Mouse Embryo Development Research Articles

VISTA Enhancer browser: an updated database of tissue-specific developmental enhancers.

Regulatory elements (enhancers) are major drivers of gene expression in mammals and harbor many genetic variants associated with human diseases. Here, we present an updated VISTA Enhancer Browser (https://enhancer.lbl.gov), a database of transgenic enhancer assays conducted in developing mouse embryos in vivo. Since the original publication in 2007, the database grew nearly 20-fold from 250 to over 4500 experiments and currently harbors over 23500 images. The updated database provides structured information on experiments conducted at different stages of embryonic development, including enhancer activities of human pathogenic and synthetic variants and sequences derived from a variety of species. In addition to manually curated results of thousands of individual experiments, the new database also features hundreds of manually curated comparisons between alleles. The VISTA Enhancer Browser provides a crucial resource for study of human genetic variation, gene regulation and developmental biology.

Maternal total sleep deprivation causes oxidative stress and mitochondrial dysfunction in oocytes associated with fertility decline in mice.

Previous studies have shown sleep deprivation is increasingly reported as one of the causes of female infertility. However, how and by what relevant mechanisms it affects female fertility remains unclear. In this study, female mice underwent 72 hours of total sleep deprivation (TSD) caused by rotating wheel or 2 different controls: a stationary wheel, or forced movement at night. Even though, there was no significant difference in the number of eggs ovulated by the TSD mice compared to the control groups. Overall levels of estrogen and FSH were lower throughout the estrus cycle. A total of 42 genes showed significant differential expression in GV oocytes after TSD by RNA sequencing (RNA-Seq). These included genes were enriched in gene ontology terms of mitochondrial protein complex, oxidoreductase activity, cell division, cell cycle G1/S phase transition, as well as others. The increased concentrations of reactive oxygen species (ROS) in germinal vesicle (GV) and metaphase II (MII) oocytes from TSD mice were observed, which might be induced by impaired mitochondrial function caused by TSD. The GV oocytes displayed increased mitochondrial DNA (mtDNA) copy number and a significant transient increase in inner mitochondrial membrane potential (Δψm) from the TSD mice probably due to compensatory effect. In contrast, MII oocytes in the TSD group showed a decrease in the mtDNA copy number and a lower Δψm compared with the controls. Furthermore, abnormal distribution of mitochondria in the GV and MII oocytes was also observed in TSD mice, suggesting mitochondrial dysfunction. In addition, abnormal spindle and abnormal arrangement of chromosomes in MII oocytes were markedly increased in the TSD mice compared with the control mice. In conclusion, our results suggest that TSD significantly alters the oocyte transcriptome, contributing to oxidative stress and disrupted mitochondrial function, which then resulted in oocyte defects and impaired early embryo development in female mice.

Selective utilization of glucose metabolism guides mammalian gastrulation

The prevailing dogma for morphological patterning in developing organisms argues that the combined inputs of transcription factor networks and signalling morphogens alone generate spatially and temporally distinct expression patterns. However, metabolism has also emerged as a critical developmental regulator1–10, independent of its functions in energy production and growth. The mechanistic role of nutrient utilization in instructing cellular programmes to shape the in vivo developing mammalian embryo remains unknown. Here we reveal two spatially resolved, cell-type- and stage-specific waves of glucose metabolism during mammalian gastrulation by using single-cell-resolution quantitative imaging of developing mouse embryos, stem cell models and embryo-derived tissue explants. We identify that the first spatiotemporal wave of glucose metabolism occurs through the hexosamine biosynthetic pathway to drive fate acquisition in the epiblast, and the second wave uses glycolysis to guide mesoderm migration and lateral expansion. Furthermore, we demonstrate that glucose exerts its influence on these developmental processes through cellular signalling pathways, with distinct mechanisms connecting glucose with the ERK activity in each wave. Our findings underscore that—in synergy with genetic mechanisms and morphogenic gradients—compartmentalized cellular metabolism is integral in guiding cell fate and specialized functions during development. This study challenges the view of the generic and housekeeping nature of cellular metabolism, offering valuable insights into its roles in various developmental contexts.

