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The intricate dynamics of Hes expression across diverse cell types in the developing vertebrate embryonic tail have remained elusive. To address this, we have developed an endogenously tagged Hes1-Achilles mouse line, enabling precise quantification of dynamics at the single-cell resolution across various tissues. Our findings reveal striking disparities in Hes1 dynamics between presomitic mesoderm (PSM) and preneural tube (pre-NT) cells. While pre-NT cells display variable, low-amplitude oscillations, PSM cells exhibit synchronized, high-amplitude oscillations. Upon the induction of differentiation, the oscillation amplitude increases in pre-NT cells. Additionally, our study of Notch inhibition on Hes1 oscillations unveils distinct responses in PSM and pre-NT cells, corresponding to differential Notch ligand expression dynamics. These findings suggest the involvement of separate mechanisms driving Hes1 oscillations. Thus, Hes1 demonstrates dynamic behaviour across adjacent tissues of the embryonic tail, yet the varying oscillation parameters imply differences in the information that can be transmitted by these dynamics., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Embryonic diapause is a reproductive adaptation that enables some mammalian species to halt the otherwise continuous pace of embryonic development. In this dormant state, the embryo exploits poorly understood regulatory mechanisms to preserve its developmental potential for prolonged periods of time. Here, using mouse embryos and single-cell RNA sequencing, we molecularly defined embryonic diapause at single-cell resolution, revealing transcriptional dynamics while the embryo seemingly resides in a state of suspended animation. Additionally, we found that the dormant pluripotent cells rely on integrin receptors to sense their microenvironment and preserve their viability via Yap/Taz-mediated prosurvival signaling., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Faithful DNA replication is essential for genome integrity 1-4 . Under-replicated DNA leads to defects in chromosome segregation, which are common during embryogenesis 5-8 . However, the regulation of DNA replication remains poorly understood in early mammalian embryos. Here we constructed a single-cell genome-wide DNA replication atlas of pre-implantation mouse embryos and identified an abrupt replication program switch accompanied by a transient period of genomic instability. In 1- and 2-cell embryos, we observed the complete absence of a replication timing program, and the entire genome replicated gradually and uniformly using extremely slow-moving replication forks. In 4-cell embryos, a somatic-cell-like replication timing program commenced abruptly. However, the fork speed was still slow, S phase was extended, and markers of replication stress, DNA damage and repair increased. This was followed by an increase in break-type chromosome segregation errors specifically during the 4-to-8-cell division with breakpoints enriched in late-replicating regions. These errors were rescued by nucleoside supplementation, which accelerated fork speed and reduced the replication stress. By the 8-cell stage, forks gained speed, S phase was no longer extended and chromosome aberrations decreased. Thus, a transient period of genomic instability exists during normal mouse development, preceded by an S phase lacking coordination between replisome-level regulation and megabase-scale replication timing regulation, implicating a link between their coordination and genome stability., (© 2024. The Author(s).)

The heterogeneity of CARM1 controls first cell fate bias during early mouse development. However, how this heterogeneity is established is unknown. Here, we show that Carm1 mRNA is of a variety of specific exon-skipping splicing (ESS) isoforms in mouse two-cell to four-cell embryos that contribute to CARM1 heterogeneity. Disruption of paraspeckles promotes the ESS of Carm1 precursor mRNAs (pre-mRNAs). LincGET, but not Neat1, is required for paraspeckle assembly and inhibits the ESS of Carm1 pre-mRNAs in mouse two-cell to four-cell embryos. We further find that LincGET recruits paraspeckles to the Carm1 gene locus through HNRNPU. Interestingly, PCBP1 binds the Carm1 pre-mRNAs and promotes its ESS in the absence of LincGET. Finally, we find that the ESS seen in mouse two-cell to four-cell embryos decreases CARM1 protein levels and leads to trophectoderm fate bias. Our findings demonstrate that alternative splicing of CARM1 has an important role in first cell fate determination., (© 2024. The Author(s).)

This study aimed to develop a method to evaluate the quality of bovine in vitro fertilized (IVF) embryos based on gene expression profiling via whole-transcriptome amplification. The expression of 11 developmentally important genes in individual bovine in vivo-derived (IVD) and IVF embryos were examined. Gene expression profiling was conducted by classifying the expression level of each gene in individual embryos as low, medium, or high. The IVF group had a higher (P < 0.01) proportion of embryos with low expression of SOX2, NANOG, and FGF4. In addition, a correlation analysis between the expression levels of each gene in individual embryos demonstrated that the relationship between gene expression differed with respect to IVD and IVF embryos. Our results suggest that the expression profiling of developmentally important genes using IVD embryos as normal controls could be a useful indicator for evaluating the quality of bovine IVF embryos.

The timing of DNA replication in mammals is crucial for minimizing errors and influenced by genome usage and chromatin states. Replication timing in the newly formed mammalian embryo remains poorly understood. Here, we have investigated replication timing in mouse zygotes and 2-cell embryos, revealing that zygotes lack a conventional replication timing program, which then emerges in 2-cell embryos. This program differs from embryonic stem cells and generally correlates with transcription and genome compartmentalization of both parental genomes. However, consistent and systematic differences existed between the replication timing of the two parental genomes, including considerably later replication of maternal pericentromeric regions compared to paternal counterparts. Moreover, maternal chromatin modified by Polycomb Repressive Complexes in the oocyte, undergoes early replication, despite belonging to the typically late-replicating B-compartment of the genome. This atypical and asynchronous replication of the two parental genomes may advance our understanding of replication stress in early human embryos and trigger strategies to reduce errors and aneuploidies., (© 2024. The Author(s).)

DNA replication in differentiated cells follows a defined program, but when and how it is established during mammalian development is not known. Here we show using single-cell sequencing, that late replicating regions are established in association with the B compartment and the nuclear lamina from the first cell cycle after fertilization on both maternal and paternal genomes. Late replicating regions contain a relative paucity of active origins and few but long genes and low G/C content. In both bovine and mouse embryos, replication timing patterns are established prior to embryonic genome activation. Chromosome breaks, which form spontaneously in bovine embryos at sites concordant with human embryos, preferentially locate to late replicating regions. In mice, late replicating regions show enhanced fragility due to a sparsity of dormant origins that can be activated under conditions of replication stress. This pattern predisposes regions with long neuronal genes to fragility and genetic change prior to separation of soma and germ cell lineages. Our studies show that the segregation of early and late replicating regions is among the first layers of genome organization established after fertilization., (© 2024. The Author(s).)

