ABSTRACT
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.
Subject(s)
Cell Differentiation , Pluripotent Stem Cells , Wnt Signaling Pathway , Animals , beta Catenin/genetics , beta Catenin/metabolism , Cell Differentiation/genetics , Chromatin/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Swine , Wnt Signaling Pathway/genetics , T-Box Domain Proteins/metabolism , Octamer Transcription Factor-3/metabolism , Cell LineABSTRACT
In mammalian growing follicles, oocytes are arrested at the diplotene stage (which resembles the G2/M boundary in mitosis), while the granulosa cells (GCs) continue to proliferate during follicular development, reflecting a cell cycle asynchrony between oocytes and GCs. Hypoxanthine (Hx), a purine present in the follicular fluid, has been shown to induce oocytes meiotic arrest, although its role in GC proliferation remains ill-defined. Here, we demonstrate that Hx indiscriminately prevents G2-to-M phase transition in porcine GCs. However, oocyte-derived paracrine factors (ODPFs), particularly GDF9 and BMP15, maintain the proliferation of GCs, partly by activating the ERK1/2 signaling and enabling the G2/M transition that is suppressed by Hx. Interestingly, GCs with lower expression of GDF9/BMP15 receptors appear to be more sensitive to Hx-induced G2/M arrest and become easily detached from the follicular wall. Importantly, Hx-mediated inhibition of G2/M progression instigates GC apoptosis, which is ameliorated in the presence of GDF9 and/or BMP15. Therefore, our data indicate that the counterbalance of intrafollicular factors, particularly Hx and oocyte-derived GDF9/BMP15, fine-tunes the development of porcine follicles by regulating the cell cycle progression of GCs.
Subject(s)
Granulosa Cells/metabolism , Hypoxanthine/metabolism , Oocytes/metabolism , Animals , Apoptosis/genetics , Apoptosis/physiology , Bone Morphogenetic Protein 15/genetics , Bone Morphogenetic Protein 15/metabolism , Cell Proliferation/genetics , Cell Proliferation/physiology , Female , G2 Phase Cell Cycle Checkpoints/genetics , G2 Phase Cell Cycle Checkpoints/physiology , Growth Differentiation Factor 9/genetics , Growth Differentiation Factor 9/metabolism , SwineABSTRACT
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.
Subject(s)
Placenta , Placentation , Pregnancy , Female , Humans , Placenta/metabolism , Nicotine/toxicity , Nicotine/metabolism , Nuclear Proteins/metabolism , Signal TransductionABSTRACT
It is well known that charge separation is crucial for efficient photocatalytic solar conversion. Although some covalent-organic frameworks (COFs) exhibit visible-light harvest, the large exciton binding energies reduce their photocatalytic efficiencies. Herein, we developed a novel method to post-treat the olefin-linked COFs with end-capping polycyclic aromatic hydrocarbons (PAHs) for spontaneous charge separation. Interestingly, a type-II heterostructure is constructed in our perylene-modified COFs which displays drastically enhanced performance for photocatalytic CO2 reduction, with an efficiency of 8-fold higher than that of unmodified COF. A combination of electrochemical, steady-state, and time-resolved spectroscopic measurements indicates that such drastically enhanced performance should be attributed to photoinduced spontaneous charge separation in the heterostructure. These results illustrate the feasibility of engineering the charge-separation properties of crystalline porous frameworks at a molecular level for artificial photosynthesis.
ABSTRACT
In developing follicles, the granulosa cells (GCs) live in a hypoxic environment due to the devoid of blood supply. Upon hypoxic conditions, several types of mammalian cells have been reported to undergo apoptosis. Follicle-stimulating hormone (FSH) is known as the primary survival factor for antral follicles by preventing GCs apoptosis. Mitophagy is a type of organelle-specific autophagy that removes damaged or stressed mitochondria to maintain cellular health. This study provides the first evidence suggesting that FSH-mediated mitophagy protected porcine GCs from hypoxia-induced apoptosis. Our data showed that the GCs apoptosis caused by mitochondrial pathway upon hypoxia stress was markedly attenuated after FSH treatment, which was correlated with enhanced activation of mitophagy. Interestingly, FSH also stimulated mitochondrial biogenesis as suggested by increased expression of mitochondrial transcription factor A and nuclear respiratory factor 1 during hypoxia exposure. Notably, the protein level of hypoxia inducible factor-1α (HIF-1α) was significantly increased in hypoxic GCs following FSH treatment, accompanied by elevated mitophagic activity and dampened apoptotic signaling. Blocking HIF-1α inhibited mitophagy and restored hypoxia-induced apoptosis despite FSH treatment. Importantly, FSH promoted the expression of serine/threonine kinase PTEN induced putative kinase 1 (PINK1) and the E3 ligase Parkin during hypoxia stress through a HIF-1α dependent manner. This induced the mitophagic clearance of damaged mitochondria, hence inhibiting apoptosis by reducing cytochrome c releasing. The inhibition of HIF-1α and/or PINK1 using inhibitor or RNAi further confirmed the role of the FSH-HIF-1α-PINK1-Parkin-mitophagy axis in suppressing GC apoptosis under hypoxic conditions. These findings highlight a novel function of FSH in preserving GCs viability against hypoxic damage by activating HIF-1α-PINK1-Parkin-mediated mitophagy.