TMEM132A regulates Wnt/β-catenin signaling through stabilizing LRP6 during mouse embryonic development

The Wnt/β-catenin signaling pathway is crucial for embryonic development and adult tissue homeostasis. Dysregulation of Wnt signaling is linked to various developmental anomalies and diseases, notably cancer. Although numerous regulators of the Wnt signaling pathway have been identified, their precise function during mouse embryo development remains unclear. Here, we revealed that TMEM132A is a crucial regulator of canonical Wnt/β-catenin signaling in mouse development. Mouse embryos lacking Tmem132a displayed a range of malformations, including open spina bifida, caudal truncation, syndactyly, and renal defects, similar to the phenotypes of Wnt/β-catenin mutants. Tmem132a knockdown in cultured cells suppressed canonical Wnt/β-catenin signaling. In developing mice, loss of Tmem132a also led to diminished Wnt/β-catenin signaling. Mechanistically, we showed that TMEM132A interacts with the Wnt co-receptor LRP6, thereby stabilizing it and preventing its lysosomal degradation. These findings shed light on a novel role for TMEM132A in regulating LRP6 stability and canonical Wnt/β-catenin signaling during mouse embryo development. This study provides valuable insights into the molecular intricacies of the Wnt signaling pathway. Further research may deepen our understanding of Wnt pathway regulation and offer its potential therapeutic applications.

Loss of Cell-Cell Contact Inhibits Cellular Differentiation of α-Catenin Knock Out P19 Embryonal Carcinoma Cells and Their Colonization into the Developing Mouse Embryos.

Cadherin-catenin cell-cell adhesion complexes, composed of cadherin, β-catenin or plakoglobin, and α-catenin (α-cat) molecules, are crucial for maintaining cell-cell contact and are commonly referred to as "adherens junctions (AJs)." Inactivating this system leads to loss of cell-cell contact and developmental arrest in early embryos. However, it remains unclear whether the loss of cell-cell contact affects the differentiation of embryonic cells. In this study, we explored the use of a murine embryonal carcinoma cell line, P19, as an in vitro model for early embryogenesis. P19 cells easily form embryoid bodies (EBs) and are susceptible to cellular differentiation in response to retinoic acid (RA) and teratoma formation. Using CRISPR/Cas9 technology to disrupt the endogenous α-cat gene in P19 cells, we generated α-cat knockout (KO) cells that exhibited a loss of cell-cell contact. When cultivated on non-coated dishes, these α-cat KO cells formed EBs, but their structures were labile. In the RA-containing medium, the α-cat KO EBs failed to produce differentiated cells on their outer layer and continued to express SSEA-1, an antigen specific to pluripotent cells. Teratoma formation assays revealed an absence of overt differentiated cells in tumors derived from α-cat KO P19 cells. Aggregation assays revealed the inability of the KO cells to colonize into the zona pellucida-denuded 8-cell embryos. These findings suggest that the AJs are essential for promoting the early stages of cellular differentiation and for the colonization of early-developing embryos.

SpaGRA: Graph augmentation facilitates domain identification for spatially resolved transcriptomics

Recent advances in spatially resolved transcriptomics (SRT) have provided new opportunities for characterizing spatial structures of various tissues. Graph-based geometric deep learning have gained widespread adoption for spatial domain identification tasks. Currently, most methods define adjacency relation between cells or spots by their spatial distance in SRT data, which overlooks key biological interactions like gene expression similarities, and leads to inaccuracies in spatial domain identification. To tackle this challenge, we propose a novel method, SpaGRA (https://github.com/sunxue-yy/SpaGRA), for automatic multi-relationship construction based on graph augmentation. SpaGRA uses spatial distance as prior knowledge and dynamically adjusts edge weights with multi-head graph attention networks (GATs). This helps SpaGRA to uncover diverse node relationships and enhance message passing in geometric contrastive learning. Additionally, SpaGRA uses these multi-view relationships to construct negative samples, addressing sampling bias posed by random selection. Experimental results show that SpaGRA demonstrates superior domain identification performance on multiple datasets generated from different protocols. Using SpaGRA, we analyzed the functional regions in the mouse hypothalamus, identified key genes related to heart development in mouse embryos, and observed cancer-associated fibroblasts enveloping cancer cells in the latest Visium HD data. Overall, SpaGRA can effectively characterize spatial structures across diverse SRT datasets.

