Nr5a2 is dispensable for zygotic genome activation but essential for morula development.

Early embryogenesis is driven by transcription factors (TFs) that first activate the zygotic genome and then specify the lineages constituting the blastocyst. Although the TFs specifying the blastocyst's lineages are well characterized, those playing earlier roles remain poorly defined. Using mouse models of the TF Nr5a2, we show that Nr5a2-/- embryos arrest at the early morula stage and exhibit altered lineage specification, frequent mitotic failure, and substantial chromosome segregation defects. Although NR5A2 plays a minor but measurable role during zygotic genome activation, it predominantly acts as a master regulator at the eight-cell stage, controlling expression of lineage-specifying TFs and genes involved in mitosis, telomere maintenance, and DNA repair. We conclude that NR5A2 coordinates proliferation, genome stability, and lineage specification to ensure correct morula development.

Branching topology of the human embryo transcriptome revealed by Entropy Sort Feature Weighting.

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.

Maternal Ezh1/2 deficiency impairs the function of mitochondria in mouse oocytes and early embryos.

Maternal histone methyltransferase is critical for epigenetic regulation and development of mammalian embryos by regulating histone and DNA modifications. Here, we reported a novel mechanism by revealing the critical effects of maternal Ezh1/2 deletion on mitochondria in MII oocytes and early embryos in mice. We found that Ezh1/2 knockout in mouse MII oocytes impaired the structure of mitochondria and decreased its number, but membrane potential and respiratory function of mitochondrion were increased. The similar effects of Ezh1/2 deletion have been observed in 2-cell and morula embryos, indicating that the effects of maternal Ezh1/2 deficiency on mitochondrion extend to early embryos. However, the loss of maternal Ezh1/2 resulted in a severe defect of morula: the number, membrane potential, respiratory function, and ATP production of mitochondrion dropped significantly. Content of reactive oxygen species was raised in both MII oocytes and early embryos, suggesting maternal Ezh1/2 knockout induced oxidative stress. In addition, maternal Ezh1/2 ablation interfered the autophagy in morula and blastocyst embryos. Finally, maternal Ezh1/2 deletion led to cell apoptosis in blastocyst embryos in mice. By analyzing the gene expression profile, we revealed that maternal Ezh1/2 knockout affected the expression of mitochondrial related genes in MII oocytes and early embryos. The chromatin immunoprecipitation-polymerase chain reaction assay demonstrated that Ezh1/2 directly regulated the expression of genes Fxyd6, Adpgk, Aurkb, Zfp521, Ehd3, Sgms2, Pygl, Slc1a1, and Chst12 by H3K27me3 modification. In conclusion, our study revealed the critical effect of maternal Ezh1/2 on the structure and function of mitochondria in oocytes and early embryos, and suggested a novel mechanism underlying maternal epigenetic regulation on early embryonic development through the modulation of mitochondrial status.

In vitro generation of mouse morula-like cells.

Generating cells with the molecular and functional properties of embryo cells and with full developmental potential is an aim with fundamental biological significance. Here we report the in vitro generation of mouse transient morula-like cells (MLCs) via the manipulation of signaling pathways. MLCs are molecularly distinct from embryonic stem cells (ESCs) and cluster instead with embryo 8- to 16-cell stage cells. A single MLC can generate a blastoid, and the efficiency increases to 80% when 8-10 MLCs are used. MLCs make embryoids directly, efficiently, and within 4 days. Transcriptomic analysis shows that day 4-5 MLC-derived embryoids contain the cell types found in natural embryos at early gastrulation. Furthermore, MLCs introduced into morulae segregate into epiblast (EPI), primitive endoderm (PrE), and trophectoderm (TE) fates in blastocyst chimeras and have a molecular signature indistinguishable from that of host embryo cells. These findings represent the generation of cells that are molecularly and functionally similar to the precursors of the first three cell lineages of the embryo.

Characterization of the long noncoding RNA transcriptome in human preimplantation embryo development.

