Identification of New Compounds against PRRSV Infection by Directly Targeting CD163.

The porcine reproductive and respiratory syndrome viruses (PRRSV) led to a global panzootic and huge economical losses to the pork industry. PRRSV targets the scavenger receptor CD163 for productive infection. However, currently no effective treatment is available to control the spread of this disease. Using bimolecular fluorescence complementation (BiFC) assays, we screened a set of small molecules potentially targeting the scavenger receptor cysteine-rich domain 5 (SRCR5) of CD163. We found that the assay examining protein-protein interactions (PPI) between PRRSV glycoprotein 4 (GP4) and the CD163-SRCR5 domain mainly identifies compounds that potently inhibit PRRSV infection, while examining the PPI between PRRSV-GP2a and the SRCR5 domain maximized the identification of positive compounds, including additional ones with various antiviral capabilities. These positive compounds significantly inhibited both types 1 and 2 PRRSV infection of porcine alveolar macrophages. We confirmed that the highly active compounds physically bind to the CD163-SRCR5 protein, with dissociation constant (KD) values ranging from 28 to 39 µM. Structure-activity-relationship (SAR) analysis revealed that although both the 3-(morpholinosulfonyl)anilino and benzenesulfonamide moieties in these compounds are critical for the potency to inhibit PRRSV infection, the morpholinosulfonyl group can be replaced by chlorine substituents without significant loss of antiviral potency. Our study established a system for throughput screening of natural or synthetic compounds highly effective on blocking of PRRSV infection and shed light on further SAR modification of these compounds. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Current vaccines cannot provide cross protection against different strains, and there are no effective treatments available to hamper the spread of this disease. In this study, we identified a group of new small molecules that can inhibit the PRRSV interaction with its specific receptor CD163 and dramatically block the infection of both types 1 and type 2 PRRSVs to host cells. We also demonstrated the physical association of these compounds with the SRCR5 domain of CD163. In addition, molecular docking and structure-activity relationship analyses provided new insights for the CD163/PRRSV glycoprotein interaction and further improvement of these compounds against PRRSV infection.

Au nano-cone array for SERS detection of associated miRNA in lymphoma patients.

Based on Au nano-cone array (Au-NCA) and a three-segment hybridization strategy, a novel SERS biosensor is proposed for the ultrasensitive detection of the microRNA miR-21. The uniform, stable, and reproducible Au-NCA was prepared by the single-layer colloidal ball template method. Subsequently, the target was hybridized with sequence 2. The resulting target-sequence 2 complex was then hybridized with sequence 1 anchored on Au-NCA. Thus, a three-segment sequence complex was formed. SERS measurements can be performed without the need for complex purification and amplification steps. Due to the ability of miR-21 to perform specific complementary hybridization with two sequences, SERS biosensors have superior specificity for miR-21 without interference from other miRNAs. Under the optimal conditions, the SERS biosensor was applied and the limit of detection (LOD) was as low as 3.02 aM. This method has been successfully used to the detection of miR-21 in the serum of lymphoma patients and healthy volunteers. The results are consistent with the traditional test methods. Therefore, this novel SERS biosensor shows excellent clinical translational potential in the detection of lymphoma.

Dynamic and aberrant patterns of H3K4me3, H3K9me3, and H3K27me3 during early zygotic genome activation in cloned mouse embryos.

Somatic cell nuclear transfer (NT) is associated with aberrant changes in epigenetic reprogramming that impede the development of embryos, particularly during zygotic genome activation. Here, we characterized epigenetic patterns of H3K4me3, H3K9me3, and H3K27me3 in mouse NT embryos up to the second cell cycle (i.e. four-celled stage) during zygotic genome activation. In vivo fertilized and parthenogenetically activated (PA) embryos served as controls. In fertilized embryos, maternal and paternal pronuclei exhibited asymmetric H3K4me3, H3K9me3, and H3K27me3 modifications, with the paternal pronucleus showing delayed epigenetic modifications. Higher levels of H3K4me3 and H3K9me3 were observed in NT and PA embryos than in fertilized embryos. However, NT embryos exhibited a lower level of H3K27me3 than PA and fertilized embryos from pronuclear stage 3 to the four-celled stage. Our finding that NT embryos exhibited aberrant H3K4me3, H3K9me3, and H3K27me3 modifications in comparison with fertilized embryos during early zygotic genome activation help to unravel the epigenetic mechanisms of methylation changes in early NT reprogramming and provide an insight into the role of histone H3 in the regulation of cell plasticity during natural reproduction and somatic cell NT.