Subject(s)
Apoptosis/drug effects , Follicle Stimulating Hormone/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mitophagy/drug effects , Protein Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Cell Survival/drug effects , Cell Survival/genetics , Female , Microscopy, Electron, Transmission , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , SwineABSTRACT
Malathion (MAL) is a common organophosphorus pesticide and affects both animal and human reproduction. However, the mechanisms regarding how MAL affects the mammalian oocyte quality and how to prevent it have not been fully investigated. In this study, we used porcine oocyte as a model and proved that MAL impaired porcine oocyte quality in a dose-dependent manner during maturation. MAL decreased the first polar body extrusion, disrupted spindle assembly and chromosome alignment, impaired cortical granules (CGs) distribution, and increased reactive oxygen species (ROS) level in oocytes. RNA-seq analysis showed that MAL exposure altered the expression of 2,917 genes in the porcine maturated oocytes and most genes were related to ROS, the lipid droplet process, and the energy supplement. Nevertheless, these defects could be remarkably ameliorated by adding melatonin (MLT) into the oocyte maturation medium. MLT increased oocyte maturation rate and decreased the abnormities of spindle assembly, CGs distribution and ROS accumulation in MAL-exposed porcine oocytes. More important, MLT upregulated the expression of genes related to lipid droplet metabolism (PPARγ and PLIN2), decreased lipid droplet size and lipid peroxidation in MAL-exposed porcine oocytes. Finally, we found that MLT increased the blastocysts formation and the cell numbers of blastocysts in MAL-exposed porcine oocytes after parthenogenetic activation, which was mediated by reduction of ROS levels and maintaining lipid droplet metabolism. Taken together, our results revealed that MLT had a protective action against MAL-induced deterioration of porcine oocyte quality.
Subject(s)
Malathion/metabolism , Melatonin/pharmacology , Oocytes/drug effects , Oogenesis/drug effects , Animals , Embryonic Development/drug effects , In Vitro Oocyte Maturation Techniques/methods , Lipid Peroxidation/drug effects , Meiosis/drug effects , Oocytes/metabolism , Parthenogenesis/drug effects , Reactive Oxygen Species/metabolism , SwineABSTRACT
As the follicle develops, the thickening of the granulosa compartment leads to progressively deficient supply of oxygen in granulosa cells (GCs) due to the growing distances from the follicular vessels. These conditions are believed to cause hypoxia in GCs during folliculogenesis. Upon hypoxic conditions, several types of mammalian cells have been reported to undergo cell cycle arrest. However, it remains unclear whether hypoxia exerts any impact on cell cycle progression of GCs. On the other hand, although the GCs may live in a hypoxic environment, their mitotic capability appears to be unaffected in growing follicles. It thus raises the question whether there are certain intraovarian factors that might overcome the inhibitory effects of hypoxia. The present study provides the first evidence suggesting that cobalt chloride (CoCl2)-mimicked hypoxia prevented G1-to-S cell cycle progression in porcine GCs. In addition, we demonstrated that the inhibitory effects of CoCl2 on GCs cell cycle are mediated through hypoxia-inducible factor-1 alpha/FOXO1/Cdkn1b pathway. Moreover, we identified insulin-like growth factor-I (IGF-I) as an intrafollicular factor required for cell cycle recovery by binding to IGF-I receptor in GCs suffering CoCl2 stimulation. Further investigations confirmed a role of IGF-I in preserving G1/S progression of CoCl2-treated GCs via activating the cyclin E/cyclin-dependent kinase2 complex through the phoshatidylinositol-3 kinase/protein kinase B (AKT)/FOXO1/Cdkn1b axis. Although the present findings were based on a hypoxia mimicking model by using CoCl2, our study might shed new light on the regulatory mechanism of GCs cell cycle upon hypoxic stimulation.