Analysis of single cell transcriptomics (scRNA-seq) data is typically performed after subsetting to highly variable genes (HVGs). Here, we show that Entropy Sorting provides an alternative mathematical framework for feature selection. On synthetic datasets, continuous Entropy Sort Feature Weighting (cESFW) outperforms HVG selection in distinguishing cell-state-specific genes. We apply cESFW to six merged scRNA-seq datasets spanning human early embryo development. Without smoothing or augmenting the raw counts matrices, cESFW generates a high-resolution embedding displaying coherent developmental progression from eight-cell to post-implantation stages and delineating 15 distinct cell states. The embedding highlights sequential lineage decisions during blastocyst development, while unsupervised clustering identifies branch point populations obscured in previous analyses. The first branching region, where morula cells become specified for inner cell mass or trophectoderm, includes cells previously asserted to lack a developmental trajectory. We quantify the relatedness of different pluripotent stem cell cultures to distinct embryo cell types and identify marker genes of naïve and primed pluripotency. Finally, by revealing genes with dynamic lineage-specific expression, we provide markers for staging progression from morula to blastocyst., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Zygotic genome activation (ZGA) after fertilization enables the maternal-to-zygotic transition. However, the global view of ZGA, particularly at initiation, is incompletely understood. Here, we develop a method to capture and sequence newly synthesized RNA in early mouse embryos, providing a view of transcriptional reprogramming during ZGA. Our data demonstrate that major ZGA gene activation begins earlier than previously thought. Furthermore, we identify a set of genes activated during minor ZGA, the promoters of which show enrichment of the Obox factor motif, and find that Obox3 or Obox5 overexpression in mouse embryonic stem cells activates ZGA genes. Notably, the expression of Obox factors is severely impaired in somatic cell nuclear transfer (SCNT) embryos, and restoration of Obox3 expression corrects the ZGA profile and greatly improves SCNT embryo development. Hence, our study reveals dynamic transcriptional reprogramming during ZGA and underscores the crucial role of Obox3 in facilitating totipotency acquisition., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Development requires coordinated interactions between the epiblast, which generates the embryo proper; the trophectoderm, which generates the placenta; and the hypoblast, which forms both the anterior signalling centre and the yolk sac. These interactions remain poorly understood in human embryogenesis because mechanistic studies have only recently become possible. Here we examine signalling interactions post-implantation using human embryos and stem cell models of the epiblast and hypoblast. We find anterior hypoblast specification is NODAL dependent, as in the mouse. However, while BMP inhibits anterior signalling centre specification in the mouse, it is essential for its maintenance in human. We also find contrasting requirements for BMP in the naive pre-implantation epiblast of mouse and human embryos. Finally, we show that NOTCH signalling is important for human epiblast survival. Our findings of conserved and species-specific factors that drive these early stages of embryonic development highlight the strengths of comparative species studies., (© 2024. The Author(s).)

Totipotency is the ability of a single cell to develop into a full organism and, in mammals, is strictly associated with the early stages of development following fertilization. This unlimited developmental potential becomes quickly restricted as embryonic cells transition into a pluripotent state. The loss of totipotency seems a consequence of the zygotic genome activation (ZGA), a process that determines the switch from maternal to embryonic transcription, which in mice takes place following the first cleavage. ZGA confers to the totipotent cell a transient transcriptional profile characterized by the expression of stage-specific genes and a set of transposable elements that prepares the embryo for subsequent development. The timely silencing of this transcriptional program during the exit from totipotency is required to ensure proper development. Importantly, the molecular mechanisms regulating the transition from totipotency to pluripotency have remained elusive due to the scarcity of embryonic material. However, the development of new in vitro totipotent-like models together with advances in low-input genome-wide technologies, are providing a better mechanistic understanding of how this important transition is achieved. This review summarizes the current knowledge on the molecular determinants that regulate the exit from totipotency., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Stem cell-based mammalian embryo models facilitate the discovery of developmental mechanisms because they are more amenable to genetic and epigenetic perturbations than natural embryos. Here, we highlight exciting recent advances that have yielded a plethora of models of embryonic development. Imperfections in these models highlight gaps in our current understanding and outline future research directions, ushering in an exciting new era for embryology., Competing Interests: Declaration of Competing Interest The authors are inventors on the following patents: 1. Patent applicant: Caltech. Inventors: Magdalena Zernicka-Goetz, Berna Sozen, and Victoria Jorgensen. Application number: 17/692,790. Specific aspect of the paper covered in patent application: Reconstructing human early embryogenesis in vitro with pluripotent stem cells. 2. Patent applicant: Caltech and Cambridge Enterprise Limited. Inventors: Magdalena Zernicka-Goetz, Gianluca Amadei, and Charlotte Handford. Application number: 63/397,630. Specific aspect of the paper covered in patent application: Synthetic embryos. 3. Patent Applicant: Caltech and Cambridge Enterprise Limited. Inventors: Magdalena Zernicka-Goetz, Bailey Weatherbee, and Carlos Gantner. Application number: 63/403,684. Specific aspect of the paper covered in patent application: Stem cell-derived model of the human embryo., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The house mouse (Mus musculus) is an exceptional model system, combining genetic tractability with close evolutionary affinity to humans 1,2 . Mouse gestation lasts only 3 weeks, during which the genome orchestrates the astonishing transformation of a single-cell zygote into a free-living pup composed of more than 500 million cells. Here, to establish a global framework for exploring mammalian development, we applied optimized single-cell combinatorial indexing 3 to profile the transcriptional states of 12.4 million nuclei from 83 embryos, precisely staged at 2- to 6-hour intervals spanning late gastrulation (embryonic day 8) to birth (postnatal day 0). From these data, we annotate hundreds of cell types and explore the ontogenesis of the posterior embryo during somitogenesis and of kidney, mesenchyme, retina and early neurons. We leverage the temporal resolution and sampling depth of these whole-embryo snapshots, together with published data 4-8 from earlier timepoints, to construct a rooted tree of cell-type relationships that spans the entirety of prenatal development, from zygote to birth. Throughout this tree, we systematically nominate genes encoding transcription factors and other proteins as candidate drivers of the in vivo differentiation of hundreds of cell types. Remarkably, the most marked temporal shifts in cell states are observed within one hour of birth and presumably underlie the massive physiological adaptations that must accompany the successful transition of a mammalian fetus to life outside the womb., (© 2024. The Author(s).)

