The PTM profiling of CTCF reveals the regulation of 3D chromatin structure by O-GlcNAcylation.

CCCTC-binding factor (CTCF), a ubiquitously expressed and highly conserved protein, is known to play a critical role in chromatin structure. Post-translational modifications (PTMs) diversify the functions of protein to regulate numerous cellular processes. However, the effects of PTMs on the genome-wide binding of CTCF and the organization of three-dimensional (3D) chromatin structure have not been fully understood. In this study, we uncovered the PTM profiling of CTCF and demonstrated that CTCF can be O-GlcNAcylated and arginine methylated. Functionally, we demonstrated that O-GlcNAcylation inhibits CTCF binding to chromatin. Meanwhile, deficiency of CTCF O-GlcNAcylation results in the disruption of loop domains and the alteration of chromatin loops associated with cellular development. Furthermore, the deficiency of CTCF O-GlcNAcylation increases the expression of developmental genes and negatively regulates maintenance and establishment of stem cell pluripotency. In conclusion, these results provide key insights into the role of PTMs for the 3D chromatin structure.

KLF4 facilitates chromatin accessibility remodeling in porcine early embryos.

Chromatin accessibility remodeling driven by pioneer factors is critical for the development of early embryos. Current studies have illustrated several pioneer factors as being important for agricultural animals, but what are the pioneer factors and how the pioneer factors remodel the chromatin accessibility in porcine early embryos is not clear. By employing low-input DNase-seq (liDNase-seq), we profiled the landscapes of chromatin accessibility in porcine early embryos and uncovered a unique chromatin accessibility reprogramming pattern during porcine preimplantation development. Our data revealed that KLF4 played critical roles in remodeling chromatin accessibility in porcine early embryos. Knocking down of KLF4 led to the reduction of chromatin accessibility in early embryos, whereas KLF4 overexpression promoted the chromatin openness in porcine blastocysts. Furthermore, KLF4 deficiency resulted in mitochondrial dysfunction and developmental failure of porcine embryos. In addition, we found that overexpression of KLF4 in blastocysts promoted lipid droplet accumulation, whereas knockdown of KLF4 disrupted this process. Taken together, our study revealed the chromatin accessibility dynamics and identified KLF4 as a key regulator in chromatin accessibility and cellular metabolism during porcine preimplantation embryo development.

The transcriptional activator Klf5 recruits p300-mediated H3K27ac for maintaining trophoblast stem cell pluripotency.

The effective proliferation and differentiation of trophoblast stem cells (TSCs) is indispensable for the development of the placenta, which is the key to maintaining normal fetal growth during pregnancy. Kruppel-like factor 5 (Klf5) is implicated in the activation of pluripotency gene expression in embryonic stem cells (ESCs), yet its function in TSCs is poorly understood. Here, we showed that Klf5 knockdown resulted in the downregulation of core TSC-specific genes, consequently causing rapid differentiation of TSCs. Consistently, Klf5-depleted embryos lost the ability to establish TSCs in vitro. At the molecular level, Klf5 preferentially occupied the proximal promoter regions and maintained an open chromatin architecture of key TSC-specific genes. Deprivation of Klf5 impaired the enrichment of p300, a major histone acetyl transferase of H3 lysine 27 acetylation (H3K27ac), and further reduced the occupancy of H3K27ac at promoter regions, leading to decreased transcriptional activity of TSC pluripotency genes. Thus, our findings highlight a novel mechanism of Klf5 in regulating the self-renewal and differentiation of TSCs and provide a reference for understanding placental development and improving pregnancy rates.

Rapid conversion of porcine pluripotent stem cells into macrophages with chemically defined conditions.

A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms.

OCT4 regulates WNT/ß-catenin signaling and prevents mesoendoderm differentiation by repressing EOMES in porcine pluripotent stem cells.