SALL2 regulates neural differentiation of mouse embryonic stem cells through Tuba1a

The spalt (Sal) gene family has four members (Sall1-4) in vertebrates, all of which play pivotal roles in various biological processes and diseases. However, the expression and function of SALL2 in development are still less clear. Here, we first charted SALL2 protein expression pattern during mouse embryo development by immunofluorescence, which revealed its dominant expression in the developing nervous system. With the establishment of Sall2 deficient mouse embryonic stem cells (ESCs), the in vitro neural differentiation system was leveraged to interrogate the function of SALL2, which showed impaired neural differentiation of Sall2 knockout (KO) ESCs. Furthermore, neural stem cells (NSCs) could not be derived from Sall2 KO ESCs and the generation of neural tube organoids (NTOs) was greatly inhibited in the absence of SALL2. Meanwhile, transgenic expression of E1 isoform of SALL2 restored the defects of neural differentiation in Sall2 KO ESCs. By chromatin immunoprecipitation sequencing (ChIP-seq), Tuba1a was identified as downstream target of SALL2, whose function in neural differentiation was confirmed by rescuing neural phenotypes of Sall2 KO ESCs when overexpressed. In sum, by elucidating SALL2 expression dynamics during early mouse development and mechanistically characterizing its indispensable role in neural differentiation, this study offers insights into SALL2’s function in human nervous system development, associated pathologies stemming from its mutations and relevant therapeutic strategy.

Dose-dependent effects of silver nanoparticles on cell death modes in mouse blastocysts induced via endoplasmic reticulum stress and mitochondrial apoptosis.

In view of the rapidly expanding medical and commercial applications of silver nanoparticles (AgNPs), their potential health risks and environmental effects are a significant growing concern. Earlier research by our group uncovered the embryotoxic potential of AgNPs, showing detrimental impacts of these nanoparticles on both pre- and post-implantation embryonic development. In the current study, we showed that low (50-100μM) and high (200-400μM) dose ranges of AgNPs trigger distinct cell death programs affecting mouse embryo development and further explored the underlying mechanisms. Treatment with low concentrations of AgNPs (50-100μM) triggered ROS generation, in turn, inducing mitochondria-dependent apoptosis, and ultimately, harmful effects on embryo implantation, post-implantation development, and fetal development. Notably, high concentrations of AgNPs (200-400μM) evoked more high-level ROS generation and endoplasmic reticulum (ER) stress-mediated necrosis. Interestingly, pre-incubation with Trolox, a strong antioxidant, reduced ROS generation in the group treated with 200-400μM AgNPs to the level induced by 50-100μM AgNPs, resulting in switching of the cell death mode from necrosis to apoptosis and a significant improvement in the impairment of embryonic development. Our findings additionally indicate that activation of PAK2 is a crucial step in AgNP-triggered apoptosis and sequent detrimental effects on embryonic development. Based on the collective results, we propose that the levels of ROS generated by AgNP treatment of embryos serve as a critical regulator of cell death type, leading to differential degrees of damage to embryo implantation, post-implantation development and fetal development through triggering apoptosis, necrosis or other cell death signaling cascades.

Single-cell RNA sequencing reveals the effects of high-fat diet on oocyte and early embryo development in female mice

BackgroundObesity is a global health issue with detrimental effects on various human organs, including the reproductive system. Observational human data and several lines of animal experimental data suggest that maternal obesity impairs ovarian function and early embryo development, but the precise pathogenesis remains unclear.MethodsWe established a high-fat diet (HFD)-induced obese female mouse model to assess systemic metabolism, ovarian morphology, and oocyte function in mice. For the first time, this study employed single-cell RNA sequencing to explore the altered transcriptomic landscape of preimplantation embryos at different stages in HFD-induced obese mice. Differential gene expression analysis, enrichment analysis and protein-protein interactions network analysis were performed.ResultsHFD-induced obese female mice exhibited impaired glucolipid metabolism and insulin resistance. The ovaries of HFD mice had a reduced total follicle number, an increased proportion of atretic follicles, and irregular granulosa cell arrangement. Furthermore, the maturation rate of embryonic development by in vitro fertilization of oocytes was significantly decreased in HFD mice. Additionally, the transcriptional landscapes of preimplantation embryos at different stages in mice induced by different diets were significantly distinguished. The maternal-to-zygotic transition was also affected by the failure to remove maternal RNAs and to turn off zygotic genome expression.ConclusionsHFD-induced obesity impaired ovarian morphology and oocyte function in female mice and further led to alterations in the transcriptional landscape of preimplantation embryos at different stages of HFD mice.