Atlas of receptor genes expressed by the bovine morula and corresponding ligand-related genes expressed by uterine endometrium.

Regulation of the mammalian embryo involves cell-signaling molecules produced by the maternal oviduct and endometrium. Here, datasets on the transcriptome of the gestational Days 5 and 6 bovine morula and Day 5 maternal endometrium were examined to identify receptor genes expressed by the morula and expression of the corresponding ligand-related genes in the endometrium. A total of 175 receptor genes were identified in the morula, including 48 encoding for growth factors or WNT signaling molecules, 25 for cytokines and chemokines, 35 involved in juxtacrine and matricellular signaling and 25 encoding for receptors for small molecules. Some of the highly-expressed pairs of endometrial ligand and embryo receptor genes included MDK and its receptors ITGB1, SDC4 and LRP2, WNT5A (RYK), VEGFA (ITGB1), GPI (AMFR), and the hedgehog proteins IHH and DHH (HHIP). The most highly expressed receptors for small molecules were GPRC5C (retinoic acid receptor), PGRMC1 (progesterone), and CHRNB2 (acetylcholine). There were also 84 genes encoding for cell signaling ligands expressed by the morula, with the most highly expressed being GPI, AIMP1, TIMP1, IK, and CCN2. The atlas of receptor and ligand genes should prove useful for understanding details of the communication between the embryo and mother that underlies optimal embryonic development.

Establishment of Bovine-Induced Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) and the other reprogramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined media of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of integration into extra-embryonic tissues. Finally, at least 24.5% cloning efficiency could be obtained in nuclear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.

JAK/STAT3 regulated global gene expression dynamics during late-stage reprogramming process.

BACKGROUND: The generation of induced pluripotent stem cells (iPSCs) has underdefined mechanisms. In addition, leukemia inhibitory factor (LIF) activated Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is the master regulator for naïve-state pluripotency achievement and maintenance. However, the regulatory process to attain naïve pluripotent iPSCs is not well understood. RESULTS: We performed transcriptome analysis to dissect the genomic expression during mouse iPSC induction, with or without blocking the JAK/STAT3 activity. We describe JAK/STAT3 signaling-specific biological events such as gametogenesis, meiotic/mitotic cell cycle, and DNA repair, and JAK/STAT3-dependent expression of key transcription factors such as the naïve pluripotency-specific genes, developmental pluripotency associated (Dppa) family, along with histone modifiers and non-coding RNAs in reprogramming. We discover that JAK/STAT3 activity does not affect early phase mesenchymal to epithelial transition (MET) but is necessary for proper imprinting of the Dlk1-Dio3 region, an essential event for pluripotency achievement at late-reprogramming stage. This correlates with the JAK/STAT3-dependent stimulation of Dppa3 and Polycomb repressive complex 2 (PRC2) genes. We further demonstrate that JAK/STAT3 activity is essential for DNA demethylation of pluripotent loci including Oct4, Nanog, and the Dlk1-Dio3 regions. These findings correlate well with the previously identified STAT3 direct targets. We further propose a model of pluripotency achievement regulated by JAK/STAT3 signaling during the reprogramming process. CONCLUSIONS: Our study illustrates novel insights for JAK/STAT3 promoted pluripotency establishment, which are valuable for further improving the naïve-pluripotent iPSC generation across different species including humans.

DNA methylomes of bovine gametes and in vivo produced preimplantation embryos.

