Changes in the N-glycosylation of porcine immune globulin G during postnatal development.

N-glycosylation influences the effectiveness of immune globulin G (IgG) and thus the immunological downstream responses of immune cells. This impact arises from the presence of N-glycans within the Fc region, which not only alters the conformation of IgG but also influences its steric hindrance. Consequently, these modifications affect the interaction between IgG and its binding partners within the immune system. Moreover, this posttranslational modification vary according to the physiological condition of each individual. In this study, we examined the N-glycosylation of IgG in pigs from birth to five months of age. Our analysis identified a total of 48 distinct N-glycan structures. Remarkably, we observed defined changes in the composition of these N-glycans during postnatal development. The presence of agalactosylated and sialylated structures increases in relation to the number of N-glycans terminated by galactose residues during the first months of life. This shift may indicate a transition from passively transferred antibodies from the colostrum of the sow to the active production of endogenous IgG by the pig's own immune system.

Bile acid profiles and mRNA abundance of bile acid-related genes in adipose tissue of dairy cows with high versus normal body condition.

Besides their lipid-digestive role, bile acids (BA) influence overall energy homeostasis, such as glucose and lipid metabolism. We hypothesized that BA along with their receptors, regulatory enzymes, and transporters are present in subcutaneous adipose tissue (scAT). In addition, we hypothesized that their mRNA abundance varies with the body condition of dairy cows around calving. Therefore, we analyzed BA in serum and scAT as well as the mRNA abundance of BA -related enzymes, transporters, and receptors in scAT during the transition period in cows with different body conditions around calving. In a previously established animal model, 38 German Holstein cows were divided into either a high (HBCS; n = 19) or normal BCS (NBCS; n = 19) group based on their body condition score (BCS) and back fat thickness (BFT). Cows were fed different diets to achieve the targeted differences in BCS and BFT (NBCS: BCS <3.5, BFT <1.2 cm; HBCS: BCS >3.75, BFT >1.4 cm) until dry-off at 7 wk ante partum. During the dry period and subsequent lactation, both groups were fed the same diets regarding their demands. Using a targeted metabolomics approach via LC-ESI-MS /MS, BA were analyzed in serum and scAT at wk -7, 1, 3, and 12 relative to parturition. In serum, 15 BA (cholic acid (CA), chenodeoxycholic acid (CDCA), glycocholic acid (GCA), taurocholic acid (TCA), glycochenodeoxycholic acid (GCDCA), taurochenodeoxycholic acid (TCDCA), deoxycholic acid (DCA), lithocholic acid (LCA), glycodeoxycholic acid (GDCA), glycolithocholic acid (GLCA), taurodeoxycholic acid (TDCA), taurolithocholic acid (TLCA), ß-muricholic acid (MCA(b)), tauromuricholic acid (sum of α and ß) (TMCA (a+b)), glycoursodeoxycholic acid (GUDCA)) were observed, whereas in scAT 7 BA (CA, GCA, TCA, GCDCA, TCDCA, GDCA, TDCA) were detected. In serum and scAT samples, the primary BA CA and its conjugate GCA were predominantly detected. Increasing serum concentrations of CA, CDCA, TCA, GCA, GCDCA, DCA, and MCA(b) with the onset of lactation might be related to the increasing DMI after parturition. Furthermore, serum concentrations of CA, CDCA, GCA, DCA, GCDCA, TCA, LCA, and GDCA were lower in HBCS cows compared with NBCS cows, concomitant with increased lipolysis in HBCS cows. The correlation between CA in serum and scAT may point to the transport of CA across cell membranes. Overall, the findings of the present study suggest a potential role of BA in lipid metabolism depending on the body condition of periparturient dairy cows.

RNA-Seq-based discovery of genetic variants and allele-specific expression of two layer lines and broiler chicken.