Development and tissue specific expression of RAPGEF1 (C3G) transcripts having exons encoding disordered segments with predicted regulatory function.

The ubiquitously expressed Guanine nucleotide exchange factor, RAPGEF1 (C3G), is essential for early development of mouse embryos. It functions to regulate gene expression and cytoskeletal reorganization, thereby controlling cell proliferation and differentiation. While multiple transcripts have been predicted, their expression in mouse tissues has not been investigated in detail. Full length RAPGEF1 isoforms primarily arise due to splicing at two hotspots, one involving exon-3, and the other involving exons 12-14 incorporating amino acids immediately following the Crk binding region of the protein. These isoforms vary in expression across embryonic and adult organs. We detected the presence of unannotated, and unpredicted transcripts with incorporation of cassette exons in various combinations, specifically in the heart, brain, testis and skeletal muscle. Isoform switching was detected as myocytes in culture and mouse embryonic stem cells were differentiated to form myotubes, and embryoid bodies respectively. The cassette exons encode a serine-rich polypeptide chain, which is intrinsically disordered, and undergoes phosphorylation. In silico structural analysis using AlphaFold indicated that the presence of cassette exons alters intra-molecular interactions, important for regulating catalytic activity. LZerD based docking studies predicted that the isoforms with one or more cassette exons differ in interaction with their target GTPase, RAP1A. Our results demonstrate the expression of novel RAPGEF1 isoforms, and predict cassette exon inclusion as an additional means of regulating RAPGEF1 activity in various tissues and during differentiation.

Chromosomal missegregation and aberrant embryo development in repro57 female mice with Rnf212 homozygous mutation.

Repro57 mice, bearing an Rnf212 gene mutation, exhibit infertility in both homozygous mutant males and females, revealing arrested spermatogenesis in males and investigating unclear mechanisms in females. The study highlights aneuploidy and altered kinetochore patterns in repro57 homozygous mutant oocytes, which impact later stages of embryo development. Repro57 mice, induced with N-ethyl-N-nitrosourea and harboring a mutation in the Rnf212 gene, exhibit infertility in both homozygous mutant males and females. Rnf212 plays a crucial role in recombination and crossover designation. In male repro57 homozygous mutants, spermatocytes often degenerate during late prophase, and mature spermatozoa are absent in the seminiferous epithelium, indicating arrested spermatogenesis as the cause of infertility. Despite reports of infertility in Rnf212-knockout female mice, the specific mechanisms underlying infertility in female repro57 homozygous mutants remain elusive. This study investigates the chromosomal and kinetochore patterns of mature oocytes and their developmental potential following in vitro fertilization in female repro57 homozygous mutant mice. While all wild-type oocytes progress to metaphase II and exhibit euploidy, all repro57 homozygous mutant mouse oocytes display aneuploidy. Additionally, kinetochore distances in repro57 homozygous mutant oocytes exceed those observed in wild-type counterparts. Although no significant differences are noted in fertilization and early embryo development rates between wild-type and repro57 homozygous mutant mice, embryos derived from repro57 homozygous mutants exhibit significantly lower morula and blastocyst rates, accompanied by frequent cytokinesis failure and vacuole formation. These findings suggest that the premature segregation of sister chromatids in repro57 homozygous mutant mice adversely impacts the later stages of embryo development.

SANTO: a coarse-to-fine alignment and stitching method for spatial omics

With the flourishing of spatial omics technologies, alignment and stitching of slices becomes indispensable to decipher a holistic view of 3D molecular profile. However, existing alignment and stitching methods are unpractical to process large-scale and image-based spatial omics dataset due to extreme time consumption and unsatisfactory accuracy. Here we propose SANTO, a coarse-to-fine method targeting alignment and stitching tasks for spatial omics. SANTO firstly rapidly supplies reasonable spatial positions of two slices and identifies the overlap region. Then, SANTO refines the positions of two slices by considering spatial and omics patterns. Comprehensive experiments demonstrate the superior performance of SANTO over existing methods. Specifically, SANTO stitches cross-platform slices for breast cancer samples, enabling integration of complementary features to synergistically explore tumor microenvironment. SANTO is then applied to 3D-to-3D spatiotemporal alignment to study development of mouse embryo. Furthermore, SANTO enables cross-modality alignment of spatial transcriptomic and epigenomic data to understand complementary interactions.