DNA methylation is an important epigenetic modification that undergoes dynamic changes in mammalian embryogenesis, during which both parental genomes are reprogrammed. Despite the many immunostaining studies that have assessed global methylation, the gene-specific DNA methylation patterns in bovine preimplantation embryos are unknown. Using reduced representation bisulfite sequencing, we determined genome-scale DNA methylation of bovine sperm and individual in vivo developed oocytes and preimplantation embryos. We show that (1) the major wave of genome-wide demethylation was completed by the 8-cell stage; (2) promoter methylation was significantly and inversely correlated with gene expression at the 8-cell and blastocyst stages; (3) sperm and oocytes have numerous differentially methylated regions (DMRs)-DMRs specific for sperm were strongly enriched in long terminal repeats and rapidly lost methylation in embryos; while the oocyte-specific DMRs were more frequently localized in exons and CpG islands (CGIs) and demethylated gradually across cleavage stages; (4) DMRs were also found between in vivo and in vitro matured oocytes; and (5) differential methylation between bovine gametes was confirmed in some but not all known imprinted genes. Our data provide insights into the complex epigenetic reprogramming of bovine early embryos, which serve as an important model for human preimplantation development.

A cross-species bi-clustering approach to identifying conserved co-regulated genes.

MOTIVATION: A growing number of studies have explored the process of pre-implantation embryonic development of multiple mammalian species. However, the conservation and variation among different species in their developmental programming are poorly defined due to the lack of effective computational methods for detecting co-regularized genes that are conserved across species. The most sophisticated method to date for identifying conserved co-regulated genes is a two-step approach. This approach first identifies gene clusters for each species by a cluster analysis of gene expression data, and subsequently computes the overlaps of clusters identified from different species to reveal common subgroups. This approach is ineffective to deal with the noise in the expression data introduced by the complicated procedures in quantifying gene expression. Furthermore, due to the sequential nature of the approach, the gene clusters identified in the first step may have little overlap among different species in the second step, thus difficult to detect conserved co-regulated genes. RESULTS: We propose a cross-species bi-clustering approach which first denoises the gene expression data of each species into a data matrix. The rows of the data matrices of different species represent the same set of genes that are characterized by their expression patterns over the developmental stages of each species as columns. A novel bi-clustering method is then developed to cluster genes into subgroups by a joint sparse rank-one factorization of all the data matrices. This method decomposes a data matrix into a product of a column vector and a row vector where the column vector is a consistent indicator across the matrices (species) to identify the same gene cluster and the row vector specifies for each species the developmental stages that the clustered genes co-regulate. Efficient optimization algorithm has been developed with convergence analysis. This approach was first validated on synthetic data and compared to the two-step method and several recent joint clustering methods. We then applied this approach to two real world datasets of gene expression during the pre-implantation embryonic development of the human and mouse. Co-regulated genes consistent between the human and mouse were identified, offering insights into conserved functions, as well as similarities and differences in genome activation timing between the human and mouse embryos. AVAILABILITY AND IMPLEMENTATION: The R package containing the implementation of the proposed method in C ++ is available at: https://github.com/JavonSun/mvbc.git and also at the R platform https://www.r-project.org/ CONTACT: jinbo@engr.uconn.edu.

WNT regulation of embryonic development likely involves pathways independent of nuclear CTNNB1.

The bovine was used to examine the potential for WNT signaling to affect the preimplantation embryo. Expression of seven key genes involved in canonical WNT signaling declined to a nadir at the morula or blastocyst stage. Expression of 80 genes associated with WNT signaling in the morula and inner cell mass (ICM) and trophectoderm (TE) of the blastocyst was also evaluated. Many genes associated with WNT signaling were characterized by low transcript abundance. Seven genes were different between ICM and TE, and all of them were overexpressed in TE as compared to ICM, including WNT6, FZD1, FZD7, LRP6, PORCN, APC and SFRP1 Immunoreactive CTNNB1 was localized primarily to the plasma membrane at all stages examined from the 2-cell to blastocyst stages of development. Strikingly, neither CTNNB1 nor non-phospho (i.e., active) CTNNB1 was observed in the nucleus of blastomeres at any stage of development even after the addition of WNT activators to culture. In contrast, CTNNB1 associated with the plasma membrane was increased by activators of WNT signaling. The planar cell polarity pathway (PCP) could be activated in the embryo as indicated by an experiment demonstrating an increase in phospho-JNK in the nucleus of blastocysts treated with the non-canonical WNT11. Furthermore, WNT11 improved development to the blastocyst stage. In conclusion, canonical WNT signaling is attenuated in the preimplantation bovine embryo but WNT can activate the PCP component JNK. Thus, regulation of embryonic development by WNT is likely to involve activation of pathways independent of nuclear actions of CTNNB1.