Extensive epigenetic reprogramming occurs during preimplantation embryonic development. However, the impact of DNA methylation in plateau yak preimplantation embryos and how epigenetic reprogramming contributes to transcriptional regulatory networks are unclear. In this study, we quantified gene expression and DNA methylation in oocytes and a series of yak embryos at different developmental stages and at single-cell resolution using single-cell bisulfite-sequencing and RNA-seq. We characterized embryonic genome activation and maternal transcript degradation and mapped epigenetic reprogramming events critical for embryonic development. Through cross-species transcriptome analysis, we identified 31 conserved maternal hub genes and 39 conserved zygotic hub genes, including SIN3A, PRC1, HDAC1/2, and HSPD1. Notably, by combining single-cell DNA methylation and transcriptome analysis, we identified 43 candidate methylation driver genes, such as AURKA, NUSAP1, CENPF, and PLK1, that may be associated with embryonic development. Finally, using functional approaches, we further determined that the epigenetic modifications associated with the histone deacetylases HDAC1/2 are essential for embryonic development and that the deubiquitinating enzyme USP7 may affect embryonic development by regulating DNA methylation. Our data represent an extensive resource on the transcriptional dynamics of yak embryonic development and DNA methylation remodeling, and provide new insights into strategies for the conservation of germplasm resources, as well as a better understanding of mammalian early embryonic development that can be applied to investigate the causes of early developmental disorders., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Dynamic metabolism is exhibited by early mammalian embryos to support changing cell fates during development. It is widely acknowledged that metabolic pathways not only satisfy cellular energetic demands, but also play pivotal roles in the process of cell signalling, gene regulation, cell proliferation and differentiation. Recently, various new technological advances have been made in metabolomics and computational analysis, deepening our understanding of the crucial role of dynamic metabolism during early mammalian embryogenesis. In this Review, we summarize recent studies on oocyte and embryo metabolism and its regulation, with a particular focus on its association with key developmental events such as fertilization, zygote genome activation and cell fate determination. In addition, we discuss the mechanisms of certain metabolites that, in addition to serving as energy sources, contribute to epigenetic modifications., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

During mammalian preimplantation development, a totipotent zygote undergoes several cell cleavages and two rounds of cell fate determination, ultimately forming a mature blastocyst. Along with compaction, the establishment of apicobasal cell polarity breaks the symmetry of an embryo and guides subsequent cell fate choice. Although the lineage segregation of the inner cell mass (ICM) and trophectoderm (TE) is the first symbol of cell differentiation, several molecules have been shown to bias the early cell fate through their inter-cellular variations at much earlier stages, including the 2- and 4-cell stages. The underlying mechanisms of early cell fate determination have long been an important research topic. In this review, we summarize the molecular events that occur during early embryogenesis, as well as the current understanding of their regulatory roles in cell fate decisions. Moreover, as powerful tools for early embryogenesis research, single-cell omics techniques have been applied to both mouse and human preimplantation embryos and have contributed to the discovery of cell fate regulators. Here, we summarize their applications in the research of preimplantation embryos, and provide new insights and perspectives on cell fate regulation., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Isolation of individual cells ensures detailed analysis of human embryos and promotes our understanding of molecular mechanisms driving embryo development and cell specification. Here, we present a protocol for the processing of human embryos for single-cell analysis. We describe steps for growing embryos and individualizing cells from the polar and the mural parts of trophectoderm at the blastocyst stage using laser dissection. We then detail embryo dissociation followed by steps to pick, wash, and dispense cells in plates., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Technologies to reproduce specific aspects of early mammalian embryogenesis in vitro using stem cells have skyrocketed over the last several years. With these advances, we have gained new perspectives on how embryonic and extraembryonic cells self-organize to form the embryo. These reductionist approaches hold promise for the future implementation of precise environmental and genetic controls to understand variables affecting embryo development. Our review discusses recent progress in cellular models of early mammalian embryo development and bioengineering advancements that can be leveraged to study the embryo-maternal interface. We summarize current gaps in the field, emphasizing the importance of understanding how intercellular interactions at this interface contribute to reproductive and developmental health., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The preimplantation mammalian (including mouse and human) embryo holds remarkable regulatory abilities, which have found their application, for example, in the preimplantation genetic diagnosis of human embryos. Another manifestation of this developmental plasticity is the possibility of obtaining chimaeras by combining either two embryos or embryos and pluripotent stem cells, which enables the verification of the cell pluripotency and generation of genetically modified animals used to elucidate gene function. Using mouse chimaeric embryos (constructed by injection of embryonic stem cells into the eight-cell embryos) as a tool, we aimed to explore the mechanisms underlying the regulatory nature of the preimplantation mouse embryo. We comprehensively demonstrated the functioning of a multi-level regulatory mechanism involving FGF4/MAPK signalling as a leading player in the communication between both components of the chimaera. This pathway, coupled with apoptosis, the cleavage division pattern and cell cycle duration controlling the size of the embryonic stem cell component and giving it a competitive advantage over host embryo blastomeres, provides a cellular and molecular basis for regulative development, ensuring the generation of the embryo characterised by proper cellular composition., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The changes in epigenetic modifications during early embryonic development significantly impact mammalian embryonic genome activation (EGA) and are species-conserved to some degree. Here, we reanalyzed the published RNA-Seq of human, mouse, and goat early embryos and found that Zfp296 (zinc finger protein 296) expression was higher at the EGA stage than at the oocyte stage in all three species (adjusted p -value < 0.05 |log2(foldchange)| ≥ 1). Subsequently, we found that Zfp296 was conserved across human, mouse, goat, sheep, pig, and bovine embryos. In addition, we identified that ZFP296 interacts with the epigenetic regulators KDM5B, SMARCA4, DNMT1, DNMT3B, HP1β, and UHRF1. The Cys 2 -His 2 (C2H2) zinc finger domain TYPE2 TYPE3 domains of ZFP296 co-regulated the modification level of the trimethylation of lysine 9 on the histone H3 protein subunit (H3K9me3). According to ChIP-seq analysis, ZFP296 was also enriched in Trim28 , Suv39h1 , Setdb1 , Kdm4a , and Ehmt2 in the mESC genome. Then, knockdown of the expression of Zfp296 at the late zygote of the mouse led to the early developmental arrest of the mouse embryos and failure resulting from a decrease in H3K9me3. Together, our results reveal that Zfp296 is an H3K9me3 modulator which is essential to the embryonic genome activation of mouse embryos.

The spatial sorting of RNA transcripts is fundamental for the refinement of gene expression to distinct subcellular regions. Although, in non-mammalian early embryogenesis, differential RNA localisation presages cell fate determination, in mammals it remains unclear. Here, we uncover apical-to-basal RNA asymmetries in outer blastomeres of 16-cell stage mouse preimplantation embryos. Basally directed RNA transport is facilitated in a microtubule- and lysosome-mediated manner. Yet, despite an increased accumulation of RNA transcripts in basal regions, higher translation activity occurs at the more dispersed apical RNA foci, demonstrated by spatial heterogeneities in RNA subtypes, RNA-organelle interactions and translation events. During the transition to the 32-cell stage, the biased inheritance of RNA transcripts, coupled with differential translation capacity, regulates cell fate allocation of trophectoderm and cells destined to form the pluripotent inner cell mass. Our study identifies a paradigm for the spatiotemporal regulation of post-transcriptional gene expression governing mammalian preimplantation embryogenesis and cell fate., (© 2023. The Author(s).)