Subject(s)
Cell Cycle Checkpoints/drug effects , Granulosa Cells/drug effects , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Hypoxia/metabolism , Insulin-Like Growth Factor I/pharmacology , Signal Transduction/drug effects , Animals , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Cycle Checkpoints/physiology , Cell Hypoxia/drug effects , Cell Hypoxia/physiology , Cobalt/pharmacology , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Estradiol/pharmacology , Female , Follicle Stimulating Hormone/pharmacology , Forkhead Box Protein O1/metabolism , Granulosa Cells/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/physiology , SwineABSTRACT
Autophagy plays an important role in embryo development; however, only limited information is available on how autophagy specifically regulates embryo development, especially under low oxygen culture conditions. In this study we used parthenogenetic activation (PA) of porcine embryos to test the hypothesis that a low oxygen concentration (5%) could promote porcine embryo development by activating autophagy. Immunofluorescence staining revealed that low oxygen tension activated autophagy and alleviated oxidative stress in porcine PA embryos. Development was significantly affected when autophagy was blocked by 3-methyladenine, even under low oxygen culture conditions, with increased reactive oxygen species levels and malondialdehyde content. Furthermore, the decreased expression of pluripotency-associated genes induced by autophagy inhibition could be recovered by treatment with the antioxidant vitamin C. Together, these results demonstrate that low oxygen-induced autophagy regulates embryo development through antioxidant mechanisms in the pig.
Subject(s)
Autophagy/physiology , Embryo Culture Techniques/veterinary , Embryonic Development/physiology , Oxygen/administration & dosage , Parthenogenesis/physiology , Swine/embryology , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Antioxidants/pharmacology , Ascorbic Acid/pharmacology , Autophagy/drug effects , Embryo Culture Techniques/methods , Oxidative Stress/drug effects , Oxidative Stress/physiologyABSTRACT
Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components. Here we showed that autophagy was essential for the glycolysis switch and energy homeostasis in mouse granulosa cells under hypoxic condition. Our data indicated that hypoxia inducible factor-1α (HIF-1α) could be largely increased in developing follicles and this remarkable upregulation of HIF-1α triggered cell autophagy and glucose uptake. We found that blocking autophagy by Atg7 knockdown and 3-methyladenine (3-MA) treatment affected the glucose metabolism, with increased glycolytic enzyme activity and decreased ATP production. We also found enhanced lactate level, which was harmful to granulosa cells and could induce cell apoptosis. Thus, our findings highlight a protective role of HIF-1α-dependent autophagy for the granulosa cell glycolysis switch in both energy supply and cell survival.
Subject(s)
Autophagy/physiology , Cell Hypoxia/physiology , Glycolysis/physiology , Granulosa Cells/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Animals , Apoptosis/drug effects , Apoptosis/physiology , Autophagy/drug effects , Cell Hypoxia/drug effects , Female , Follicle Stimulating Hormone/pharmacology , Glucose/metabolism , Glycolysis/drug effects , Granulosa Cells/drug effects , MiceABSTRACT
The insecticide cypermethrin has been considered as an endocrine-disrupting chemicals (EDCs) with anti-androgenic activity by interfering with interleukin-6 (IL-6) - induced ligand-independent AR signaling. The purpose of this study was to clarify whether the signal transducer and activator of transcription 3 (STAT3) was involved in the antagonism effect of cypermethrin. In this study, the Western blot was to test the level of STAT3 phosphorylation and the mammalian two-hybrid assay was developed to assess the AR-STAT3 interaction. The date showed that IL-6 increased the phosphorylation level of STAT3 and enhanced the AR-STAT3 interaction. Cypermethrin did not affect the phosphorylation level of STAT3 induced by IL-6, while suppressed the AR-STAT3 interaction induced by IL-6 significantly at the concentration of 10-5 M (p < 0.05). The study indicates cypermethrin inhibits IL-6-induced AR signaling by suppressing the interaction between the AR and STAT3. We provide a novel mechanism of cypermethrin-mediated antagonism on IL-6-induced AR activation associated with STAT3.