Differential effects of Akt isoforms on somatic cell reprogramming.

Akt plays an important role in cell growth, proliferation and survival. The specific roles of the three Akt isoforms in somatic cell reprogramming have not been investigated. Here we report that, during iPSC generation, enhanced Akt1 activity promotes complete reprogramming mainly through increased activation of Stat3 in concert with leukemia inhibitory factor (LIF) and, to a lesser extent, through promotion of colony formation. Akt1 augments Stat3 activity through activation of mTOR and upregulation of LIF receptor expression. Similarly, enhanced Akt2 or Akt3 activation also promotes reprogramming and coordinates with LIF to activate Stat3. Blocking Akt1 or Akt3 but not Akt2 expression prohibits cell proliferation and reprogramming. Furthermore, the halt in cell proliferation and reprogramming caused by mTOR and Akt inhibitors can be reversed by inhibition of GSK3. Finally, we found that expressing the GSK3ß target Esrrb overrides inhibition of Akt and restores reprogramming. Our data demonstrated that during reprogramming, Akt promotes establishment of pluripotency through co-stimulation of Stat3 activity with LIF. Akt1 and Akt3 are essential for the proliferation of reprogrammed cells, and Esrrb supports cell proliferation and complete reprogramming during Akt signaling.

Transcriptional profiles of bovine in vivo pre-implantation development.

BACKGROUND: During mammalian pre-implantation embryonic development dramatic and orchestrated changes occur in gene transcription. The identification of the complete changes has not been possible until the development of the Next Generation Sequencing Technology. RESULTS: Here we report comprehensive transcriptome dynamics of single matured bovine oocytes and pre-implantation embryos developed in vivo. Surprisingly, more than half of the estimated 22,000 bovine genes, 11,488 to 12,729 involved in more than 100 pathways, is expressed in oocytes and early embryos. Despite the similarity in the total numbers of genes expressed across stages, the nature of the expressed genes is dramatically different. A total of 2,845 genes were differentially expressed among different stages, of which the largest change was observed between the 4- and 8-cell stages, demonstrating that the bovine embryonic genome is activated at this transition. Additionally, 774 genes were identified as only expressed/highly enriched in particular stages of development, suggesting their stage-specific roles in embryogenesis. Using weighted gene co-expression network analysis, we found 12 stage-specific modules of co-expressed genes that can be used to represent the corresponding stage of development. Furthermore, we identified conserved key members (or hub genes) of the bovine expressed gene networks. Their vast association with other embryonic genes suggests that they may have important regulatory roles in embryo development; yet, the majority of the hub genes are relatively unknown/under-studied in embryos. We also conducted the first comparison of embryonic expression profiles across three mammalian species, human, mouse and bovine, for which RNA-seq data are available. We found that the three species share more maternally deposited genes than embryonic genome activated genes. More importantly, there are more similarities in embryonic transcriptomes between bovine and humans than between humans and mice, demonstrating that bovine embryos are better models for human embryonic development. CONCLUSIONS: This study provides a comprehensive examination of gene activities in bovine embryos and identified little-known potential master regulators of pre-implantation development.

Jak/Stat3 signaling promotes somatic cell reprogramming by epigenetic regulation.