Polycomb function in early mouse development.

Epigenetic factors are crucial for ensuring proper chromatin dynamics during the initial stages of embryo development. Among these factors, the Polycomb group (PcG) of proteins plays a key role in establishing correct transcriptional programmes during mouse embryogenesis. PcG proteins are classified into two complexes: Polycomb repressive complex 1 (PRC1) and PRC2. Both complexes decorate histone proteins with distinct post-translational modifications (PTMs) that are predictive of a silent transcriptional chromatin state. In recent years, a critical adaptation of the classical techniques to analyse chromatin profiles and to study biochemical interactions at low-input resolution has allowed us to deeply explore PcG molecular mechanisms in the very early stages of mouse embryo development- from fertilisation to gastrulation, and from zygotic genome activation(ZGA) to specific lineages differentiation. These advancements provide a foundation for a deeper understanding of the fundamental role Polycomb complexes play in early development and have elucidated the mechanistic dynamics of PRC1 and PRC2. In this review, we discuss the functions and molecular mechanisms of both PRC1 and PRC2 during early mouse embryo development, integrating new studies with existing knowledge. Furthermore, we highlight the molecular functionality of Polycomb complexes from ZGA through gastrulation, with a particular focus on non-canonical imprinted and bivalent genes, and Hox cluster regulation.

P-200 Vitrification induces a deviation in cleavage morphokinetic patterns and transcriptomic signatures along mouse embryo development

Abstract Study question Are there cellular, molecular and/or epigenetic risks associated with cryopreservation that may affect embryo development and compromise the health of future newborns? Summary answer Cryopreservation significantly impacted mice pre-implantation embryo morphokinetics, altered transcriptomic profiles, and cell lineage distribution. However, global DNA methylation remained unaffected by cryopreservation. What is known already Cryopreservation is currently widely used in assisted reproduction. However, epidemiological studies suggest a negative impact in the long-term health of children born after frozen-embryo transfer (FET). Recent population-based register studies have reported: 1) an increased risk of infant mortality from birth up to 1 year of life in singletons conceived using cryopreserved embryos vs natural conceptions; 2) high birth weights for gestational age associated with FET; 3) an increased risk of obstetrical complications in singletons conceived using cryopreserved embryos; 4) a higher incidence of childhood cancer and imprinting disorders associated with FET. Study design, size, duration F1 hybrid (B6/CBA) mice zygotes were collected following uterus dissection (n = 200 embryos/collection), and half of those were vitrified for comparison. Each embryo’s morphokinetics was assessed through time-lapse imaging using GERI, at 37 °C, 6% CO2, 5% O2, up to 105 hours. Extended culture beyond blastocyst stage was also performed. Fresh and cryopreserved blastocysts at E5.0 were cultured up to E9.0-E10 in static conditions (IVC1 and IVC2 medium). Participants/materials, setting, methods Fresh and cryopreserved embryos at several developmental stages were used for: 1) immunofluorescence staining, 2) gene expression analysis through RT-qPCR and 3) single-cell RNAseq. Global methylation activity, cell lineage distribution and single-cell transcriptome were assessed using one of the previously described methodologies. Main results and the role of chance The morphokinetics of preimplantation embryos were examined by tracking the time required to reach 4-cell, 8-cell, compactation, morula, full blastocyst, and expanded blastocyst stage. A comprehensive analysis revealed notable delays in the developmental rhythm of cryopreserved embryos compared to fresh embryos (p < 0.01). Applying single-cell RNA sequencing (scRNAseq), transcriptomic profiles were generated for fresh and cryopreserved embryos, covering the zygote to blastocyst stages. Notably, distinct transcriptomic signatures for fresh and frozen embryos emerged within separate clusters at different developmental stages. These findings suggest a potential functional impact of cryopreservation on embryo development. To complement this, gene expression analysis of DNA methyltransferases and MAT enzymes was conducted through RT-qPCR at 8-cell and blastocyst stages. No significant differences were detected between fresh and cryopreserved conditions. Furthermore, immunofluorescence staining of nuclear 5mC and DNMT1 in 8-cell embryos and blastocysts demonstrated similar levels between fresh and cryopreserved embryos. Additionally, differential staining of embryonic and extraembryonic cells (OCT4, GATA6 and GATA3) in extended embryos (E9 and E10) revealed distinct cell distribution patterns between fresh and cryopreserved conditions. Limitations, reasons for caution Our research, conducted using a mouse experimental model, has limited applicability to humans due to considerable species differences in embryonic development. Additional research is needed to validate our findings and establish their relevance to human embryos, facilitating translational approaches. Wider implications of the findings Our study highlights a noticeable functional impact of vitrification on mouse embryos. Given the global incorporation of this method in fertility clinics and its potential association with increased disease risks, our findings emphasize the need for increased research to reduce uncertainties regarding the health of children born from cryopreservation methods. Trial registration number Not applicable