Subject(s)
Insecticides/pharmacology , Interleukin-6/physiology , Pyrethrins/pharmacology , Receptors, Androgen/genetics , STAT3 Transcription Factor/metabolism , Transcriptional Activation/physiology , Animals , Cell Line , Humans , Phosphorylation , Receptors, Androgen/metabolismABSTRACT
BACKGROUND: Non-syndromic cleft lip with or without cleft palate (NSCL/P) is among the most common congenital malformations. The etiology of NSCL/P remains poorly characterized owing to its complex genetic heterogeneity. The objective of this study was to identify genetic variants that increase susceptibility to NSCL/P. MATERIAL AND METHODS: Whole-exome sequencing (WES) was performed in 8 fetuses with NSCL/P in China. Bioinformatics analysis was performed using commercially available software. Variants detected by WES were validated by Sanger sequencing. RESULTS: By filtering out synonymous variants in exons, we identified average 8575 nonsynonymous single nucleotide variants (SNVs). We subsequently compared the SNVs against public databases including NCBI dbSNP build 135 and 1000 Genomes Project and obtained an average of 203 SNVs. Total 12 reported candidate genes were verified by Sanger sequencing. Sanger sequencing also confirmed 16 novel SNVs shared by two or more samples. CONCLUSIONS: We have found and confirmed 16 susceptibility genes responsible for NSCL/P, which may play important role in the etiology of NSCL/P. The susceptibility genes identified in this study will not only be useful in revealing the etiology of NSCL/P but also in diagnosis and treatment of the patients with NSCL/P.
Subject(s)
Cleft Lip/genetics , Cleft Palate/genetics , Genetic Predisposition to Disease , Polymorphism, Single Nucleotide , Base Sequence , Exome , HumansABSTRACT
Sma- and Mad-related protein 4 (SMAD4) is the central mediator of the transforming growth factor beta signaling pathway and is closely related to mammalian reproductive ability and the development of ovarian follicles. However, little is currently known about the role of SMAD4 in mammalian follicular granulosa cell (GC) apoptosis or its regulation by miRNAs. Here, we found that the porcine SMAD4 protein was expressed at high levels in GCs and oocytes from primary, preantral, and antral follicles, and only slightly expressed in theca cells; its expression level was down-regulated in apoptotic ovarian GCs, suggesting that SMAD4 may be involved in ovary development and selection. Overexpression and knockdown of SMAD4 increased the proliferation and apoptosis of cultured porcine GCs, respectively. In addition, the use of miRNA mimics and luciferase reporter assays revealed that miRNA-26b (miR-26b) functions as a proapoptotic factor in porcine follicular GCs by targeting the 3'-untranslated region of the SMAD4 gene. Overexpression of miR-26b in follicular GCs suppressed SMAD4 mRNA and protein levels, resulting in down-regulation of the antiapoptotic BCL-2 gene and the promotion of GC apoptosis. Furthermore, transforming growth factor beta 1 (TGF-beta1) down-regulates miR-26b expression in porcine GCs. Taken together, these data suggest that SMAD4 plays a critical role in porcine follicular GC apoptosis and follicular atresia and that miR-26b may have a proapoptotic role in GCs by regulating the expression of SMAD4 in the transforming growth factor beta signaling pathway.
Subject(s)
Apoptosis/genetics , Granulosa Cells/metabolism , MicroRNAs/physiology , Smad4 Protein/genetics , Swine/genetics , Animals , Cells, Cultured , Female , Follicular Phase/genetics , Follicular Phase/metabolism , Gene Expression Regulation , Ovarian Follicle/metabolism , Ovary/metabolism , Smad4 Protein/metabolismABSTRACT
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.
Subject(s)
Placenta , Transcription Factors , Female , Pregnancy , Humans , Placenta/metabolism , Transcription Factors/metabolism , Trophoblasts/metabolism , Cell Differentiation/genetics , Embryonic Stem Cells/metabolismABSTRACT
BACKGROUND: The Luchuan pig is an indigenous breed from Luchuan County, China, with cultural and genetic significance. However, traditional knowledge and conservation status have not been systematically documented. METHODS: Using ethnobiological methods, we surveyed 72 Luchuan pig farmers in 7 townships during 2021-2023. Semi-structured interviews and participant observation were conducted to document traditional knowledge and management practices. RESULTS: The locals reported 51 plant species used as pig feed, with 30 wild species. Growth-stage-specific feeding and seasonal adjustment practices were documented. We recorded 62 ethnoveterinary plant uses, mainly for treating pigs' heat stress and skin conditions. Luchuan pigs play central roles in local Hakka customs, rituals, and cuisine. Additonally, the new ecological farming models minimize the environmental impacts to the local community. However, there are still some challenges remained for conserving and promoting Luchuan pigs. CONCLUSIONS: The Luchuan Hakka people possess rich traditional knowledge and management experience in raising Luchuan pigs. Our study provides extensive documentation of traditional knowledge and recommends integrating cultural and genetic aspects for sustaining this biocultural heritage. Findings can inform initiatives supporting local breed conservation globally.
