RNA surveillance by the RNA helicase MTR4 determines volume of mouse oocytes.

Oocytes are the largest cell type in multicellular animals. Here, we show that mRNA transporter 4 (MTR4) is indispensable for oocyte growth and functions as part of the RNA surveillance mechanism, which is responsible for nuclear waste RNA clearance. MTR4 ensures the normal post-transcriptional processing of maternal RNAs, their nuclear export to the cytoplasm, and the accumulation of properly processed transcripts. Oocytes with Mtr4 knockout fail to accumulate sufficient and normal transcripts in the cytoplasm and cannot grow to normal sizes. MTR4-dependent RNA surveillance has a previously unrecognized function in maintaining a stable nuclear environment for the establishment of non-canonical histone H3 lysine-4 trimethylation and chromatin reorganization, which is necessary to form a nucleolus-like structure in oocytes. In conclusion, MTR4-dependent RNA surveillance activity is a checkpoint that allows oocytes to grow to a normal size, undergo nuclear and cytoplasmic maturation, and acquire developmental competence.

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.

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.

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.

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.

Defective chromatin architectures in embryonic stem cells derived from somatic cell nuclear transfer impair their differentiation potentials.

Nuclear transfer embryonic stem cells (ntESCs) hold enormous promise for individual-specific regenerative medicine. However, the chromatin states of ntESCs remain poorly characterized. In this study, we employed ATAC-seq and Hi-C techniques to explore the chromatin accessibility and three-dimensional (3D) genome organization of ntESCs. The results show that the chromatin accessibility and genome structures of somatic cells are re-arranged to ESC-like states overall in ntESCs, including compartments, topologically associating domains (TADs) and chromatin loops. However, compared to fertilized ESCs (fESCs), ntESCs show some abnormal openness and structures that have not been reprogrammed completely, which impair the differentiation potential of ntESCs. The histone modification H3K9me3 may be involved in abnormal structures in ntESCs, including incorrect compartment switches and incomplete TAD rebuilding. Moreover, ntESCs and iPSCs show high similarity in 3D genome structures, while a few differences are detected due to different somatic cell origins and reprogramming mechanisms. Through systematic analyses, our study provides a global view of chromatin accessibility and 3D genome organization in ntESCs, which can further facilitate the understanding of the similarities and differences between ntESCs and fESCs.

TDG is a pig-specific epigenetic regulator with insensitivity to H3K9 and H3K27 demethylation in nuclear transfer embryos.

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.

Targeted overexpression of PPARγ in skeletal muscle by random insertion and CRISPR/Cas9 transgenic pig cloning enhances oxidative fiber formation and intramuscular fat deposition.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulator of adipogenesis and lipogenesis. To understand its roles in fiber formation and fat deposition in skeletal muscle, we successfully generated muscle-specific overexpression of PPARγ in two pig models by random insertion and CRISPR/Cas9 transgenic cloning procedures. The content of intramuscular fat was significantly increased in PPARγ pigs while had no changes on lean meat ratio. PPARγ could promote adipocyte differentiation by activating adipocyte differentiating regulators such as FABP4 and CCAAT/enhancer-binding protein (C/EBP), along with enhanced expression of LPL, FABP4, and PLIN1 to proceed fat deposition. Proteomics analyses demonstrated that oxidative metabolism of fatty acids and respiratory chain were activated in PPARγ pigs, thus, gathered more Ca2+ in PPARγ pigs. Raising of Ca2+ could result in increased phosphorylation of CAMKII and p38 MAPK in PPARγ pigs, which can stimulate MEF2 and PGC1α to affect fiber type and oxidative capacity. These results support that skeletal muscle-specific overexpression of PPARγ can promote oxidative fiber formation and intramuscular fat deposition in pigs.

Maternal Cytokines CXCL12, VEGFA, and WNT5A Promote Porcine Oocyte Maturation via MAPK Activation and Canonical WNT Inhibition.