Although leukemia inhibitory factor (LIF) maintains the ground state pluripotency of mouse embryonic stem cells and induced pluripotent stem cells (iPSCs) by activating the Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) pathway, the mechanism remained unclear. Stat3 has only been shown to promote complete reprogramming of epiblast and neural stem cells and partially reprogrammed cells (pre-iPSCs). We investigated if and how Jak/Stat3 activation promotes reprogramming of terminally differentiated mouse embryonic fibroblasts (MEFs). We demonstrated that activated Stat3 not only promotes but also is essential for the pluripotency establishment of MEFs during reprogramming. We further demonstrated that during this process, inhibiting Jak/Stat3 activity blocks demethylation of Oct4 and Nanog regulatory elements in induced cells, which are marked by suppressed endogenous pluripotent gene expression. These are correlated with the significant upregulation of DNA methyltransferase (Dnmt) 1 and histone deacetylases (HDACs) expression as well as the increased expression of lysine-specific histone demethylase 2 and methyl CpG binding protein 2. Inhibiting Jak/Stat3 also blocks the expression of Dnmt3L, which is correlated with the failure of retroviral transgene silencing. Furthermore, Dnmt or HDAC inhibitor but not overexpression of Nanog significantly rescues the reprogramming arrested by Jak/Stat3 inhibition or LIF deprivation. Finally, we demonstrated that LIF/Stat3 signal also represents the prerequisite for complete reprogramming of pre-iPSCs. We conclude that Jak/Stat3 activity plays a fundamental role to promote pluripotency establishment at the epigenetic level, by facilitating DNA demethylation/de novo methylation, and open-chromatin formation during late-stage reprogramming.

Intergenic transcription in in vivo developed bovine oocytes and pre-implantation embryos.

Background: Intergenic transcription, either failure to terminate at the transcription end site (TES), or transcription initiation at other intergenic regions, is present in cultured cells and enhanced in the presence of stressors such as viral infection. Transcription termination failure has not been characterized in natural biological samples such as pre-implantation embryos which express more than 10,000 genes and undergo drastic changes in DNA methylation. Results: Using Automatic Readthrough Transcription Detection (ARTDeco) and data of in vivo developed bovine oocytes and embryos, we found abundant intergenic transcripts that we termed as read-outs (transcribed from 5 to 15 kb after TES) and read-ins (transcribed 1 kb up-stream of reference genes, extending up to 15 kb up-stream). Read-throughs (continued transcription from TES of expressed reference genes, 4-15 kb in length), however, were much fewer. For example, the numbers of read-outs and read-ins ranged from 3,084 to 6,565 or 33.36-66.67% of expressed reference genes at different stages of embryo development. The less copious read-throughs were at an average of 10% and significantly correlated with reference gene expression (P < 0.05). Interestingly, intergenic transcription did not seem to be random because many intergenic transcripts (1,504 read-outs, 1,045 read-ins, and 1,021 read-throughs) were associated with common reference genes across all stages of pre-implantation development. Their expression also seemed to be regulated by developmental stages because many were differentially expressed (log2 fold change ≥ 2, P < 0.05). Additionally, while gradual but un-patterned decreases in DNA methylation densities 10 kb both up- and down-stream of the intergenic transcribed regions were observed, the correlation between intergenic transcription and DNA methylation was insignificant. Finally, transcription factor binding motifs and polyadenylation signals were found in 27.2% and 12.15% of intergenic transcripts, respectively, suggesting considerable novel transcription initiation and RNA processing. Conclusion: In summary, in vivo developed oocytes and pre-implantation embryos express large numbers of intergenic transcripts, which are not related to the overall DNA methylation profiles either up- or down-stream.

Effects of intramuscularly injected plant-derived antimicrobials in the mouse model.