P-548 An investigation into the mechanisms of active enhancers regulating the initial lineage differentiation in mammals

Abstract Study question What role do active enhancers play in determining the fate of early embryonic cells? Are there differences in active enhancers between mouse and human blastocysts? Summary answer Tissue-specific active enhancers in inner cell mass (ICM) and trophoblast (TE) are essential for blastocyst formation, regulating gene expression through interactive loops with promoters. What is known already At the two-cell stage of nuclear transfer embryos, the absence of interaction between Zscan4d promoter and distal active enhancer leads to reduced Zscan4d expression, resulting in lower developmental rates. Overexpressing the demethylase Kdm4d alleviates reprogramming-resistant H3K9me3 modification, restoring the structural loop between the Zscan4d promoter and active enhancer and enhancing developmental efficiency. These findings strongly suggest a close association between the formation of regulatory chromatin interactions in early embryonic development and histone modifications. Indicating active enhancers marked with H3K27ac may regulate differentiation gene expression through distal interactions in blastocyst. Study design, size, duration Analyze variations in histone H3K27ac modification, chromatin accessibility, higher-order structures, and transcriptomes during mouse embryo differentiation from 8-cell stage to ICM and TE formation. Identify tissue-specific active enhancers in ICM and TE, validate their gene expression regulation, explore their role in early differentiation-associated genes in mice. Compare and clarify similarities and differences in enhancer regulation mechanisms during early embryo development in humans and mice. Participants/materials, setting, methods The 8-cell, morula and blastocyst stage embryos were collected from the oviducts and uterus of super-ovulated female ICR mice mated with male mice. The clinical human blastocysts were collected and separated the ICM and TE. Low-input multi-omics sequencing, including Cut-Tag, ATAC-seq, Hi-C, and RNA-seq, was performed on the embryonic samples. After analyzing and identifying active enhancers, tissue-specific ones were selected and validated for their impact on gene expression via CRISPR-Cas9 knockout in embryos. Main results and the role of chance Inhibiting ep300 expression in mouse embryos through siRNA interference reduces blastocyst formation from 90% to approximately 40%. Introducing the small molecule inhibitor A485 at the 8-cell stage, blocking the active center of CBP/p300 enzymes, completely arrests embryos at the morula stage, preventing their development into blastocysts. Emphasizing the crucial role of correctly establishing active enhancers marked by H3K27ac modification in embryonic differentiation. Multi-omics data demonstrates high repeatability and quality. PCA clustering analysis reveals a clear differentiation path in the distribution of H3K27ac-modified histones, chromatin accessibility sites, and the transcriptome from the 8-cell stage to the blastocyst. Differences in H3K27ac histone modification distribution between the ICM and TE indicate the presence of tissue-specific active enhancers in blastocyst. The disparities in H3K27ac modification distribution between blastocyst ICM and TE primarily occur in distal gene regions rather than proximal ones. Moreover, these distal enhancers exhibit a positive correlation with the expression of lineage-specific genes, suggesting a probable regulatory role of distal enhancers in gene modulation during embryonic differentiation. Tissue-specific enhancer-promoter (EP) interaction loops emerge during embryonic differentiation, allowing direct interaction between tissue-specific active enhancers and promoters, thereby activating the expression of tissue-specific genes. Limitations, reasons for caution While DNA sequences with H3K27ac modification are currently considered markers for active enhancers, H3K4me modification is another histone marker for enhancers. However, enhancers associated with H3K4me modification may not necessarily be in an activated state. Future research can simultaneously incorporate appropriate detection methods for H3K4me modification. Wider implications of the findings After elucidating the mechanisms through which active enhancers regulate mouse differentiation, comparing the differences in active enhancers during the first lineage differentiation process between mice and humans can help identify factors that enhance human blastocyst quality. This can potentially improve clinical outcomes in assisted reproduction. Trial registration number not applicable