Study Question: Does mitochondrial deficiency affect human embryonic preimplantation development?, Summary Answer: The presence of a pathogenic mitochondrial variant triggers changes in the gene expression of preimplantation human embryos, compromising their development, cell differentiation, and survival., What Is Known Already: Quantitative and qualitative anomalies of mitochondrial DNA (mtDNA) are reportedly associated with impaired human embryonic development, but the underlying mechanisms remain unexplained., Study Design, Size, Duration: Taking advantage of the preimplantation genetic testing for mitochondrial disorders in at-risk couples, we have compared gene expression of 9 human embryos carrying pathogenic variants in either mtDNA genes or nuclear genes encoding mitochondrial protein to 33 age-matched control embryos., Participants/materials, Setting, Methods: Single-embryo transcriptomic analysis was performed on whole human blastocyst embryos donated to research., Main Results and the Role of Chance: Specific pathogenic mitochondrial variants downregulate gene expression in preimplantation human embryos [566 genes in oxidative phosphorylation (OXPHOS)-deficient embryos], impacting transcriptional regulators, differentiation factors, and nuclear genes encoding mitochondrial proteins. These changes in gene expression primarily alter OXPHOS and cell survival pathways., Limitations, Reasons for Caution: The number of OXPHOS-deficient embryos available for the study was limited owing to the rarity of this material. However, the molecular signature shared by all these embryos supports the relevance of the findings., Wider Implications of the Findings: While identification of reliable markers of normal embryonic development is urgently needed in ART, our study prompts us to consider under-expression of the targeted genes reported here, as predictive biomarkers of mitochondrial dysfunction during preimplantation development., Study Funding/competing Interest(s): This work was supported by the 'Association Française contre les Myopathies (AFM-Téléthon)' and the 'La Fondation Maladies Rares'. No competing interests to declare., Trial Registration Number: N/A., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A wide-ranging review study regarding the molecular characterization of the first cell lineages of the developmental embryo is lacking, especially for the primary events during earliest differentiation which leads to the determination of cellular fate. Here, a systematic review and meta-analysis were conducted according to PRISMA guidelines. MEDLINE-PubMed was searched based on an established search strategy through April 2021. Thirty-six studies fulfilling the inclusion criteria were subjected to qualitative and quantitative analysis. Among the studies, 50 % (18/36) used mice as an animal model, 22.2 % (8/36) pigs, 16.7 % (6/36) cattle, 5.5 % (2/36) humans, and 2.8 % (1/36) goats as well as 2.8 % (1/36) equine. Our results demonstrated that each of the first cell lineages of embryos requires a certain pattern of expression to establish the cellular determination of fate. Moreover, these patterns are shared by many species, particularly for those molecules that have already been identified in the literature as biomarkers. In conclusion, the present study integrated carefully chosen studies regarding embryonic development and first cellular decisions in mammalian species and summarized the information about the differential characterization of the first cell lineages and their possible relationship with specific gene expression., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Gamete (sperm and oocyte) genomes are transcriptionally silent until embryonic genome activation (EGA) following fertilization. EGA in humans had been thought to occur around the eight-cell stage, but recent findings suggest that it is triggered in one-cell embryos, by fertilization. Phosphorylation and other post-translational modifications during fertilization may instate transcriptionally favorable chromatin and activate oocyte-derived transcription factors (TFs) to initiate EGA. Expressed genes lay on cancer-associated pathways and their identities predict upregulation by MYC and other cancer-associated TFs. One interpretation of this is that the onset of EGA, and the somatic cell trajectory to cancer, are mechanistically related: cancer initiates epigenetically. We describe how fertilization might be linked to the initiation of EGA and involve distinctive processes recapitulated in cancer., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The embryo-maternal interaction occurs during the early stages of embryo development and is essential for the implantation and full-term development of the embryo. In bovines, the secretion of interferon Tau (IFNT) during elongation is the main signal for pregnancy recognition, but its expression starts around the blastocyst stage. Embryos release extracellular vesicles (EVs) as an alternative mechanism of embryo-maternal communication. The aim of the study was to determine whether EVs secreted by bovine embryos during blastulation (D5-D7) could induce transcriptomic modifications, activating IFNT signaling in endometrial cells. Additionally, it aims to assess whether the EVs secreted by embryos produced in vivo (EVs-IVV) or in vitro (EVs-IVP) have different effects on the transcriptomic profiles of the endometrial cells. In vitro- and in vivo-produced bovine morulae were selected and individually cultured for 48 h to collect embryonic EVs (E-EVs) secreted during blastulation. E-EVs stained with PKH67 were added to in vitro-cultured bovine endometrial cells to assess EV internalization. The effect of EVs on the transcriptomic profile of endometrial cells was determined by RNA sequencing. EVs from both types of embryos induced several classical and non-classical IFNT-stimulated genes (ISGs) and other pathways related to endometrial function in epithelial endometrial cells. Higher numbers of differentially expressed genes (3552) were induced by EVs released by IVP embryos compared to EVs from IVV (1838). Gene ontology analysis showed that EVs-IVP/IVV induced the upregulation of the extracellular exosome pathway, the cellular response to stimulus, and the protein modification processes. This work provides evidence regarding the effect of embryo origin (in vivo or in vitro) on the early embryo-maternal interaction mediated by extracellular vesicles.