With increasing antibiotic resistance, the use of plant derived antimicrobials (PDAs) has gained momentum. Here, we investigated the toxicity of trans-cinnamaldehyde, eugenol, and carvacrol after intramuscular injection in mice. Two doses of each PDA-300 and 500 mg/kg body weight-and vehicle controls were injected into the muscle of the right hind limb of CD-1 adult mice (n = 8/treatment). Ten physical/behavioral parameters were monitored hourly for 2 h and twice daily for 4 days post-injection together with postmortem examination of leg muscles and organs. Within the first 2 days of carvacrol treatment, one male died in each dose level and a third male receiving 500 mg/kg was removed from the study. No mortality was seen with any other treatment. Among all 81 parameters examined, significant higher relative liver weights (300 and 500 mg/kg eugenol groups; P < 0.05) and relative kidney weights (300 mg/kg carvacrol group; P < 0.001) were observed. Taken together, little to mild toxicity was seen for trans-cinnamaldehyde and eugenol, respectively, while carvacrol exerted more toxicity in males. This study lays the foundation for future extensive work with large sample size, varied treatment durations, and additional treatment levels.

Transcriptomic Responses of Mycoplasma bovis Upon Treatments of trans-Cinnamaldehyde, Carvacrol, and Eugenol.

Mycoplasma bovis (M. bovis) is an insidious, wall-less primary bacterial pathogen that causes bovine pneumonia, mid-ear infection, mastitis, and arthritis. The economic losses caused by M. bovis due to culling, diminished milk production, and feed conversion are underestimated because of poor diagnosis/recognition. Treatment with common antibiotics targeting the cell wall is ineffective. Plant-derived antimicrobials (PDAs) such as food-grade trans-cinnamaldehyde (TC), eugenol (EU), and carvacrol (CAR) are inexpensive and generally regarded as safe for humans and animals yet possess strong anti-bacterial properties. In preliminary studies, we found that all three PDAs inhibited the growth of M. bovis in vitro. Through RNA sequencing, we report here that CAR affected the expression of 153 genes which included the downregulation of energy generation-related proteins, pentose phosphate pathway, and upregulation of ribosomes and translation-related proteins. Few differentially expressed genes were found when M. bovis was treated with TC, EU, or when the three PDAs were double or triple combined. Our results suggest that, as opposed to the effect of CAR, the growth-inhibitory effects of TC and EU at levels tested may be exerted through mechanisms other than gene expression regulations.

Cytotoxic effects on cancerous and non-cancerous cells of trans-cinnamaldehyde, carvacrol, and eugenol.

Essential oils and their active components, referred here as plant derived antimicrobials (PDAs), have been used for their antimicrobial, anti-inflammatory and antioxidant properties. Many reports also document PDAs' cytotoxic effects on cancerous cells, raising the hope that they could be used for cancer treatments. Due to the lack of specificity, we hypothesize that PDAs are cytotoxic to both cancerous and non-cancerous cells. Trans-cinnamaldehyde (TCA), carvacrol, and eugenol were assessed for their cytotoxicity on cancerous HeLa cells and normal skin fibroblasts (CCD-1123Sk, CCD) by MTT and LDH assays, flow cytometry, and reverse transcription quantitative PCR (RT-qPCR). After 24 h of treatment, carvacrol and TCA significantly decreased cell viability (by more than 50%) at 100 µg/ml, whereas eugenol was ineffective up to 400 µg/ml. Cell detachment and significantly increased apoptosis were observed with 100 µg/ml of TCA on both cell types. RT-qPCR for apoptotic genes (BCL2, CASP3 and CASP8) and necrosis genes (MLKL, RIPK1 and RIPK3) did not show significant differences between control and treated cells of both types, with the exception of eugenol-treated HeLa cells in which expression of BCL2, MLKL and RIPK1 was significantly higher than controls. Taken together, we conclude that the three PDAs studied here exhibited similar cytotoxic effects on both cancerous and non-cancerous cells.

Methylome Dynamics of Bovine Gametes and in vivo Early Embryos.