P-117 Extracellular vesicles from fallopian tubal fluid benefit human embryo development in vitro

Abstract Study question Do extracellular vesicles (EVs) from Fallopian tubes benefit human embryo development, and how do they affect preimplantation embryo development? Summary answer The Fallopian tubal EVs can be internalized by human embryos, and they contain miR-21-5p that downregulated P53 in blastocysts. What is known already EVs in the Fallopian tubes are identified as key mediators in embryo-oviduct interactions that contribute to a successful pregnancy in vivo. We previously showed Fallopian tubal EVs can benefit mouse embryo development in vitro. However, if a similar phenomenon can be observed in human embryos and what is the key component in Fallopian tubal EVs remains elusive. Study design, size, duration The EVs were isolated from the luminal fluid of human Fallopian tubes (n = 23). Immature human oocytes were collected and in vitro matured and fertilized, and 2-pronuclei embryos were included in this study and randomly divided (n = 87). We cocultured these zygotes with Fallopian tubal EVs until the blastocyst stage. Further, two-cell mouse embryos were used to explore the expression levels of miRNAs in Fallopian tubal EVs. The effects of miR-21-5p on mouse embryos were analyzed. Participants/materials, setting, methods EVs were isolated using ultracentrifugation. The uptake of labeled EVs by human embryos was analyzed using confocal microscopy. The fragmentation of the day-3 embryos and the blastocyst rate were compared between the control and the EV-treatment group. Then the stage-specific expression of miRNAs was compared in mouse embryos that were either developed in vivo or cultured in vitro. miR-21-5p were supplemented to the culture medium and the p53-related apoptosis in embryos was evaluated. Main results and the role of chance EVs were measured less than 200 nm in diameter with a typical bi-layer membrane structure and expressed protein markers Heat shock protein 70 (HSP70) and CD9. Most of the fluorescence-labeled EVs were attached to the zona pellucida within 2 h of co-culture. However, human embryos internalized EV within 4 h, with fluorescence signals observed in embryonic cells. The fragmentation rate was decreased and the blastocyst rate was increased by EVs. miR-21-5p, which was highly enriched in EVs, was found to be expressed at a significantly lower level in in-vitro-cultured mouse embryos compared to the in-vivo-developed embryos, through the two-cell stage to the eight-cell stage. miR-21-5p agomir supplementation to the culture medium was found to increase the mouse blastocyst formation rate, decrease apoptosis, and downregulate the P53 expression level in mouse blastocysts. Limitations, reasons for caution Though we observed that Fallopian tubal EVs could be internalized by human embryos and improve embryo development morphologically, the subsequent mechanism exploration was conducted using mouse embryos. Whether the stage-specific expression level of miR-21-5p and the effects of miR-21-5p are the same in human embryos remains to be determined. Wider implications of the findings This study showed miRNAs in Fallopian tubal EVs can act as embryonic survival signals, which opens the insight into further explorations to demonstrate the molecular mechanisms during early embryo development, as well as holds the potential to optimize the culture medium used in ART in the future. Trial registration number 2022YFC2702503

Poissonian Cellular Potts Models Reveal Nonequilibrium Kinetics of Cell Sorting

Cellular Potts models are broadly applied across developmental biology and cancer research. We overcome limitations of the traditional approach, which reinterprets a modified Metropolis sampling as dynamics, by introducing a physical timescale through Poissonian kinetics and by applying principles of stochastic thermodynamics to separate thermal and relaxation effects from athermal noise and nonconservative forces. Our method accurately describes cell-sorting dynamics in mouse-embryo development and identifies the distinct contributions of nonequilibrium processes, e.g., cell growth and active fluctuations. Published by the American Physical Society 2024

Reproductive chemical database: a curated database of chemicals that modulate protein targets regulating important reproductive biological processes