The regulatory network between signaling pathways and transcription factors (TFs) is crucial for the maintenance of pluripotent stem cells. However, little is known about how the key TF OCT4 coordinates signaling pathways to regulate self-renewal and lineage differentiation of porcine pluripotent stem cells (pPSCs). Here, we explored the function of OCT4 in pPSCs by transcriptome and chromatin accessibility analysis. The TFs motif enrichment analysis revealed that, following OCT4 knockdown, the regions of increased chromatin accessibility were enriched with EOMES, GATA6, and FOXA1, indicating that pPSCs differentiated toward the mesoendoderm (ME) lineage. Besides, pPSCs rapidly differentiated into ME when the WNT/ß-catenin inhibitor XAV939 was removed. However, the ME differentiation of pPSCs caused by OCT4 knockdown did not rely on the activation of WNT/ß-catenin signaling because the target gene of WNT/ß-catenin signaling, AXIN2 was not upregulated after OCT4 knockdown, despite significant upregulation of WLS and some WNT ligands. Importantly, OCT4 is directly bound to the promoter and enhancers of EOMES and repressed its transcription. Overexpression of EOMES was sufficient to induce ME differentiation in the presence of XAV939. These results demonstrate that OCT4 can regulate WNT/ß-catenin signaling and prevent ME differentiation of pPSCs by repressing EOMES transcription.

ELF1/PRR11/ARP2/3 promoted trophoblast cells proliferation and motility in early pregnancy.

BACKGROUND/OBJECTIVE: Early pregnancy loss (EPL) is a common adverse pregnancy outcome with an incidence of approximately 10-30%. There are many factors that cause EPL, among which the lack of proliferation and invasive properties of trophoblast cells can lead to embryonic development. Therefore, in this study, the molecular biology of trophoblast cells was investigated. METHODS: Placental villous tissues from EPL patients were collected to explore ELF1 and PRR11 gene expression. The proliferation and migration of trophoblast cells were assessed by MTT, crystalline violet staining, and traswell assays, respectively. Western blotting and RT-qPCR were performed to investigate the relationship between ELF1, PRR11, and ARP2/3. F-actin polymerization and FAK activation were evaluated by immunofluorescence and western blotting. Ultimately, ELF1/PRR11/ARP2/3 expression was verified in the EPL mice model RESULTS: ELF1 and PRR11 were lowly expressed in placental villous tissues from EPL. The overexpression of ELF1 and PRR11 promoted proliferation and migration of trophoblast cells. Moreover, while ELF1 bound to the PRR11 promoter and promoted transcriptional activation. Finally, ELF1/PRR11/ARP2/3 showed low expression in the placental tissue of EPL mice. CONCLUSION: Our study suggested that PRR11 promoted the motility of trophoblast cells by binding to the ARP2/3 complex to promote F-actin polymerization and FAK activation. In addition, ELF1 bound to the initiation site of PRR11 to promote its transcription. ELF1/PRR11/ARP2/3 may play an important role in EPL.

Nicotine exposure disrupts placental development via the Notch signaling pathway.

In brief: Normal gene expression during early embryonic development and in the placenta is crucial for a successful pregnancy. Nicotine can disrupt normal gene expression during development, leading to abnormal embryonic and placental development. Abstract: Nicotine is a common indoor air pollutant that is present in cigarette fumes. Due to its lipophilic nature, nicotine can rapidly transport through membrane barriers and spread throughout the body, which can lead to the development of diseases. However, the impact of nicotine exposure during early embryonic development on subsequent development remains elusive. In this study, we found that nicotine significantly elevated reactive oxygen species, DNA damage and cell apoptosis levels with the decrease of blastocyst formation during early embryonic development. More importantly, nicotine exposure during early embryonic development increased placental weight and disrupted placental structure. In molecular level, we also observed that nicotine exposure could specifically cause the hypermethylation of Phlda2 promoter (a maternally expressed imprinted gene associated with placental development) and reduce the mRNA expression of Phlda2. By RNA sequencing analysis, we demonstrated that nicotine exposure affected the gene expression and excessive activation of the Notch signaling pathway thereby affecting placental development. Blocking the Notch signaling pathway by DAPT treatment could recover abnormal placental weight and structure induced by nicotine exposure. Taken together, this study indicates that nicotine causes the declining quality of early embryos and leads to placental abnormalities related to over-activation of the Notch signaling pathway.

ISG15 suppresses ovulation and female fertility by ISGylating ADAMTS1.

BACKGROUND: ISGylation is a post-translational protein modification that regulates many life activities, including immunomodulation, antiviral responses, and embryo implantation. The exact contribution of ISGylation to folliculogenesis remains largely undefined. RESULTS: Here, Isg15 knockout in mice causes hyperfertility along with sensitive ovarian responses to gonadotropin, such as increases in cumulus expansion and ovulation rate. Moreover, ISG15 represses the expression of ovulation-related genes in an ISGylation-dependent manner. Mechanistically, ISG15 binds to ADAMTS1 via the ISG15-conjugating system (UBA7, UBE2L6, and HERC6), ISGylating ADAMTS1 at the binding sites Lys309, Lys593, Lys597, and Lys602, resulting in ADAMTS1 degradation via a 20S proteasome-dependent pathway. CONCLUSION: Taken together, the present study demonstrates that covalent ISG15 conjugation produces a novel regulatory axis of ISG15-ADAMTS1 that enhances the degradation of ADAMTS1, thereby compromising ovulation and female fertility.