DNA methylation undergoes drastic fluctuation during early mammalian embryogenesis. The dynamics of global DNA methylation in bovine embryos, however, have mostly been studied by immunostaining. We adopted the whole genome bisulfite sequencing (WGBS) method to characterize stage-specific genome-wide DNA methylation in bovine sperm, immature oocytes, oocytes matured in vivo and in vitro, as well as in vivo developed single embryos at the 2-, 4-, 8-, and 16-cell stages. We found that the major wave of genome-wide DNA demethylation was complete by the 8-cell stage when de novo methylation became prominent. Sperm and oocytes were differentially methylated in numerous regions (DMRs), which were primarily intergenic, suggesting that these non-coding regions may play important roles in gamete specification. DMRs were also identified between in vivo and in vitro matured oocytes, suggesting environmental effects on epigenetic modifications. In addition, virtually no (less than 1.5%) DNA methylation was found in mitochondrial DNA. Finally, by using RNA-seq data generated from embryos at the same developmental stages, we revealed a weak inverse correlation between gene expression and promoter methylation. This comprehensive analysis provides insight into the critical features of the bovine embryo methylome, and serves as an important reference for embryos produced in vitro, such as by in vitro fertilization and cloning. Lastly, these data can also provide a model for the epigenetic dynamics in human early embryos.

Dosage Compensation of the X Chromosomes in Bovine Germline, Early Embryos, and Somatic Tissues.

Dosage compensation of the mammalian X chromosome (X) was proposed by Susumu Ohno as a mechanism wherein the inactivation of one X in females would lead to doubling the expression of the other. This would resolve the dosage imbalance between eutherian females (XX) versus male (XY) and between a single active X versus autosome pairs (A). Expression ratio of X- and A-linked genes has been relatively well studied in humans and mice, despite controversial results over the existence of upregulation of X-linked genes. Here we report the first comprehensive test of Ohno's hypothesis in bovine preattachment embryos, germline, and somatic tissues. Overall an incomplete dosage compensation (0.5 < X:A < 1) of expressed genes and an excess X dosage compensation (X:A > 1) of ubiquitously expressed "dosage-sensitive" genes were seen. No significant differences in X:A ratios were observed between bovine female and male somatic tissues, further supporting Ohno's hypothesis. Interestingly, preimplantation embryos manifested a unique pattern of X dosage compensation dynamics. Specifically, X dosage decreased after fertilization, indicating that the sperm brings in an inactive X to the matured oocyte. Subsequently, the activation of the bovine embryonic genome enhanced expression of X-linked genes and increased the X dosage. As a result, an excess compensation was exhibited from the 8-cell stage to the compact morula stage. The X dosage peaked at the 16-cell stage and stabilized after the blastocyst stage. Together, our findings confirm Ohno's hypothesis of X dosage compensation in the bovine and extend it by showing incomplete and over-compensation for expressed and "dosage-sensitive" genes, respectively.

Dosage Compensation and Gene Expression of the X Chromosome in Sheep.

Ohno's hypothesis predicts that the expression of the single X chromosome in males needs compensatory upregulation to balance its dosage with that of the diploid autosomes. Additionally, X chromosome inactivation ensures that quadruple expression of the two X chromosomes is avoided in females. These mechanisms have been actively studied in mice and humans but lag behind in domestic species. Using RNA sequencing data, we analyzed the X chromosome upregulation in sheep fetal tissues from day 135 of gestation under control, over or restricted maternal diets (100%, 140% and 60% of National Research Council Total Digestible Nutrients), and in conceptuses, juvenile, and adult somatic tissues. By computing the mean expression ratio of all X-linked genes to all autosomal genes (X:A), we found that all samples displayed some levels of X chromosome upregulation. The degrees of X upregulation were not significant (P-value = 0.74) between ovine females and males in the same somatic tissues. Brain, however, displayed complete X upregulation. Interestingly, the male and female reproduction-related tissues exhibited divergent X dosage upregulation. Moreover, expression upregulation of the X chromosome in fetal tissues was not affected by maternal diets. Maternal nutrition, however, did change expression levels of several X-linked genes, such as sex determination genes SOX3 and NR0B1 In summary, our results showed that X chromosome upregulation occurred in nearly all sheep somatic tissues analyzed, thus support Ohno's hypothesis in a new species. However, the levels of upregulation differed by different subgroups of genes such as those that are house-keeping and "dosage-sensitive".

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming.

The regulatory process of naïve-state induced pluripotent stem cell (iPSC) generation is not well understood. Leukemia inhibitory factor (LIF)-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb) serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs). However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs), and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation.