest blastocyst formation rate making this condition sub-optimal for the in vitro culture of embryos. Limitations, reasons for caution The limitation of this study is the use of bovine as an animal model instead of human embryos. Due to this, the direct translation of the results to humans should be taken with caution. Wider implications of the findings This study supports previous literature on hypoxic culture conditions being the most suitable for in vitro embryo culture. We provide new insights on the reason normoxia embryos don't have the same success rate as hypoxia embryos. We didn't observe any benefits of lowering O2 to 2%. Trial registration number No

The first lineage allocation in mouse and human embryos separates the inner cell mass (ICM) from the outer trophectoderm (TE). This symmetry breaking event is executed through polarization of cells at the 8-cell stage and subsequent asymmetric divisions, generating polar (TE) and apolar (ICM) cells. Here, we show that embryo polarization is unexpectedly asynchronous. Cells polarizing at the early and late 8-cell stage have distinct molecular and morphological properties that direct their following lineage specification, with early polarizing cells being biased towards producing the TE lineage. More recent studies have also implicated heterogeneities between cells prior to the 8-cell stage in the first lineage allocation: cells exhibiting reduced methyltransferase CARM1 activity at the 4-cell stage are predisposed towards the TE fate. Here, we demonstrate that reduced CARM1 activity and upregulation of its substrate BAF155 promote early polarization and TE specification. These findings provide a link between asymmetries at the 4-cell stage and polarization at the 8-cell stage, mechanisms of the first lineage allocation that had been considered separate.

During mammalian pre-implantation embryo development, when the first asymmetry emerges and how it develops to direct distinct cell fates remain longstanding questions. Here, by analyzing single-blastomere transcriptome data from mouse and human pre-implantation embryos, we revealed that the initial blastomere-to-blastomere biases emerge as early as the first embryonic cleavage division, following a binomial distribution pattern. The subsequent zygotic transcriptional activation further elevated overall blastomere-to-blastomere biases during the two- to 16-cell embryo stages. The trends of transcriptional asymmetry fell into two distinct patterns: for some genes, the extent of asymmetry was minimized between blastomeres (monostable pattern), whereas other genes, including those known to be lineage specifiers, showed ever-increasing asymmetry between blastomeres (bistable pattern), supposedly controlled by negative or positive feedbacks. Moreover, our analysis supports a scenario in which opposing lineage specifiers within an early blastomere constantly compete with each other based on their relative ratio, forming an inclined 'lineage strength' that pushes the blastomere onto a predisposed, yet flexible, lineage track before morphological distinction.

Obituary: Dame Dr Anne McLaren