Subject(s)
Agriculture , Breeding , Humans , Swine , Animals , China , Culture , FarmersABSTRACT
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.
Subject(s)
Arsenites , Zygote , Pregnancy , Female , Humans , Animals , Mice , Zygote/metabolism , Arsenites/toxicity , Arsenites/metabolism , Embryonic Development/genetics , Mammals/genetics , Mammals/metabolism , Gene Expression Regulation, DevelopmentalABSTRACT
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.
Subject(s)
ATP Citrate (pro-S)-Lyase , Macroautophagy , Female , Humans , Animals , Swine , Sequestosome-1 Protein/metabolism , ATP Citrate (pro-S)-Lyase/metabolism , Citric Acid/metabolism , Autophagy , Oocytes/metabolism , Citrates/metabolism , Acyltransferases/metabolism , Ubiquitin/metabolism , HomeostasisABSTRACT
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.
Subject(s)
Autophagy , Embryonic Stem Cells , Embryonic Stem Cells/metabolism , Cell Differentiation , Germ Layers/metabolism , Chromatin/metabolismABSTRACT
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.
Subject(s)
Histone Code , Histones , Animals , Chromatin/genetics , Chromatin/metabolism , Embryo, Mammalian/metabolism , Histone Code/genetics , Histones/genetics , Histones/metabolism , Mammals/genetics , Mammals/metabolism , Mice , Protein Processing, Post-TranslationalABSTRACT
Pig cloning by somatic cell nuclear transfer (SCNT) frequently undergoes incomplete epigenetic remodeling during the maternal-to-zygotic transition, which leads to a significant embryonic loss before implantation. Here, we generated the first genome-wide landscapes of histone methylation in pig SCNT embryos. Excessive H3K9me3 and H3K27me3, but not H3K4me3, were observed in the genomic regions with unfaithful embryonic genome activation and donor-cell-specific gene silencing. A combination of H3K9 demethylase KDM4A and GSK126, an inhibitor of H3K27me3 writer, were able to remove these epigenetic barriers and restore the global transcriptome in SCNT embryos. More importantly, thymine DNA glycosylase (TDG) was defined as a pig-specific epigenetic regulator for nuclear reprogramming, which was not reactivated by H3K9me3 and H3K27me3 removal. Both combined treatment and transient TDG overexpression promoted DNA demethylation and enhanced the blastocyst-forming rates of SCNT embryos, thus offering valuable methods to increase the cloning efficiency of genome-edited pigs for agricultural and biomedical purposes.
Subject(s)
Embryo, Mammalian/metabolism , Epigenesis, Genetic , Gene Expression Regulation , Histones/metabolism , Nuclear Transfer Techniques , Thymine DNA Glycosylase/genetics , Animals , Blastocyst/cytology , Blastocyst/metabolism , DNA Methylation , Demethylation , Embryo, Mammalian/drug effects , Embryo, Mammalian/embryology , Gene Expression Profiling/methods , Histone Demethylases/genetics , Histone Demethylases/metabolism , Indoles/pharmacology , Lysine/metabolism , Methylation , Pyridones/pharmacology , Swine , Thymine DNA Glycosylase/metabolismABSTRACT
Signaling pathways and transcription factors are involved in porcine embryonic development. Here, we demonstrate that glycogen synthase kinase-3 (GSK3) inhibitor, CHIR99021 and recombinant porcine interleukin-6 (rpIL6) significantly promote porcine parthenogenetic blastocyst formation (49.23 ± 8.40% vs 32.34 ± 4.15%), with increased inner cell mass (ICM) cell numbers (7.72 ± 2.30 vs 4.28 ± 1.60) and higher expression of pluripotent genes, such as OCT4, SOX2 and NANOG. Furthermore, CHIR99021 and rpIL6 improve blastocyst quality with increased blastocyst hatching percentage (16.19 ± 1.96% vs 10.25 ± 1.12%) and subsequently porcine pluripotent stem cells (pPSCs) derivation efficiency. These results advance the understanding of porcine pre-implantation development and provide evidences in improving the blastocyst quality.