At the moment of union in fertilization, sperm and oocyte are transcriptionally silent. The ensuing onset of embryonic transcription (embryonic genome activation [EGA]) is critical for development, yet its timing and profile remain elusive in any vertebrate species. We here dissect transcription during EGA by high-resolution single-cell RNA sequencing of precisely synchronized mouse one-cell embryos. This reveals a program of embryonic gene expression (immediate EGA [iEGA]) initiating within 4 h of fertilization. Expression during iEGA produces canonically spliced transcripts, occurs substantially from the maternal genome, and is mostly downregulated at the two-cell stage. Transcribed genes predict regulation by transcription factors (TFs) associated with cancer, including c-Myc. Blocking c-Myc or other predicted regulatory TF activities disrupts iEGA and induces acute developmental arrest. These findings illuminate intracellular mechanisms that regulate the onset of mammalian development and hold promise for the study of cancer., Competing Interests: Declaration of interests The authors declare no conflicting interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Naive human pluripotent stem cells (hPSCs) are defined as the in vitro counterpart of the human preimplantation embryo's epiblast and are used as a model system to study developmental processes. In this study, we report the discovery and characterization of distinct cell populations coexisting with epiblast-like cells in 5iLAF naive human induced PSC (hiPSC) cultures. It is noteworthy that these populations closely resemble different cell types of the human embryo at early developmental stages. While epiblast-like cells represent the main cell population, interestingly we detect a cell population with gene and transposable element expression profile closely resembling the totipotent eight-cell (8C)-stage human embryo, and three cell populations analogous to trophectoderm cells at different stages of their maturation process: transition, early, and mature stages. Moreover, we reveal the presence of cells resembling primitive endoderm. Thus, 5iLAF naive hiPSC cultures provide an excellent opportunity to model the earliest events of human embryogenesis, from the 8C stage to the peri-implantation period., Competing Interests: Conflict of interests J.P.R. is an employee and shareholder of 10x Genomics., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The emerging importance of embryonic development research rapidly increases the volume for a professional resource related to multi-omics data. However, the lack of global embryogenesis repository and systematic analysis tools limits the preceding in stem cell research, human congenital diseases and assisted reproduction. Here, we developed the EmAtlas, which collects the most comprehensive multi-omics data and provides multi-scale tools to explore spatiotemporal activation during mammalian embryogenesis. EmAtlas contains data on multiple types of gene expression, chromatin accessibility, DNA methylation, nucleosome occupancy, histone modifications, and transcription factors, which displays the complete spatiotemporal landscape in mouse and human across several time points, involving gametogenesis, preimplantation, even fetus and neonate, and each tissue involves various cell types. To characterize signatures involved in the tissue, cell, genome, gene and protein levels during mammalian embryogenesis, analysis tools on these five scales were developed. Additionally, we proposed EmRanger to deliver extensive development-related biological background annotations. Users can utilize these tools to analyze, browse, visualize, and download data owing to the user-friendly interface. EmAtlas is freely accessible at http://bioinfor.imu.edu.cn/ematlas., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Research on early postimplantation mammalian development has been limited by the small size and intrauterine confinement of the developing embryos. Owing to the inability to observe and manipulate living embryos at these stages in utero, the establishment of robust ex utero embryo-culture systems that capture prolonged periods of mouse development has been an important research goal. In the last few years, these methods have been significantly improved by the optimization and enhancement of in vitro culture systems sustaining embryo development during peri-implantation stages for several species, and more recently, proper growth of natural mouse embryos from pregastrulation to late organogenesis stages and of embryonic stem cell (ES)-derived synthetic embryo models until early organogenesis stages. Here, we discuss the most recent ex utero embryo-culture systems established to date for rodents, nonhuman primates, and humans. We emphasize their technical aspects and developmental timeframe and provide insights into the new opportunities that these methods will contribute to the study of natural and synthetic mammalian embryogenesis and the stem-cell field., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Synthetic embryology aims to develop embryo-like structures from stem cells to provide new insight into early stages of mammalian development. Recent advances in synthetic embryology have highlighted the remarkable capacity of stem cells to self-organize under certain biochemical or biophysical stimulations, generating structures that recapitulate the fate and form of early mouse/human embryos, in which symmetry breaking, pattern formation, or proper morphogenesis can be observed spontaneously. Here we review recent progress on the design principles for different types of embryoids and discuss the impact of different biochemical and biophysical factors on the process of stem-cell self-organization. We also offer our thoughts about the principal future challenges., (Copyright © 2022. Published by Elsevier Ltd.)

Inter-species somatic cell nuclear transfer (iSCNT) is significant in the study of biological problems such as embryonic genome activation and the mitochondrial function of embryos. Here, we used iSCNT as a model to determine whether abnormal embryo genome activation was caused by mitochondrial dysfunction. First, we found the ovine-bovine iSCNT embryos were developmentally blocked at the 8-cell stage. The reactive oxygen species level, mitochondrial membrane potential, and ATP level in ovine-bovine cloned embryos were significantly different from both bovine-bovine and IVF 8-cell stage embryos. RNA sequencing and q-PCR analysis revealed that mitochondrial transport, mitochondrial translational initiation, mitochondrial large ribosomal subunit, and mitochondrial outer membrane genes were abnormally expressed in the ovine-bovine embryos, and the mitochondrial outer membrane and mitochondrial ribosome large subunit genes, mitochondrial fusion gene 1, and ATPase Na+/K+ transporting subunit beta 3 gene were expressed at lower levels in the ovine-bovine cloned embryos. Furthermore, we found that overexpression and knockdown of Mfn1 significantly affected mitochondrial fusion and subsequent biological functions such as production of ATP, mitochondrial membrane potential, reactive oxygen species and gene expressions in cloned embryos. These findings enhance our understanding of the mechanism by which the Mfn1 gene regulates embryonic development and embryonic genome activation events.

During early mammalian embryo development, different epigenetic marks undergo reprogramming and play crucial roles in the mediation of gene expression. Currently, several databases provide multi-omics information on early embryos. However, how interconnected epigenetic markers function together to coordinate the expression of the genetic code in a spatiotemporal manner remains difficult to analyze, markedly limiting scientific and clinical research. Here, we present dbEmbryo, an integrated and interactive multi-omics database for human and mouse early embryos. dbEmbryo integrates data on gene expression, DNA methylation, histone modifications, chromatin accessibility, and higher-order chromatin structure profiles for human and mouse early embryos. It incorporates customized analysis tools, such as "multi-omics visualization," "Gene&Peak annotation," "ZGA gene cluster," " cis -regulation," "synergistic regulation," "promoter signal enrichment," and "3D genome." Users can retrieve gene expression and epigenetic profile patterns to analyze synergistic changes across different early embryo developmental stages. We showed the uniqueness of dbEmbryo among extant databases containing data on early embryo development and provided an overview. Using dbEmbryo, we obtained a phase-separated model of transcriptional control during early embryo development. dbEmbryo offers web-based analytical tools and a comprehensive resource for biologists and clinicians to decipher molecular regulatory mechanisms of human and mouse early embryo development., (© 2022 Huang et al.; Published by Cold Spring Harbor Laboratory Press.)