Lipidomic Characterization of Oocytes at Single-Cell Level Using Nanoflow Chromatography-Trapped Ion Mobility Spectrometry-Mass Spectrometry.

Mass spectrometry (MS)-based lipidomic has become a powerful tool for studying lipids in biological systems. However, lipidome analysis at the single-cell level remains a challenge. Here, we report a highly sensitive lipidomic workflow based on nanoflow liquid chromatography and trapped ion mobility spectrometry (TIMS)-MS. This approach enables the high-coverage identification of lipidome landscape at the single-oocyte level. By using the proposed method, comprehensive lipid changes in porcine oocytes during their maturation were revealed. The results provide valuable insights into the structural changes of lipid molecules during porcine oocyte maturation, highlighting the significance of sphingolipids and glycerophospholipids. This study offers a new approach to the single-cell lipidomic.

Arsenite exposure disturbs maternal-to-zygote transition by attenuating H3K27ac during mouse preimplantation development.

Arsenite is commonly used as an insecticide, antiseptic and herbicide. It can enter the food chain via through soil contamination, and harm human health, including the reproductive systems. Early embryos, as the initial stage of mammalian life, are very sensitive to the environmental toxins and pollutants. However, whether and how arsenite disturbs the early embryo development remains unclear. Our study used mouse early embryos as a model and revealed that arsenite exposure did not cause reactive oxygen species production, DNA damage or apoptosis. However, arsenite exposure arrested embryonic development at the 2-cell stage by altering gene expression patterns. The transcriptional profile in the disrupted embryos showed abnormal maternal-to-zygote transition (MZT). More importantly, arsenite exposure attenuated H3K27ac modification enrichment at the promoter region of Brg1, a key gene for MZT, which inhibited its transcription, and further affected MZT and early embryonic development. In conclusion our study highlights arsenite exposure affects MZT by reducing the enrichment of H3K27ac on the embryonic genome, and ultimately induces early embryonic development arrest at the 2-cell stage.

Preserving Porcine Genetics: A Simple and Effective Method for On-Site Cryopreservation of Ear Tissue Using Direct Cover Vitrification.

Cell cryopreservation is widely used for porcine genetic conservation; however, isolating and freezing primary cells in farms without adequate experimental equipment and environment poses a significant challenge. Therefore, it is necessary to establish a quick and simple method to freeze tissues on-site, which can be used for deriving primary fibroblasts when needed to achieve porcine genetic conservation. In this study, we explored a suitable approach for porcine ear tissue cryopreservation. The porcine ear tissues were cut into strips and frozen by direct cover vitrification (DCV) in the cryoprotectant solution with 15% EG, 15% DMSO and 0.1 M trehalose. Histological analysis and ultrastructural evaluation revealed that thawed tissues had normal tissue structure. More importantly, viable fibroblasts could be derived from these tissues frozen in liquid nitrogen for up to 6 months. Cells derived from thawed tissues did not show any cell apoptosis, had normal karyotypes and could be used for nuclear transfer. These results suggest that this quick and simple ear tissue cryopreservation method can be applied for porcine genetic conservation, especially in the face of a deadly emerging disease in pigs.

Acute exposure of triclocarban affects early embryo development in mouse through disrupting maternal-to-zygotic transition and epigenetic modifications.