PURPOSE: Infertility remains a human health burden globally. Only a fraction of embryos produced via assisted reproductive technologies (ARTs) develop to the blastocyst stage in vitro. lncRNA abundance changes significantly during human early embryonic development, indicating vital regulatory roles of lncRNAs in this process. The aim of this study is to obtain insights into the transcriptional basis of developmental events. METHODS: scRNA-seq data and SUPeR-seq data were used to investigate the lncRNA profiles of human preimplantation embryos. The top 50 highly expressed unique and shared lncRNAs in each stage of preimplantation development were identified. Comparative analysis of the two datasets was used to verify the consistent expression patterns of the lncRNAs. Differentially expressed lncRNAs were identified and subjected to functional enrichment analysis. RESULTS: The lncRNA profiles of human preimplantation embryos in the E-MTAB-3929 dataset were similar to those in the GSE71318 dataset. The ratios of overlap among the top 50 highly expressed lncRNAs between two pairs of stages (2-cell stage vs. 4-cell stage and 8-cell stage vs. morula) were aberrantly low compared with those between other stages. Each stage of preimplantation development exhibited unique and shared lncRNAs among the top 50 highly expressed lncRNAs. Among the between-group comparisons, the 2-cell stage vs. 4-cell stage showed the highest number of differentially expressed lncRNAs. Functional enrichment analysis revealed that differentially expressed lncRNAs and their associated super enhancers and RNA binding proteins (RBPs) are closely involved in regulating embryonic development. These lncRNAs could function as important cell markers for distinguishing fetal germ cells. CONCLUSIONS: Our study paves the way for understanding the regulation of developmental events, which might be beneficial for improved reproductive outcomes.

The proteomic analysis of bovine embryos developed in vivo or in vitro reveals the contribution of the maternal environment to early embryo.

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.

High-resolution ribosome profiling reveals translational selectivity for transcripts in bovine preimplantation embryo development.

High-resolution ribosome fractionation and low-input ribosome profiling of bovine oocytes and preimplantation embryos has enabled us to define the translational landscapes of early embryo development at an unprecedented level. We analyzed the transcriptome and the polysome- and non-polysome-bound RNA profiles of bovine oocytes (germinal vesicle and metaphase II stages) and early embryos at the two-cell, eight-cell, morula and blastocyst stages, and revealed four modes of translational selectivity: (1) selective translation of non-abundant mRNAs; (2) active, but modest translation of a selection of highly expressed mRNAs; (3) translationally suppressed abundant to moderately abundant mRNAs; and (4) mRNAs associated specifically with monosomes. A strong translational selection of low-abundance transcripts involved in metabolic pathways and lysosomes was found throughout bovine embryonic development. Notably, genes involved in mitochondrial function were prioritized for translation. We found that translation largely reflected transcription in oocytes and two-cell embryos, but observed a marked shift in the translational control in eight-cell embryos that was associated with the main phase of embryonic genome activation. Subsequently, transcription and translation become more synchronized in morulae and blastocysts. Taken together, these data reveal a unique spatiotemporal translational regulation that accompanies bovine preimplantation development.

Establishment of 3D chromatin structure after fertilization and the metabolic switch at the morula-to-blastocyst transition require CTCF.

The eukaryotic genome is organized in 3D at different scales. This structure is driven and maintained by different chromatin states and by architectural factors, such as the zinc finger protein CTCF. Zygotic genome structure is established de novo after fertilization, but its impact during the first stages of mammalian development is unclear. We show that deletion of Ctcf in mouse embryos impairs the establishment of chromatin structure, but the first cell fate decision is unperturbed and embryos are viable until the late blastocyst. Furthermore, maternal CTCF is not necessary for development. Gene expression changes in metabolic and protein homeostasis programs that occur during the morula-to-blastocyst transition depend on CTCF. However, these changes do not correlate with disruption of chromatin but with binding of CTCF to the promoter of downregulated genes. Our results show that CTCF regulates both 3D genome organization and transcription during mouse preimplantation development, but as independent processes.

The trophectoderm acts as a niche for the inner cell mass through C/EBPα-regulated IL-6 signaling.

IL-6 has been shown to be required for somatic cell reprogramming into induced pluripotent stem cells (iPSCs). However, how Il6 expression is regulated and whether it plays a role during embryo development remains unknown. Here, we describe that IL-6 is necessary for C/EBPα-enhanced reprogramming of B cells into iPSCs but not for B cell to macrophage transdifferentiation. C/EBPα overexpression activates both Il6 and Il6ra genes in B cells and in PSCs. In embryo development, Cebpa is enriched in the trophectoderm of blastocysts together with Il6, while Il6ra is mostly expressed in the inner cell mass (ICM). In addition, Il6 expression in blastocysts requires Cebpa. Blastocysts secrete IL-6 and neutralization of the cytokine delays the morula to blastocyst transition. The observed requirement of C/EBPα-regulated IL-6 signaling for pluripotency during somatic cell reprogramming thus recapitulates a physiologic mechanism in which the trophectoderm acts as niche for the ICM through the secretion of IL-6.