Mammalian embryogenesis relies on glycolysis and oxidative phosphorylation to balance the generation of biomass with energy production. However, the dynamics of metabolic regulation in the postimplantation embryo in vivo have remained elusive due to the inaccessibility of the implanted conceptus for biochemical studies. To address this issue, we compiled single-cell embryo profiling data in six mammalian species and determined their metabolic dynamics through glycolysis and oxidative phosphorylation associated gene expression. Strikingly, we identify a conserved switch from bivalent respiration in the late blastocyst towards a glycolytic metabolism in early gastrulation stages across species, which is independent of embryo implantation. Extraembryonic lineages followed the dynamics of the embryonic lineage, except visceral endoderm. Finally, we demonstrate that in vitro primate embryo culture substantially impacts metabolic gene regulation by comparison to in vivo samples. Our work reveals a conserved metabolic programme despite different implantation modes and highlights the need to optimise postimplantation embryo culture protocols., (© 2022. The Author(s).)

The preimplantation period of embryogenesis is crucial during mammalian ontogenesis. During this period, the mitotic cycles are initiated, the embryonic genome is activated, and the primary differentiation of embryonic cells occurs. All cellular abnormalities occurring in this period are the primary cause of fetal developmental disorders. DNA damage is a serious cause of developmental failure. In the context of DNA damage response on the cellular level, we analyzed the course of embryogenesis and phenotypic changes during the cleavage of a preimplantation embryo. Our results document that DNA damage induced before the resumption of DNA synthesis in a zygote can significantly affect the preimplantation development of the embryo. This developmental ability is related to the level of the DNA damage. We showed that one-cell embryos can correct the first cleavage cycle despite low DNA damage and incomplete replication. It seems that the phenomenon creates a predisposition to a segregation disorder of condensed chromatin that results in the formation of micronuclei in the developmental stages following the first cleavage. We conclude that zygote tolerates a certain degree of DNA damage and considers its priority to complete the first cleavage stage and continue embryogenesis as far as possible.

Embryo transfer is a crucial step in IVF cycle, with increasing trend during the last decade of transferring a single embryo, preferably at the blastocyst stage. Despite increasing evidence supporting Day 5 blastocyst-stage transfer, the optimal day of embryo transfer remains controversial. The crucial questions are therefore, whether the mechanisms responsible to embryos arrest are embryo aneuploidy or others, and whether those embryos arrested in-vitro between the cleavage to the blastocyst stage would survive in-vivo if transferred on the cleavage-stage. We therefore aim to explore whether aneuploidy can directly contribute to embryo development to the blastocyst stage. Thirty Day-5 embryos, that their Day-3 blastomere biopsy revealed a single-gene defect, were donated by 10 couples undergoing preimplantation genetic testing treatment at our center. Affected high quality Day-3 embryos were cultured to Day-5, and were classified to those that developed to the blastocyst-stage and those that were arrested. Each embryo underwent whole genome amplification. Eighteen (60%) embryos were arrested, did not develop to the blastocyst stage and 12 (40%) have developed to the blastocyst stage. Nineteen embryos (63.3%) were found to be euploid. Of them, 12 (66.6%) were arrested embryos and 7 (58.3%) were those that developed to the blastocyst-stage. These figures were not statistically different (p = 0.644). Our observation demonstrated that the mechanism responsible to embryos arrest in vitro is not embryo aneuploidy, but rather other, such as culture conditions. If further studies will confirm that Day-5 blastocyst transfer might cause losses of embryos that would have been survived in vivo, cleavage-stage embryo transfer would be the preferred timing. This might reduce the cycle cancellations due to failure of embryo to develop to the blastocyst stage and will provide the best cumulative live birth-rate per started cycle., (© 2022. The Author(s).)

Upon fertilization, the mammalian embryo must switch from dependence on maternal transcripts to transcribing its own genome, and in mice this involves the transient up-regulation of MERVL transposons and MERVL-driven genes at the two-cell stage. The mechanisms and requirement for MERVL and two-cell (2C) gene up-regulation are poorly understood. Moreover, this MERVL-driven transcriptional program must be rapidly shut off to allow two-cell exit and developmental progression. Here, we report that robust ribosomal RNA (rRNA) synthesis and nucleolar maturation are essential for exit from the 2C state. 2C-like cells and two-cell embryos show similar immature nucleoli with altered structure and reduced rRNA output. We reveal that nucleolar disruption via blocking RNA polymerase I activity or preventing nucleolar phase separation enhances conversion to a 2C-like state in embryonic stem cells (ESCs) by detachment of the MERVL activator Dux from the nucleolar surface. In embryos, nucleolar disruption prevents proper nucleolar maturation and Dux silencing and leads to two- to four-cell arrest. Our findings reveal an intriguing link between rRNA synthesis, nucleolar maturation, and gene repression during early development., (© 2022 Xie et al.; Published by Cold Spring Harbor Laboratory Press.)

The primordia of the post-otic mouse embryo forms largely from a bipotential cell population containing neuromesodermal progenitors (NMP) which reside in the tail bud and contribute to the elaboration of the major body axis after gastrulation. The mechanisms by which the NMP population is both maintained and subsequently directed down mesodermal and neural lineages is incompletely understood. The vertebrate transcription factor Cdx2, is essential for axial elongation and has been implicated in maintaining the NMP niche and in specification of NMP derivatives. To better understand the role of the Cdx family in axial elongation, we employed a conditional mutant allele which evokes total loss of Cdx function, and enriched for tail bud progenitors through the use of a Pax2-GFP transgenic reporter. Using this approach, we identified 349 Cdx-dependent genes by RNA sequencing (RNA-seq). From these, Gene Ontology and chromatin immunoprecipitation analysis further revealed a number of putative direct Cdx candidate target genes implicated in axial elongation, including Sp8, Isl1, Evx1, Zic3 and Nr2f1. Additional analysis of available single-cell RNA-seq data from mouse tail buds revealed the co-expression of Sp8, Isl1, Evx1 and Zic3 with Cdx2 in putative NMP cells, while Nr2f1 was excluded from this population. These findings identify a number of novel Cdx targets and provide further insight into the critical roles for Cdx in elaborating the post-otic embryo., Competing Interests: Declaration of competing interest The authors declare that they have no potential conflicts of interest with respect to the research, authorship or publication of this article., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Totipotency emerges in early embryogenesis, but its molecular underpinnings remain poorly characterized. In the present study, we employed DNA fiber analysis to investigate how pluripotent stem cells are reprogrammed into totipotent-like 2-cell-like cells (2CLCs). We show that totipotent cells of the early mouse embryo have slow DNA replication fork speed and that 2CLCs recapitulate this feature, suggesting that fork speed underlies the transition to a totipotent-like state. 2CLCs emerge concomitant with DNA replication and display changes in replication timing (RT), particularly during the early S-phase. RT changes occur prior to 2CLC emergence, suggesting that RT may predispose to gene expression changes and consequent reprogramming of cell fate. Slowing down replication fork speed experimentally induces 2CLCs. In vivo, slowing fork speed improves the reprogramming efficiency of somatic cell nuclear transfer. Our data suggest that fork speed regulates cellular plasticity and that remodeling of replication features leads to changes in cell fate and reprogramming., (© 2022. The Author(s).)

Since the development of ART, embryos have been cultured at 37 °C in an attempt to mimic the in vivo conditions and the average body temperature of an adult. However, a gradient of temperatures within the reproductive tract has been demonstrated in humans and several other mammalian species. Therefore, the aim of this study was to evaluate the effects of temperature variation treatments on mouse embryo quality through morphokinetic events, blastocyst morphology, the relative gene expression of Igf2, Bax, Bcl2 and Apaf1 and the metabolomics of individual culture media. Study groups consisted of 2 circadian treatments, T1 with embryos being cultured at 37 °C during the day and 35.5 °C during the night, T2 with 38.5 °C during the day and 37 °C during the night and a control group with constant 37 °C. Our main findings are that the lower-temperature group (T1) showed a consistent negative effect on mouse embryo development with "slow" cleaving embryos, poor-quality blastocysts, a higher expression of the apoptotic gene Apaf1, and a significantly different set of amino acids representing a more stressed metabolism. On the other hand, our higher-temperature group (T2) showed similar results to the control group, with no adverse effects on blastocyst viability., (© 2022. The Author(s).)

In human embryos, the initiation of transcription (embryonic genome activation [EGA]) occurs by the eight-cell stage, but its exact timing and profile are unclear. To address this, we profiled gene expression at depth in human metaphase II oocytes and bipronuclear (2PN) one-cell embryos. High-resolution single-cell RNA sequencing revealed previously inaccessible oocyte-to-embryo gene expression changes. This confirmed transcript depletion following fertilization (maternal RNA degradation) but also uncovered low-magnitude upregulation of hundreds of spliced transcripts. Gene expression analysis predicted embryonic processes including cell-cycle progression and chromosome maintenance as well as transcriptional activators that included cancer-associated gene regulators. Transcription was disrupted in abnormal monopronuclear (1PN) and tripronuclear (3PN) one-cell embryos. These findings indicate that human embryonic transcription initiates at the one-cell stage, sooner than previously thought. The pattern of gene upregulation promises to illuminate processes involved at the onset of human development, with implications for epigenetic inheritance, stem-cell-derived embryos, and cancer., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Embryo fragmentation represents a phenomenon generally characterized by the presence of membrane-bound extracellular cytoplasm into the perivitelline space. Recent evidence supports the cellular and molecular heterogeneity of embryo fragments. In this narrative review, we described the different embryo fragment-like cellular structures in their morphology, molecular content, and supposed function and have reported the proposed theories on their origin over the years. We identified articles related to characterization of embryo fragmentation with a specific literature search string. The occurrence of embryo fragmentation has been related to various mechanisms, of which the most studied are apoptotic cell death, membrane compartmentalization of altered DNA, cytoskeletal disorders, and vesicle formation. These phenomena are thought to result in the extrusion of entire blastomeres, release of apoptotic bodies and other vesicles, and micronuclei formation. Different patterns of fragmentation may have different etiologies and effects on embryo competence. Removal of fragments from the embryo before embryo transfer with the aim to improve implantation potential should be reconsidered on the basis of the present observations.