Triclocarban (TCC) is a broad-spectrum antibacterial agent used globally, and high concentrations of this harmful chemical exist in the environment. The human body is directly exposed to TCC through skin contact. Moreover, TCC is also absorbed through diet and inhaled through breathing, which results in its accumulation in the body. The safety profile of TCC and its potential impact on human health are still not completely clear; therefore, it becomes imperative to evaluate the reproductive toxicity of TCC. Here, we explored the effect of TCC on the early embryonic development of mice and its associated mechanisms. We found that acute exposure of TCC affected the early embryonic development of mice in a dose-dependent manner. Approximately 7600 differentially expressed genes (DEGs) were obtained by sequencing the transcriptome of 2-cell mouse embryos; of these, 3157 genes were upregulated and 4443 genes were downregulated in the TCC-treated embryos. GO and KEGG analysis revealed that the enriched genes were mainly involved in redox processes, RNA synthesis, DNA damage, apoptosis, mitochondria, endoplasmic reticulum, Golgi apparatus, cytoskeleton, peroxisome, RNA polymerase, and other components or processes. Moreover, the Venn analysis showed that the zygotic genome activation (ZGA) was affected and the degradation of maternal effector genes was inhibited. TCC induced changes in the epigenetic modification of 2-cell embryos. The level of DNA methylation increased significantly. Further, the levels of H3K27ac, H3K9ac, and H3K27me3 histone modifications decreased significantly, whereas those of H3K4me3 and H3K9me3 modifications increased significantly. Additionally, TCC induced oxidative stress and DNA damage in the 2-cell embryos. In conclusion, acute exposure of TCC affected early embryo development, destroyed early embryo gene expression, interfered with ZGA and maternal gene degradation, induced changes in epigenetic modification of early embryos, and led to oxidative stress and DNA damage in mouse early embryos.

Selective autophagic degradation of ACLY (ATP citrate lyase) maintains citrate homeostasis and promotes oocyte maturation.

Macroautophagy/autophagy is a cellular and energy homeostatic mechanism that contributes to maintain the number of primordial follicles, germ cell survival, and anti-ovarian aging. However, it remains unknown whether autophagy in granulosa cells affects oocyte maturation. Here, we show a clear tendency of reduced autophagy level in human granulosa cells from women of advanced maternal age, implying a potential negative correlation between autophagy levels and oocyte quality. We therefore established a co-culture system and show that either pharmacological inhibition or genetic ablation of autophagy in granulosa cells negatively affect oocyte quality and fertilization ability. Moreover, our metabolomics analysis indicates that the adverse impact of autophagy impairment on oocyte quality is mediated by downregulated citrate levels, while exogenous supplementation of citrate can significantly restore the oocyte maturation. Mechanistically, we found that ACLY (ATP citrate lyase), which is a crucial enzyme catalyzing the cleavage of citrate, was preferentially associated with K63-linked ubiquitin chains and recognized by the autophagy receptor protein SQSTM1/p62 for selective autophagic degradation. In human follicles, the autophagy level in granulosa cells was downregulated with maternal aging, accompanied by decreased citrate in the follicular fluid, implying a potential correlation between citrate metabolism and oocyte quality. We also show that elevated citrate levels in porcine follicular fluid promote oocyte maturation. Collectively, our data reveal that autophagy in granulosa cells is a beneficial mechanism to maintain a certain degree of citrate by selectively targeting ACLY during oocyte maturation.Abbreviations: 3-MA: 3-methyladenine; ACLY: ATP citrate lyase; AMA: advanced maternal age; CG: cortical granule; CHX: cycloheximide; CQ: chloroquine; CS: citrate synthase; COCs: cumulus-oocyte-complexes; GCM: granulosa cell monolayer; GV: germinal vesicle; MII: metaphase II stage of meiosis; PB1: first polar body; ROS: reactive oxygen species; shRNA: small hairpin RNA; SQSTM1/p62: sequestosome 1; TCA: tricarboxylic acid; TOMM20/TOM20: translocase of outer mitochondrial membrane 20; UBA: ubiquitin-associated domain; Ub: ubiquitin; WT: wild-type.

'Multiple and short-range' cross-linking of dialdehyde carboxymethyl cellulose contributes to regulating the physicochemical property of collagen fibril.

Collagen fibril hydrogel (CH), with controllable micro-structure, sufficient modifying sites and excellent biocompatibility, has received widely attention in the regulation of biomacromolecules. Herein, dialdehyde carboxymethyl cellulose (DCMC) in different -CHO contents and molecular weights demonstrated two types of cross-linking behaviors to CH, 'limited and long-range' or 'multiple and short range' cross-linking, corresponding to -CHO content ranged from 0 to 53 % and 53 to 90 %, respectively. In regard of structure, non-destroying effect of DCMC on collagen was supported by FT-IR and XRD analysis. CH cross-linked by DCMC (CH-DC) showed declining porosity and aggregating fibrils as -CHO content of DCMC rising. In regard of physicochemical properties, DCMC with >53 % -CHO strengthened the hydrophilicity, thermal stability and degradation resistance of CH-DC. Also, there was 110 % growth on gel strength, 86 Pa enhancements on storage modulus, and 4.6 times decrease on the swelling ratio of CH-DC. Results indicated that DCMC with 79 % -CHO remarkably improved the physicochemical properties of CH via developing sufficient Schiff-base bonds with collagen fibril in a short distance. This study distinguished two patterns of DCMC cross-linking from physicochemical view. In other words, DCMC is potential to meet the requirement of protein-based materials with different expectations by adjusting its -CHO content and molecular weight.