Recent studies have shifted the spotlight from adult disease to gametogenesis and embryo developmental events, and these are greatly affected by various environmental chemicals, such as drugs, metabolites, pollutants, and others. Growing research has highlighted the critical importance of identifying and understanding the roles of chemicals in reproductive biology. However, the functions and mechanisms of chemicals in reproductive processes remain incomplete. We developed a comprehensive database called the Reproductive Chemical Database (RCDB) (https://yu.life.sjtu.edu.cn/ChenLab/RCDB) to facilitate research on chemicals in reproductive biology. This resource is founded on rigorous manual literature extraction and precise protein target prediction methodologies. This database focuses on the delineation of chemicals associated with phenotypes, diseases, or endpoints intricately associated with four important reproductive processes: female and male gamete generation, fertilization, and embryo development in human and mouse. The RCDB encompasses 93 sub-GO processes, and it revealed 1447 intricate chemical–biological process interactions. To date, the RCDB has meticulously cataloged and annotated 830 distinct chemicals, while also predicting 614 target proteins from a selection of 3800 potential candidates. Additionally, the RCDB offers an online predictive tool that empowers researchers to ascertain whether specific chemicals play discernible functional roles in these reproductive processes. The RCDB is an exhaustive, cross-platform, manually curated database, which provides a user-friendly interface to search, browse, and use reproductive processes modulators and their comprehensive related information. The RCDB will help researchers to understand the whole reproductive process and related diseases and it has the potential to promote reproduction research in the pharmacological and pathophysiological areas.Graphical

Are the class 18 myosins Myo18A and Myo18B specialist sarcomeric proteins?

Initially, the two members of class 18 myosins, Myo18A and Myo18B, appeared to exhibit highly divergent functions, complicating the assignment of class-specific functions. However, the identification of a striated muscle-specific isoform of Myo18A, Myo18Aγ, suggests that class 18 myosins may have evolved to complement the functions of conventional class 2 myosins in sarcomeres. Indeed, both genes, Myo18a and Myo18b, are predominantly expressed in the heart and somites, precursors of skeletal muscle, of developing mouse embryos. Genetic deletion of either gene in mice is embryonic lethal and is associated with the disorganization of cardiac sarcomeres. Moreover, Myo18Aγ and Myo18B localize to sarcomeric A-bands, albeit the motor (head) domains of these unconventional myosins have been both deduced and biochemically demonstrated to exhibit negligible ATPase activity, a hallmark of motor proteins. Instead, Myo18Aγ and Myo18B presumably coassemble with thick filaments and provide structural integrity and/or internal resistance through interactions with F-actin and/or other proteins. In addition, Myo18Aγ and Myo18B may play distinct roles in the assembly of myofibrils, which may arise from actin stress fibers containing the α-isoform of Myo18A, Myo18Aα. The β-isoform of Myo18A, Myo18Aβ, is similar to Myo18Aα, except that it lacks the N-terminal extension, and may serve as a negative regulator through heterodimerization with either Myo18Aα or Myo18Aγ. In this review, we contend that Myo18Aγ and Myo18B are essential for myofibril structure and function in striated muscle cells, while α- and β-isoforms of Myo18A play diverse roles in nonmuscle cells.

Plasma-derived extracellular vesicles improve mice embryo development.

To investigate the effect of plasma-derived extracellular vesicles (EVs) or conventional medium in fertilization and early embryo development rate in mice. MII oocytes (matured in vivo or in vitro conditions) were obtained from female mice. The extracellular vesicles were isolated by ultracentrifugation of plasma and were analyzed and measured for size and morphology by dynamic light scattering (DLS) and transmission electron microscopy (TEM). By western blotting analysis, the EVs proteins markers such as CD82 protein and heat shock protein 90 (HSP90) were investigated. Incorporating DiI-labeled EVs within the oocyte cytoplasm was visible at 23h in oocyte cytoplasm. Also, the effective proteins in the early reproductive process were determined in isolated EVs by western blotting. These EVs had a positive effect on the fertilization rate (P < 0.05). The early embryo development (8 cell, morula and blastocyst stages) was higher in groups supplemented with EVs (P < 0.01). Our findings showed that supplementing in vitro maturation media with EVs derived- plasma was beneficial for mice's embryo development.