Embryonic genome activation and dosage compensation are major genetic events in early development. Combined analysis of single embryo RNA-seq data and parental genome sequencing was used to evaluate parental contributions to early development and investigate X-chromosome dynamics. In addition, we evaluated dimorphism in gene expression between male and female embryos. Evaluation of parent-specific gene expression revealed a minor increase in paternal expression at the 4-cell stage that increased at the 8-cell stage. We also detected eight genes with allelic expression bias that may have an important role in early development, notably NANOGNB. The main actor in X-chromosome inactivation, XIST, was significantly upregulated at the 8-cell, morula, and blastocyst stages in female embryos, with high expression at the latter. Sexual dimorphism in gene expression was identified at all stages, with strong representation of the X-chromosome in females from the 16-cell to the blastocyst stage. Female embryos showed biparental X-chromosome expression at all stages after the 4-cell stage, demonstrating the absence of imprinted X-inactivation at the embryo level. The analysis of gene dosage showed incomplete dosage compensation (0.5 < X:A < 1) in MII oocytes and embryos up to the 4-cell stage, an increase of the X:A ratio at the 16-cell and morula stages after genome activation, and a decrease of the X:A ratio at the blastocyst stage, which might be associated with the beginning of X-chromosome inactivation. This study represents the first critical analysis of parent- and sex-specific gene expression in early equine embryos produced in vitro., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: To investigate whether inhibition of LINE-1 affects telomere reprogramming during 2-cell embryo development., Methods: Mouse zygotes were cultured with or without 1 µM azidothymidine (AZT) for up to 15 h (early 2-cell, G1/S) or 24 h (late 2-cell, S/G2). Gene expression and DNA copy number were determined by RT-qPCR and qPCR respectively. Immunostaining and telomeric PNA-FISH were performed for co-localization between telomeres and ZSCAN4 or LINE-1-Orf1p., Results: LINE-1 copy number was remarkably reduced in later 2-cell embryos by exposure to 1 µM AZT, and telomere lengths in late 2-cell embryos with AZT were significantly shorter compared to control embryos (P = 0.0002). Additionally, in the absence of LINE-1 inhibition, Dux, Zscan4, and LINE-1 were highly transcribed in early 2-cell embryos, as compared to late 2-cell embryos (P < 0.0001), suggesting that these 2-cell genes are activated at the early 2-cell stage. However, in early 2-cell embryos with AZT treatment, mRNA levels of Dux, Zscan4, and LINE-1 were significantly decreased. Furthermore, both Zscan4 and LINE-1 encoded proteins localized to telomere regions in 2-cell embryos, but this co-localization was dramatically reduced after AZT treatment (P < 0.001)., Conclusions: Upon inhibition of LINE-1 retrotransposition in mouse 2-cell embryos, Dux, Zscan4, and LINE-1 were significantly downregulated, and telomere elongation was blocked. ZSCAN4 foci and their co-localization with telomeres were also significantly decreased, indicating that ZSCAN4 is an essential component of the telomere reprogramming that occurs in mice at the 2-cell stage. Our findings also suggest that LINE-1 may directly contribute to telomere reprogramming in addition to regulating gene expression., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Recurrent pregnancy loss (RPL) is defined as the loss of two or more consecutive pregnancies before the 20 weeks of gestation. RPL affects about 1-2% of couples trying to conceive; however, the mechanisms leading to this complication are largely unknown. Our previous studies using comparative proteomics identified 314 differentially expressed proteins (DEPs) in the placental villous. In this study, we identified 5479 proteins from a total of 34 157 peptides in decidua of patients with early RPL (data are available via ProteomeXchange with identifier PXD023849). Further analysis identified 311 DEPs in the decidua tissue; and 159 proteins were highly expressed, whereas 152 proteins were lowly expressed. These 311 proteins were further analyzed by using Ingenuity Pathway Analysis. The results suggested that 50 DEPs played important roles in the embryonic development. Upstream analysis of these DEPs revealed that angiotensinogen was the most important upstream regulator. Furthermore, protein-protein interaction analysis of the embryonic development DEPs from the placental villous and decidua was performed in the STRING database. This study identified several proteins specifically associated with embryonic development in decidua of patients with early RPL. Therefore, these results provide new insights into potential biological mechanisms, which may ultimately inform RPL., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Two-cell-like (2C-like) embryonic stem cells (ESCs) are a small group of ESCs that spontaneously express zygotic genome activation (ZGA) genes and repeats, such as Zscan4 and murine endogenous retrovirus with leucine (MERVL), and are specifically expressed in 2-cell-stage mouse embryos. Although numerous types of treatment and agents elevate the transition of ESCs to 2C-like ESCs, Dux serves as a critical factor in this transition by increasing the expression of Zscan4 and MERVL directly. However, the loss of Dux did not impair the birth of mice, suggesting that Dux may not be the primary transitioning factor in fertilized embryos. It has been reported that for 2-cell embryos derived from somatic cell nuclear transfer (SCNT) and whose expression of ZGA genes and repeats was aberrant, Dux improved the reprogramming efficiency by correcting aberrant H3K9ac modification via its C-terminal domain. We confirmed that the overexpression of full-length Dux mRNA in SCNT embryos improved the efficiency of preimplantation development (62.16% vs. 41.26% with respect to controls) and also increased the expression of Zscan4 and MERVL. Furthermore, we found that the N-terminal double homeodomains of Dux were indispensable for Dux localization and function. The intermediate region was essential for MERVL and Zscan4 activation, and the C-terminal domain was important for elevating level of H3K27ac. Mutant Dux mRNA containing N-terminal double homeodomains with the intermediate region or the C-terminal domain also improved the preimplantation development of SCNT embryos. This is the first report focusing on distinguishing functional domains of Dux in embryos derived from SCNT., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Elongation of pig conceptuses is a dynamic process, requiring adequate nutrient provisions. Glutamine is used as an energy substrate and is involved in the activation of mechanistic target of rapamycin complex 1 (mTORC1) during porcine preimplantation development. However, the roles of glutamine have not been extensively studied past the blastocyst stage. Therefore, the objective of the current study was to determine if glutaminase (GLS), which is the rate-limiting enzyme in glutamine metabolism, was necessary for conceptus elongation to proceed and was involved in mTORC1 activation. The CRISPR/Cas9 system was used to induce loss-of-function mutations in the GLS gene of porcine fetal fibroblasts. Wild type (GLS+/+) and knockout (GLS-/-) fibroblasts were used as donor cells for somatic cell nuclear transfer, and GLS+/+ and GLS-/- blastocyst-stage embryos were transferred into surrogates. On day 14 of gestation, GLS+/+ conceptuses primarily demonstrated filamentous morphologies, and GLS-/- conceptuses exhibited spherical, ovoid, tubular, and filamentous morphologies. Thus, GLS-/- embryos were able to elongate despite the absence of GLS protein and minimal enzyme activity. Furthermore, spherical GLS-/- conceptuses had increased abundance of transcripts related to glutamine and glutamate metabolism and transport compared to filamentous conceptuses of either genotype. Differences in phosphorylation of mTORC1 components and targets were not detected regarding conceptus genotype or morphology, but abundance of two transcriptional targets of mTORC1, cyclin D1, and peroxisome proliferator-activated receptor gamma coactivator 1-alpha was increased in spherical conceptuses. Therefore, porcine GLS is not essential for conceptus elongation and is not required for mTORC1 activation at this developmental timepoint., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The efficiency of somatic cell nuclear transfer (scNT) for production of viable offspring is relatively low as compared to in vitro fertilization (IVF), presumably due to deficiencies in epigenetic reprogramming of the donor cell genome. Such defects may also involve the population of small non-coding RNAs (sncRNAs), which are important during early embryonic development. The objective of this study was to examine dynamic changes in relative abundance of sncRNAs during the maternal-to-embryonic transition (MET) in bovine embryos produced by scNT as compared to IVF by using RNA sequencing. When comparing populations of miRNA in scNT versus IVF embryos, only miR-2340, miR-345, and miR34a were differentially expressed in morulae, though many more miRNAs were differentially expressed when comparing across developmental stages. Also of interest, distinct populations of piwi-interacting like RNAs (pilRNAs) were identified in bovine embryos prior to and during embryonic genome activation (EGA) as compared bovine embryos post-EGA and differentiated cells. Overall, sncRNA sequencing analysis of preimplantation embryos revealed largely similar profiles of sncRNAs for IVF and scNT embryos at the 2-cell, 8-cell, morula, and blastocyst stages of development. However, these sncRNA profiles, including miRNA, piRNA, and tRNA fragments, were notably distinct prior to and after completion of the MET., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Developmental arrest of somatic cell nuclear transfer (SCNT) embryos first occurs at zygotic/embryonic genome activation (ZGA/EGA), which is critical for preimplantation development. However, study on transcriptome of SCNT embryos during ZGA/EGA is limited. In the present study, we performed RNA sequencing (RNA-seq) of the eight-cell SCNT embryos in goat and provide cross-species analysis of transcriptional activity of SCNT embryos during ZGA/EGA in mice, human, bovine, and goat. RNA-seq data revealed 3966 differentially expressed genes (DEGs) failed to be reprogrammed or activated during EGA of SCNT embryos in goat. Series test of cluster analysis showed four clusters of DEGs and similar changes of the clusters in the four species. Specifically, genes in cluster 3 were somehow upregulated compared with the donor cells and the in vitro fertilization embryo. Moreover, the histone methylation key players and N6-methyladenosine modifiers (SUV39H1, SETDB1, SETD2, KDM5B, IGF2BP1, and YTHDF2) were differentially expressed in SCNT embryos of all species. Finally, we identified three modules correlated with the development of SCNT embryos in mice and screened 288 genes (such as BTG4, WEE1, KLF3, and USP21) that are likely critical for SCNT reprogramming using weighted gene correlation network analysis. Our data will broaden the current understanding of transcriptome activity during stochastic reprogramming events and provide an excellent source for future studies., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)