Coordination of zygotic genome activation entry and exit by H3K4me3 and H3K27me3 in porcine early embryos.

Histone modifications are critical epigenetic indicators of chromatin state associated with gene expression. Although the reprogramming patterns of H3K4me3 and H3K27me3 have been elucidated in mouse and human preimplantation embryos, the relationship between these marks and zygotic genome activation (ZGA) remains poorly understood. By ultra-low-input native chromatin immunoprecipitation and sequencing, we profiled global H3K4me3 and H3K27me3 in porcine oocytes and in vitro fertilized (IVF) embryos. We found that promoters of ZGA genes occupied sharp H3K4me3 peaks in oocytes, and these peaks became broader after fertilization, and reshaped into sharp again during ZGA. By simultaneous depletion of H3K4me3 demethylase KDM5B and KDM5C, we determined that broad H3K4me3 domain maintenance impaired ZGA gene expression, suggesting its function to prevent premature ZGA entry. By contrast, broad H3K27me3 domains underwent global removal upon fertilization, followed by a re-establishment for H3K4me3/H3K27me3 bivalency in morulae. We also found that bivalent marks were deposited at promoters of ZGA genes, and inhibiting this deposition was correlated with the activation of ZGA genes. It suggests that promoter bivalency contributes to ZGA exit in porcine embryos. Moreover, we demonstrated that aberrant reprogramming of H3K4me3 and H3K27me3 triggered ZGA dysregulation in somatic cell nuclear transfer (SCNT) embryos, whereas H3K27me3-mediated imprinting did not exist in porcine IVF and SCNT embryos. Our findings highlight two previously unknown epigenetic reprogramming modes coordinated with ZGA in porcine preimplantation embryos. Finally, the similarities observed between porcine and human histone modification dynamics suggest that the porcine embryo may also be a useful model for human embryo research.

BCL2-associated athanogene 6 exon24 contributes to testosterone synthesis and male fertility in mammals.

OBJECTIVES: BCL2-associated athanogene 6 (BAG6) plays critical roles in spermatogenesis by maintaining testicular cell survival. Our previous data showed porcine BAG6 exon24-skipped transcript is highly expressed in immature testes compared with mature testes. The objective of this study is to reveal the functional significance of BAG6 exon24 in mammalian spermatogenesis. MATERIALS AND METHODS: CRISPR/Cas9 system was used to generate Bag6 exon24 knockout mice. Testes and cauda epididymal sperm were collected from mice. TMT proteomics analysis was used to discover the protein differences induced by Bag6 exon24 deletion. Testosterone enanthate was injected into mice to generate a high-testosterone mice model. H&E staining, qRT-PCR, western blotting, vector/siRNA transfection, immunofluorescence, immunoprecipitation, transmission electron microscopy, TUNEL and ELISA were performed to investigate the phenotypes and molecular basis. RESULTS: Bag6 exon24 knockout mice show sub-fertility along with partially impaired blood-testis barrier, increased apoptotic testicular cell rate and abnormal sperm morphology. Endoplasmic reticulum stress occurs in Bag6 exon24-deficient testes and sterol regulatory element-binding transcription factor 2 is activated; as a result, cytochrome P450 family 51 subfamily A member 1 expression is up-regulated, which causes a high serum testosterone level. Additionally, serine/arginine-rich splicing factor 1 down-regulates BAG6 exon24-skipped transcripts in porcine Sertoli cells by binding to 35-51 nt on BAG6 exon24 via its N-terminal RNA-recognition domain. CONCLUSIONS: Our findings reveal the critical roles of BAG6 exon24 in testosterone biosynthesis and male fertility, which provides new insights into the regulation of spermatogenesis and pathogenesis of subfertility in mammals.

Hierarchical Accumulation of Histone Variant H2A.Z Regulates Transcriptional States and Histone Modifications in Early Mammalian Embryos.