Maternal DDB1 regulates apoptosis and lineage differentiation in porcine preimplantation embryos.

CONTEXT: Maternal-effect genes (MEGs) play a critical role in modulating both cellular and molecular biology events in preimplantation embryonic development. Damage-specific DNA binding protein 1 (DDB1) is a gene that participates in meiotic resumption, ovulation, and embryonic stem cell maintenance. Its function in preimplantation development is not well-studied. AIMS: We aimed to explore the expression pattern, genomic heritage, and potential molecular mechanisms of DDB1 in preimplantation embryos in porcine. METHODS: In this study, RNA interference, microinjection, RT-qPCR, immunofluorescence staining and single-cell RNA sequencing were used to explore the molecular function of DDB1 in porcine preimplantation embryos. KEY RESULTS: DDB1 was found to be expressed in germinal vesicle (GV) and Meiosis II (MII) oocytes and in preimplantation embryos. We confirmed it is a MEG. DDB1 -deficient blastocysts had a significantly reduced number of trophectoderm cells, an increased apoptotic cell number and increased apoptosis index. According to a next-generation sequencing (NGS) analysis, 236 genes (131 upregulated and 105 downregulated) significantly changed in the DDB1 -deficient morula. The myeloid leukaemia factor 1 (MLF1 ) and yes-associated protein 1 (YAP1 ) expressions were significantly upregulated and downregulated respectively, in the DDB1 -deficient morula. In combination with the decreased expression of TEAD4 , CDX2 , GATA3 , OCT4 , and NANOG and the increased expression of SOX2 in the blastocyst, DDB1 may play a role in determining lineage differentiation and pluripotency maintenance. CONCLUSIONS: DDB1 is a MEG and it plays a crucial role in porcine preimplantation embryonic development. IMPLICATIONS: This study provides a theoretical basis for further understanding the molecular mechanisms of preimplantation embryo development.

Regulation of Tjp1 mRNA by CPEB2 for tight junction assembly in mouse blastocyst.

Cytoplasmic polyadenylation element-binding protein 2 (CPEB2) is an mRNA-binding protein that regulates the cytoplasmic polyadenylation of mRNA and is required for tight junction (TJ) assembly in the trophectoderm epithelium during porcine preimplantation development. However, the regulatory mechanism underlying TJ assembly by CPEB2 has not been examined. The aim of this study was to elucidate how Cpeb2 regulates the subcellular localisation and stabilisation of Tjp1 mRNA for TJ biogenesis during mouse preimplantation. CPEB2 was detected in nuclei during the early stages of development and was localised at apical cell membranes from the morula stage onwards. In the Cpeb2 knockdown (KD), we observed reduced blastocyst formation with impaired TJs, defective inner cell mass development in the blastocyst outgrowth assay, and loss of pregnancy after embryo transfer. More importantly, Tjp1 mRNA was localised apically in the outer cells of control morulae but not in the Cpeb2 KD embryos, indicating that CPEB2 mediated the translocalisation of Tjp1 mRNA from the nuclei. Finally, in the control embryos, the length of the Tjp1 mRNA poly (A) tail was varied, while only a single peak was detected in the Cpeb2 KD embryos. These findings suggest that the binding of CPEB2 to the cytoplasmic polyadenylation element in the 3'-UTR can confer stability on Tjp1 mRNA and translational regulation. In summary, we demonstrated for the first time that CPEB2 mediates Tjp1 mRNA stabilisation and subcellular localisation for TJ assembly during mouse blastocyst formation.

EXOSC10/Rrp6 is essential for the eight-cell embryo/morula transition.

The conserved 3'-5' exoribonuclease EXOSC10/Rrp6 is required for gametogenesis, brain development, erythropoiesis and blood cell enhancer function. The human ortholog is essential for mitosis in cultured cancer cells. Little is known, however, about the role of Exosc10 during embryo development and organogenesis. We generated an Exosc10 knockout model and find that Exosc10-/- mice show an embryonic lethal phenotype. We demonstrate that Exosc10 maternal wild type mRNA is present in mutant oocytes and that the gene is expressed during all stages of early embryogenesis. Furthermore, we observe that EXOSC10 early on localizes to the periphery of nucleolus precursor bodies in blastomeres, which is in keeping with the protein's role in rRNA processing and may indicate a function in the establishment of chromatin domains during initial stages of embryogenesis. Finally, we infer from genotyping data for embryonic days e7.5, e6.5 and e4.5 and embryos cultured in vitro that Exosc10-/- mutants arrest at the eight-cell embryo/morula transition. Our results demonstrate a novel essential role for Exosc10 during early embryogenesis, and they are consistent with earlier work showing that impaired ribosome biogenesis causes a developmental arrest at the morula stage.

Cross-species meta-analysis of transcriptome changes during the morula-to-blastocyst transition: metabolic and physiological changes take center stage.

The morula-to-blastocyst transition (MBT) culminates with formation of inner cell mass (ICM) and trophectoderm (TE) lineages. Recent studies identified signaling pathways driving lineage specification, but some features of these pathways display significant species divergence. To better understand evolutionary conservation of the MBT, we completed a meta-analysis of RNA sequencing data from five model species and ICMTE differences from four species. Although many genes change in expression during the MBT within any given species, the number of shared differentially expressed genes (DEGs) is comparatively small, and the number of shared ICMTE DEGs is even smaller. DEGs related to known lineage determining pathways (e.g., POU5F1) are seen, but the most prominent pathways and functions associated with shared DEGs or shared across individual species DEG lists impact basic physiological and metabolic activities, such as TCA cycle, unfolded protein response, oxidative phosphorylation, sirtuin signaling, mitotic roles of polo-like kinases, NRF2-mediated oxidative stress, estrogen receptor signaling, apoptosis, necrosis, lipid and fatty acid metabolism, cholesterol biosynthesis, endocytosis, AMPK signaling, homeostasis, transcription, and cell death. We also observed prominent differences in transcriptome regulation between ungulates and nonungulates, particularly for ICM- and TE-enhanced mRNAs. These results extend our understanding of shared mechanisms of the MBT and formation of the ICM and TE and should better inform the selection of model species for particular applications.

Extracellular matrix mediates moruloid-blastuloid morphodynamics in malignant ovarian spheroids.

Ovarian cancer metastasizes into peritoneum through dissemination of transformed epithelia as multicellular spheroids. Harvested from the malignant ascites of patients, spheroids exhibit startling features of organization typical to homeostatic glandular tissues: lumen surrounded by smoothly contoured and adhered epithelia. Herein, we demonstrate that cells of specific ovarian cancer lines in suspension, aggregate into dysmorphic solid "moruloid" clusters that permit intercellular movement, cell penetration, and interspheroidal coalescence. Moruloid clusters subsequently mature into "blastuloid" spheroids with smooth contours, a temporally dynamic lumen and immotile cells. Blastuloid spheroids neither coalesce nor allow cell penetration. Ultrastructural examination reveals a basement membrane-like extracellular matrix coat on the surface of blastuloid, but not moruloid, spheroids. Quantitative proteomics reveals down-regulation in ECM protein Fibronectin-1 associated with the moruloid-blastuloid transition; immunocytochemistry also confirms the relocalization of basement membrane ECM proteins: collagen IV and laminin to the surface of blastuloid spheroids. Fibronectin depletion accelerates, and enzymatic basement membrane debridement impairs, lumen formation, respectively. The regulation by ECM dynamics of the morphogenesis of cancer spheroids potentially influences the progression of the disease.

Enhanced Synergistic-Antioxidant Activity of Melatonin and Tretinoin by Co-encapsulation into Amphiphilic Chitosan Nanocarriers: During Mice In Vitro Matured Oocyte/Morula-Compact Stage Embryo Culture Model.

The use of exogenous antioxidants or the combination of them during in vitro oocyte/embryo culture media is reasonable. Co-delivery by nanocarrier has been designed to overcome the limitations of combining them traditionally. In this work, amphiphilic chitosan nanocarrier (ACN) was applied to co-encapsulate melatonin (Mel) and tretinoin (TTN) by the self-assembled method and evaluate their synergistic antioxidant efficacy in mice oocytes/embryos. The formation of single/dual-ACN was confirmed by Fourier-transformed infrared spectroscopy (FT-IR). The average particle diameter, size distribution, polydispersity index (PDI), and zeta potential of them were measured by dynamic light scattering (DLS), and the morphology was evaluated by TEM and SEM technologies. Also, the encapsulation efficiency (EE%) and drug loading content (DL%) of the nanocapsules were determined by UV-vis spectrophotometry. Studies of the in vitro release showed a continued drug release without any bursting effect of Mel+TTN-ACNs compared with single Mel/TTN-ACNs. Then, in both experiments, nuclear staining (Aceto-orcein and Hoechst 33342), fluorescent staining of H2DCFDA, chemiluminescence test, and qRT-PCR technique were performed as in vitro toxicity studies. The results of all these evaluations demonstrated that the dual delivery of Mel and TTN could accumulate a safety (without high-dose toxicity) synergistic anti-oxidative effect in oocyte/embryo by passive controlled, and inhibit intra/extracellular ROS levels by an enhanced intracellular penetration.

Reverse transcription priming methods affect normalisation choices for gene expression levels in oocytes and early embryos.

Mammalian oocytes and embryos rely exclusively on maternal mRNAs to accomplish early developmental processes. Since oocytes and early embryos are transcriptionally silent after meiotic resumption, most of the synthesised maternal mRNA does not undergo immediate translation but is instead stored in the oocyte. Quantitative RT-PCR is commonly used to quantify mRNA levels, and correct quantification relies on reverse transcription and the choice of reference genes. Different methods for reverse transcription may affect gene expression determination in oocytes. In this study, we examined the suitability of either random or oligo(dT) primers for reverse transcription to be used for quantitative RT-PCR. We further looked for changes in poly(A) length of the maternal mRNAs during oocyte maturation. Our data indicate that depending on the method of reverse transcription, the optimal combination of reference genes for normalisation differed. Surprisingly, we observed a shortening of the poly(A) tail lengths of maternal mRNA as oocytes progressed from germinal vesicle to metaphase II. Overall, our findings suggest dynamic maternal regulation of mRNA structure and gene expression during oocyte maturation and early embryo development.

Abnormal cleavage is involved in the self-correction of bovine preimplantation embryos.

Chromosome instability leading to aneuploidy during early cleavage is well known in humans and cattle. Partial compaction (PC), which occurs only in some blastomeres, is suggested as a self-correction mechanism through which human embryos avoid aneuploid mosaicism. Partially compacted embryos show abnormal cleavages more frequently during early development; however, the mechanism by which blastomeres are excluded has not been elucidated. Here, we confirmed PC in approximately half of the tested bovine embryos, similar to that in human embryos. DNA sequencing of single-cell and intact embryos revealed that the morulae that excluded some blastomeres had euploidy, but many of the excluded blastomeres had aneuploidy. Time-lapse imaging of zygotes without the zona pellucida revealed that the excluded blastomeres underwent reverse and direct cleavages, which are abnormal cleavages, more frequently than the blastomeres involved in compaction. These results suggest the potential role of abnormal cleavage in the self-correction mechanism during the development of mammalian preimplantation embryos.

Mitochondrial reactive oxygen species regulate mitochondrial biogenesis in porcine embryos.

The number of mitochondria in blastocysts is a potential marker of embryo quality. However, the molecular mechanisms governing the mitochondrial number in embryos are unclear. This study was conducted to investigate the effect of reduced mitochondrial reactive oxygen species (ROS) levels on mitochondrial biogenesis in porcine embryos. Oocytes were collected from gilt ovaries and activated to generate over 4 cell-stage embryos at day 2 after activation. These embryos were cultured in media containing either 0.1 µM MitoTEMPOL (MitoT), 0.5 µM Mitoquinol (MitoQ), or vehicle (ethanol) for 5 days to determine the rate of development to the blastocyst stage. The mitochondrial number in blastocysts was evaluated by real-time polymerase chain reaction (PCR). Five days after activation, the embryos (early morula stage) were subjected to immunostaining to determine the expression levels of NRF2 in the nucleus. In addition, the expression levels of PGC1α and TFAM in the embryos were examined by reverse transcription PCR. One day of incubation with the antioxidants reduced the ROS content in the embryos but did not affect the rate of development to the blastocyst stage. Blastocysts developed in medium containing MitoT had lower mitochondrial DNA copy numbers and ATP content, whereas MitoQ showed similar but insignificantly trends. Treatment of embryos with either MitoT or MitoQ decreased the expression levels of NRF2 in the nucleus and levels of PGC1α and TFAM. These findings indicate that reductions in mitochondrial ROS levels are associated with low mitochondrial biogenesis in embryos.

Live visualisation of electrolytes during mouse embryonic development using electrolyte indicators.

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.

Small Noncoding RNA Signatures for Determining the Developmental Potential of an Embryo at the Morula Stage.

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.