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Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac 1 . Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human 2 – 5 and cow 6 . Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical–basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos. Comparative analysis of human, cow and mouse embryos shows that a mechanism involving atypical protein kinase C initiates the trophectoderm program during the morula stage in these three species.

Mammalian sperm and oocytes have different epigenetic landscapes and are organized in different fashions. After fertilization, the initially distinct parental epigenomes become largely equalized with the exception of certain loci, including imprinting control regions. How parental chromatin becomes equalized and how imprinting control regions escape from this reprogramming is largely unknown. Here we profile parental allele-specific DNase I hypersensitive sites in mouse zygotes and morula embryos, and investigate the epigenetic mechanisms underlying these allelic sites. Integrated analyses of DNA methylome and tri-methylation at lysine 27 of histone H3 (H3K27me3) chromatin immunoprecipitation followed by sequencing identify 76 genes with paternal allele-specific DNase I hypersensitive sites that are devoid of DNA methylation but harbour maternal allele-specific H3K27me3. Interestingly, these genes are paternally expressed in preimplantation embryos, and ectopic removal of H3K27me3 induces maternal allele expression. H3K27me3-dependent imprinting is largely lost in the embryonic cell lineage, but at least five genes maintain their imprinted expression in the extra-embryonic cell lineage. The five genes include all paternally expressed autosomal imprinted genes previously demonstrated to be independent of oocyte DNA methylation. Thus, our study identifies maternal H3K27me3 as a DNA methylation-independent imprinting mechanism. Analysis of parental allele-specific chromatin accessibility genome-wide in mouse zygotes and morula embryos, and investigation of the epigenetic mechanisms underlying these allelic sites, identifying maternal H3K27me3 as a DNA methylation-independent mechanism for genomic imprinting. Non-canonical genomic imprinting After fertilization in mammals, there is asymmetry in the epigenetic landscapes of paternal and maternal chromatin, which becomes largely equalized during subsequent development with the exception of imprinted genes. Here, Yi Zhang and colleagues have profiled parental allele-specific chromatin accessibility genome-wide in zygotes and morula embryos, together with analysing DNA methylation and histone H3K27me3 patterns. They find that H3K27me3 is a DNA-methylation-independent mechanism for repression of the maternal allele in genomic imprinting and can identify novel imprinted genes that are dependent on this non-canonical mechanism.

Single-cell RNA sequencing and weighted gene co-expression network analysis are used to study transcriptome change in pre-implantation embryos and oocytes; this reveals a conserved genetic program between human and mouse but with different developmental specificity and timing, and conserved hub genes that may be key in pre-implantation development. The early embryonic transcriptome This study of early embryonic development uses single-cell RNA sequencing and weighted gene co-expression network analysis (WGCNA) to obtain a detailed gene expression profile of human and mouse pre-implantation embryos and oocytes. The authors identify a small number of key functional modules that shape a sequential order of transcriptional changes in various pathways. They also found key hub genes that are conserved between human and mouse networks and argue that these genes may be key players in driving mammalian pre-implantation. Mammalian pre-implantation development is a complex process involving dramatic changes in the transcriptional architecture 1 , 2 , 3 , 4 . We report here a comprehensive analysis of transcriptome dynamics from oocyte to morula in both human and mouse embryos, using single-cell RNA sequencing. Based on single-nucleotide variants in human blastomere messenger RNAs and paternal-specific single-nucleotide polymorphisms, we identify novel stage-specific monoallelic expression patterns for a significant portion of polymorphic gene transcripts (25 to 53%). By weighted gene co-expression network analysis 5 , 6 , we find that each developmental stage can be delineated concisely by a small number of functional modules of co-expressed genes. This result indicates a sequential order of transcriptional changes in pathways of cell cycle, gene regulation, translation and metabolism, acting in a step-wise fashion from cleavage to morula. Cross-species comparisons with mouse pre-implantation embryos reveal that the majority of human stage-specific modules (7 out of 9) are notably preserved, but developmental specificity and timing differ between human and mouse. Furthermore, we identify conserved key members (or hub genes) of the human and mouse networks. These genes represent novel candidates that are likely to be key in driving mammalian pre-implantation development. Together, the results provide a valuable resource to dissect gene regulatory mechanisms underlying progressive development of early mammalian embryos.

Autophagy is a system that contributes to cellular homeostasis by degrading intracellular proteins and organelles. Autophagy is essential for the preimplantation development of mammalian embryos, lack of which results in developmental arrest at the 4/8-cell stages. The role of autophagy beyond the compaction stage remains insufficiently explored. In this study, we investigated the role of autophagy after the 4/8-cell stages in mice using chloroquine (CQ), an autophagy inhibitor. CQ treatment from the 4/8-cell to morula stage impaired development, reducing the number of Cdx2-positive cells, an effect rescued by amino acid (AA) supplementation. CQ treatment also downregulated TFAP2C, an upstream regulator of Cdx2,which was similarly restored by AA supplementation. Consistently, autophagy at this stage showed higher activity in the outer cells and lower activity in the inner cells of the embryo. Treatment with XMU-MP-1, an MST1/2 inhibitor targeting the Hippo signaling pathway, disrupted this spatial regulation by inducing autophagy in the inner cells. Stage-specific staining revealed temporal and positional regulation of autophagy activity. These findings illustrate that autophagy during the morula stage promotes differentiation into the trophectoderm by supplying AAs, a process regulated by the Hippo signaling pathway.

Background Despite many improvements with in vitro culture systems, the quality and developmental ability of mammalian embryos produced in vitro are still lower than their in vivo counterparts. Though previous studies have evidenced differences in gene expression between in vivo- and in vitro -derived bovine embryos, there is no comparison at the protein expression level. Results A total of 38 pools of grade-1 quality bovine embryos at the 4–6 cell, 8–12 cell, morula, compact morula, and blastocyst stages developed either in vivo or in vitro were analyzed by nano-liquid chromatography coupled with label-free quantitative mass spectrometry, allowing for the identification of 3,028 proteins. Multivariate analysis of quantified proteins showed a clear separation of embryo pools according to their in vivo or in vitro origin at all stages. Three clusters of differentially abundant proteins (DAPs) were evidenced according to embryo origin, including 463 proteins more abundant in vivo than in vitro across development and 314 and 222 proteins more abundant in vitro than in vivo before and after the morula stage, respectively. The functional analysis of proteins found more abundant in vivo showed an enrichment in carbohydrate metabolism and cytoplasmic cellular components. Proteins found more abundant in vitro before the morula stage were mostly localized in mitochondrial matrix and involved in ATP-dependent activity, while those overabundant after the morula stage were mostly localized in the ribonucleoprotein complex and involved in protein synthesis. Oviductin and other oviductal proteins, previously shown to interact with early embryos, were among the most overabundant proteins after in vivo development. Conclusions The maternal environment led to higher degradation of mitochondrial proteins at early developmental stages, lower abundance of proteins involved in protein synthesis at the time of embryonic genome activation, and a global upregulation of carbohydrate metabolic pathways compared to in vitro production. Furthermore, embryos developed in vivo internalized large amounts of oviductin and other proteins probably originated in the oviduct as soon as the 4–6 cell stage. These data provide new insight into the molecular contribution of the mother to the developmental ability of early embryos and will help design better in vitro culture systems.

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity. In fast proliferating embryonic stem cells (ESC) the DNA damage response is activated by mechanisms that are as yet elusive. Here, Ahuja et al. link the DNA damage response to replication stress in mouse ESCs, caused by a short G1 phase, and propose fork remodelling as maintaining genome stability in embryos.

In human embryos, major zygotic genome activation (ZGA) initiates at the 8-cell (8C) blastomere stage, marking the start of the ontogenesis program. Recent advancements have shown that primed human pluripotent stem cells (hPSCs) can be reprogrammed to 8C-like cells (8CLCs) with totipotent characteristics in vitro. However, the key regulators driving this transition remain largely unexplored. In this study, we identify OTX2 as a key factor that establishes a repressive barrier to the induction of 8CLCs from primed hPSCs. Our findings reveal that OTX2 deletion greatly enhances the generation of TPRX1-EGFP + 8CLCs, which closely resemble the transcriptomic profiles and epigenetic landscape of 8C/morula embryos. Notably, these OTX2-deleted 8CLCs exhibit improved bidirectional differentiation potential and contribute to both embryonic and extraembryonic tissues in chimeric embryos. Mechanistically, OTX2 regulates both naive and totipotent state transition, but exerts its predominant effect on the latter by binding to loci of key 8C-specific regulators. Collectively, our findings define a critical role for OTX2 in regulating totipotency and establish a foundational framework for generating 8CLCs from primed hPSCs in vitro, offering significant insights for stem cell biology and regenerative medicine. OTX2 acts as a barrier to the formation of human 8-cell-like and morula-like cells in vitro. Deleting OTX2 enhances their induction efficiency and developmental potential, uncovering key mechanisms of totipotency regulation and early human development.

Studies have shown that some electrolytes, including Na + and K + , play important roles in embryonic development. However, these studies evaluated these electrolytes by using inhibitors or knockout mice, with no mention on the changes in the intracellular electrolyte concentrations during embryogenesis. In this study, we used the electrolyte indicators CoroNa Green AM and ION Potassium Green-2 AM to directly visualise intracellular concentrations of Na + and K + , respectively, at each embryonic developmental stage in mouse embryos. We directly observed intracellular electrolyte concentrations at the morula, blastocyst, and hatching stages. Our results revealed dynamic changes in intracellular electrolyte concentrations; we found that the intracellular Na + concentration decreased, while K + concentration increased during blastocoel formation. The degree of change in intensity in response to ouabain, an inhibitor of Na + /K + ATPase, was considered to correspond to the degree of Na + /K + ATPase activity at each developmental stage. Additionally, after the blastocyst stage, trophectoderm cells in direct contact with the blastocoel showed higher K + concentrations than in direct contact with inner cell mass, indicating that Na + /K + ATPase activity differs depending on the location in the trophectoderm. This is the first study to use CoroNa Green AM and ION Potassium Green-2 AM in mouse embryos and visualise electrolytes during embryonic development. The changes in electrolyte concentration observed in this study were consistent with the activity of Na + /K + ATPase reported previously, and it was possible to image more detailed electrolyte behaviour in embryo cells. This method can be used to improve the understanding of cell physiology and is useful for future embryonic development studies.

Somatic cell nuclear transfer (SCNT) is a valuable tool in regenerative medicine, yet its efficiency remains limited by epigenetic reprogramming barriers that have been partially corrected by global regulation of epigenetic enzymes. However, these approaches lack gene locus specificity and may disrupt normal gene regulation. Therefore, new strategies capable of broadly enhancing reprogramming fidelity are needed. Here, we demonstrate that overexpression of the pioneer transcription factor Nr5a2 in mouse SCNT embryos improves both zygotic genome activation and the morula-to-blastocyst transition, two major developmental barriers in SCNT, and enhances birth rates. Mechanistically, Nr5a2 recruits P300 to increase H3K27ac at genes with low expression, restoring transcriptional activity and promoting SCNT embryo development.

As part of the optimization of assisted reproductive technology programs, the aim of the study was to identify key small noncoding RNA (sncRNA) molecules that participate in maternal-to-zygotic transition and determine development potential and competence to form a healthy fetus. Small RNA deep sequencing followed by quantitative real-time RT-PCR was used to profile sncRNAs in 50 samples of spent culture medium from morula with different development potentials (no potential (degradation/developmental arrest), low potential (poor-quality blastocyst), and high potential (good/excellent quality blastocyst capable of implanting and leading to live birth)) obtained from 27 subfertile couples who underwent in vitro fertilization. We have shown that the quality of embryos at the morula stage is determined by secretion/uptake rates of certain sets of piRNAs and miRNAs, namely hsa_piR_011291, hsa_piR_019122, hsa_piR_001311, hsa_piR_015026, hsa_piR_015462, hsa_piR_016735, hsa_piR_019675, hsa_piR_020381, hsa_piR_020485, hsa_piR_004880, hsa_piR_000807, hsa-let-7b-5p, and hsa-let-7i-5p. Predicted gene targets of these sncRNAs included those globally decreased at the 8-cell–morula–blastocyst stage and critical to early embryo development. We show new original data on sncRNA profiling in spent culture medium from morula with different development potential. Our findings provide a view of a more complex network that controls human embryogenesis at the pre-implantation stage. Further research is required using reporter analysis to experimentally confirm interactions between identified sncRNA/gene target pairs.