Maternal regulatory factors endow the oocyte with developmental competence in vivo, which might be absent in current in vitro maturation (IVM) systems, thereby compromising oocyte quality. In the present study, by employing RNA sequencing data analysis, we expect to identify potential contributing factors to support porcine oocyte maturation through binding to their receptors on the oolemma. Here, C-X-C motif chemokine ligand 12 (CXCL12), vascular endothelial growth factor A (VEGFA), and Wingless-type MMTV integration site family member 5A (WNT5A), termed CVW, are selected and confirmed to be important maternal cytokines for porcine oocyte maturation. Combined supplementation of CVW promotes the nuclear maturation percentage from 57.2% in controls to 75.9%. More importantly, these maternal cytokines improve the developmental potential of matured oocytes by parthenogenesis, fertilization, and cloning, as their blastocyst formation efficiencies and total cell numbers are increased. CVW supplementation also enlarges perivitelline space and promotes cumulus expansion, which results in a more complete transzonal projection retraction on the zona pellucida, and a reduced incidence of polyspermy in fertilized oocytes. Meanwhile, inhibiting the CVW receptor-mediated signaling pathways severely impairs oocyte meiotic resumption and cumulus expansion during IVM. We further determine that maturation improvement by CVW is achieved through activating the MAPK pathway in advance and inhibiting the canonical WNT pathway at the end of the IVM period. These findings provide a new combination of three cytokines to promote the porcine IVM process, which also holds potential to be used in human assisted reproduction technologies as well as in other species.

Muscle fiber-type conversion in the transgenic pigs with overexpression of PGC1α gene in muscle.

The peroxisome proliferator-activated receptor gamma, co-activator 1 alpha(PGC1α) effectively induced the biosynthesis of the mitochondria and the energy metabolism, and also regulated the muscle fiber-type shift. Overexpression of PGC1α gene in mice led to higher oxidative muscle fiber composition in muscle. However, no researches about the significant differences of muscle fiber phenotype in pigs after PGC1α overexpression had been reported. The composition of muscle fiber-types which were distinguished by four myosin heavy chain(MYHC) isoforms, can significantly affect the muscle functions. In our study, we generated the transgenic pigs to investigate the effect of overexpression of PGC1α gene on muscle fiber-type conversion. The results showed that the number of oxidative muscle fiber(type1 muscle fiber) was increased and the number of glycolytic muscle fiber(type2b muscle fiber) was decreased in the transgenic pigs. Furthermore, we found that PGC1α overexpression up-regulated the expression of MYHC1 and MYHC2a and down-regulated the expression of MYHC2b.The analysis of genes expression demonstrated the main differentially expressed genes were MSTN, Myog and FOXO1. In conclusion, the overexpression of PGC1α gene can promote the glycolytic muscle fiber transform to the oxidative muscle fiber in pigs.

Transcriptional regulation analysis of FAM3A gene and its effect on adipocyte differentiation.

FAM3A (family with sequence similarity 3, member A) is regulated by PPARG and participates in the metabolism of lipid in liver. However, the transcriptional regulation analysis of FAM3A is very little and biological function of FAM3A still unclear. In this study, the core promoter region and transcription factor binding sites of FAM3A gene were identified and characterized using dual luciferase report experiments and electrophoretic mobility shift assays (EMSA). The promoter activity of FAM3A was dramatically decreased after the mutation of C/EBPß binding sites, suggesting that C/EBPß is a transcriptional activator of FAM3A. Overexpression of FAM3A significantly inhibited the efficiency of preadipocytes to differentiate into adipocytes as indicated by Western Blot and Oil Red O staining assay. These results suggest that C/EBPß plays an important role in regulating FAM3A promoter activity and FAM3A inhibits adipocyte differentiation.

The Expression of Porcine Prdx6 Gene Is Up-Regulated by C/EBPß and CREB.

Peroxiredoxin6 (Prdx6) is one of the peroxiredoxin (Prdxs) family members that play an important role in maintaining cell homeostasis. Our previous studies demonstrated that Prdx6 was significantly associated with pig meat quality, especially meat tenderness. However, the transcriptional regulation of porcine Prdx6 remains unclear. In this study, we determined the transcription start site (TSS) of porcine Prdx6 gene by 5' rapid-amplification of cDNA ends (5' RACE). Several regulatory elements including CCAAT/enhancer-binding proteinß (C/EBPß), Myogenic Differentiation (MyoD), cAMP response element binding protein (CREB), stimulating protein1 (Sp1) and heat shock factor (HSF) binding sites were found by computational analyses together with luciferase reporter system. Overexpression and RNA interference experiments showed that C/EBPß or CREB could up-regulate the expression of porcine Prdx6 gene at both mRNA and protein level. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation assays (ChIP) confirmed that C/EBPß and CREB could interact with Prdx6 promoter. Immuoprecipitation results also showed that C/EBPß could interact with Prdx6 in vivo. Taken together, our findings identified C/EBPß and CREB as the important regulators of porcine Prdx6 gene expression, and offered clues for further investigation of Prdx6 gene function.