Early embryos undergo extensive epigenetic reprogramming to achieve gamete-to-embryo transition, which involves the loading and removal of histone variant H2A.Z on chromatin. However, how does H2A.Z regulate gene expression and histone modifications during preimplantation development remains unrevealed. Here, by using ultra-low-input native chromatin immunoprecipitation and sequencing, the genome-wide distribution of H2A.Z is delineated in mouse oocytes and early embryos. These landscapes indicate that paternal H2A.Z is removed upon fertilization, followed by unbiased accumulation on parental genomes during zygotic genome activation (ZGA). Remarkably, H2A.Z exhibits hierarchical accumulation as different peak types at promoters: promoters with double H2A.Z peaks are colocalized with H3K4me3 and indicate transcriptional activation; promoters with a single H2A.Z peak are more likely to occupy bivalent marks (H3K4me3+H3K27me3) and indicate development gene suppression; promoters with no H2A.Z accumulation exhibit persisting gene silencing in early embryos. Moreover, H2A.Z depletion changes the enrichment of histone modifications and RNA polymerase II binding at promoters, resulting in abnormal gene expression and developmental arrest during lineage commitment. Furthermore, similar transcription and accumulation patterns between mouse and porcine embryos indicate that a dual role of H2A.Z in regulating the epigenome required for proper gene expression is conserved during mammalian preimplantation development.

ATG7-mediated autophagy facilitates embryonic stem cell exit from naive pluripotency and marks commitment to differentiation.

Macroautophagy/autophagy is a conserved cellular mechanism to degrade unneeded cytoplasmic proteins and organelles to recycle their components, and it is critical for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Whereas autophagy is essential for early development of embryos, no information exists regarding its functions during the transition from naive-to-primed pluripotency. Here, by using an in vitro transition model of ESCs to epiblast-like cells (EpiLCs), we find that dynamic changes in ATG7-dependent autophagy are critical for the naive-to-primed transition, and are also necessary for germline specification. RNA-seq and ATAC-seq profiling reveal that NANOG acts as a barrier to prevent pluripotency transition, and autophagy-dependent NANOG degradation is important for dismantling the naive pluripotency expression program through decommissioning of naive-associated active enhancers. Mechanistically, we found that autophagy receptor protein SQSTM1/p62 translocated into the nucleus during the pluripotency transition period and is preferentially associated with K63 ubiquitinated NANOG for selective protein degradation. In vivo, loss of autophagy by ATG7 depletion disrupts peri-implantation development and causes increased chromatin association of NANOG, which affects neuronal differentiation by competitively binding to OTX2-specific neuroectodermal development-associated regions. Taken together, our findings reveal that autophagy-dependent degradation of NANOG plays a critical role in regulating exit from the naive state and marks distinct cell fate allocation during lineage specification.Abbreviations: 3-MA: 3-methyladenine; EpiLC: epiblast-like cell; ESC: embryonic stem cell; PGC: primordial germ cell.

Glycomics reveal that ST6GAL1-mediated sialylation regulates uterine lumen closure during implantation.

OBJECTIVES: Implantation failure is a major cause of prenatal mortality. The uterine lumen closure contributes to embryo adhesion to the uterus, but its underlying mechanisms are largely unknown. Our previous study has reported that endometrial fold extension can lead to uterine lumen closure in pigs. The objective of this study was to reveal molecular mechanisms of the uterine lumen closure by characterizing the molecular basis of the endometrial fold extension during implantation in pigs. MATERIALS AND METHODS: Uterine and endometrium tissues during implantation were collected in pigs. MALDI-TOF MS was used to characterize the N-glycomic profiles. Histochemistry, siRNA transfection, Western blotting, lectin immumoprecipitation, mass spectrometry and assays of wounding healing and cell aggregation were performed to investigate the molecular basis. RESULTS: We observed that uterine luminal epithelium (LE) migrated collectively during endometrial fold extension. For the first time, we identified a large number of N-glycan compositions from endometrium during implantation using MALDI-TOF MS. Notably, the α2,6-linked sialic acid and ST6GAL1 were highly expressed in uterine LE when the endometrial folds extended greatly. Subsequently, the role of ST6GAL1-mediated 2,6-sialylation in collective epithelial migration was demonstrated. Finally, we found that ST6GAL1-mediated α2,6-sialylation of E-cadherin may participate in collective migration of uterine LE. CONCLUSIONS: The study reveals a mechanism of uterine lumen closure by identifying that ST6GAL1-mediated α2,6-sialylation of cell adhesion molecules contributes to endometrial fold extension through regulating collective migration of uterine LE.

Generation and characterization of stable pig pregastrulation epiblast stem cell lines.

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.