Recent advances in the selective breeding of broilers and layers have made poultry production one of the fastest-growing industries. In this study, a transcriptome variant calling approach from RNA-seq data was used to determine population diversity between broilers and layers. In total, 200 individuals were analyzed from three different chicken populations (Lohmann Brown (LB), n = 90), Lohmann Selected Leghorn (LSL, n = 89), and Broiler (BR, n = 21). The raw RNA-sequencing reads were pre-processed, quality control checked, mapped to the reference genome, and made compatible with Genome Analysis ToolKit for variant detection. Subsequently, pairwise fixation index (F ST) analysis was performed between broilers and layers. Numerous candidate genes were identified, that were associated with growth, development, metabolism, immunity, and other economically significant traits. Finally, allele-specific expression (ASE) analysis was performed in the gut mucosa of LB and LSL strains at 10, 16, 24, 30, and 60 weeks of age. At different ages, the two-layer strains showed significantly different allele-specific expressions in the gut mucosa, and changes in allelic imbalance were observed across the entire lifespan. Most ASE genes are involved in energy metabolism, including sirtuin signaling pathways, oxidative phosphorylation, and mitochondrial dysfunction. A high number of ASE genes were found during the peak of laying, which were particularly enriched in cholesterol biosynthesis. These findings indicate that genetic architecture as well as biological processes driving particular demands relate to metabolic and nutritional requirements during the laying period shape allelic heterogeneity. These processes are considerably affected by breeding and management, whereby elucidating allele-specific gene regulation is an essential step towards deciphering the genotype to phenotype map or functional diversity between the chicken populations. Additionally, we observed that several genes showing significant allelic imbalance also colocalized with the top 1% of genes identified by the FST approach, suggesting a fixation of genes in cis-regulatory elements.

Exploration of genotype-by-environment interactions affecting gene expression responses in porcine immune cells.

As one of the keys to healthy performance, robustness of farm animals is gaining importance, and with this comes increasing interest in genetic dissection of genotype-by-environment interactions (G×E). Changes in gene expression are among the most sensitive responses conveying adaptation to environmental stimuli. Environmentally responsive regulatory variation thus likely plays a central role in G×E. In the present study, we set out to detect action of environmentally responsive cis-regulatory variation by the analysis of condition-dependent allele specific expression (cd-ASE) in porcine immune cells. For this, we harnessed mRNA-sequencing data of peripheral blood mononuclear cells (PBMCs) stimulated in vitro with lipopolysaccharide, dexamethasone, or their combination. These treatments mimic common challenges such as bacterial infection or stress, and induce vast transcriptome changes. About two thirds of the examined loci showed significant ASE in at least one treatment, and out of those about ten percent exhibited cd-ASE. Most of the ASE variants were not yet reported in the PigGTEx Atlas. Genes showing cd-ASE were enriched in cytokine signaling in immune system and include several key candidates for animal health. In contrast, genes showing no ASE featured cell-cycle related functions. We confirmed LPS-dependent ASE for one of the top candidates, SOD2, which ranks among the major response genes in LPS-stimulated monocytes. The results of the present study demonstrate the potential of in vitro cell models coupled with cd-ASE analysis for the investigation of G×E in farm animals. The identified loci may benefit efforts to unravel the genetic basis of robustness and improvement of health and welfare in pigs.

Glucocorticoid receptor hypersensitivity enhances inflammatory signaling and inhibits cell cycle progression in porcine PBMCs.

The consequences of glucocorticoid receptor (GR) hypersensitivity during infection have so far received little attention. We previously discovered that a natural gain-of-function Ala610Val substitution in the porcine GR aggravates response of pigs to lipopolysaccharide (LPS)-induced endotoxemia, which can be alleviated by dexamethasone (DEX) pretreatment. In this work, we investigated the relevant molecular basis of these phenotypes by transcriptomic profiling of porcine peripheral blood mononuclear cells (PBMCs) carrying different GR genotypes, in unstimulated conditions or in response to DEX and/or LPS in vitro. The Val allele differentially regulated abunda+nt genes in an additive-genetic manner. A subset of more than 200 genes was consistently affected by the substitution across treatments. This was associated with upregulation of genes related i.a. to endo-lysosomal system, lipid and protein catabolism, and immune terms including platelet activation, and antigen presentation, while downregulated genes were mainly involved in cell cycle regulation. Most importantly, the set of genes constitutively upregulated by Val includes members of the TLR4/LPS signaling pathway, such as LY96. Consequently, when exposing PBMCs to LPS treatment, the Val variant upregulated a panel of additional genes related to TLR4 and several other pattern recognition receptors, as well as cell death and lymphocyte signaling, ultimately amplifying the inflammatory responses. In contrast, when stimulated by DEX treatment, the Val allele orchestrated several genes involved in anti-inflammatory responses during infection. This study provides novel insights into the impact of GR hypersensitivity on the fate and function of immune cells, which may be useful for endotoxemia therapy.

Regulation of Porcine Oviduct Epithelium Functions via Progesterone and Estradiol Is Influenced by Cortisol.

Preimplantation maternal stress, characterized by elevated glucocorticoids (GCs), has been linked to reproductive failures caused by impaired oviduct functionality, which is known to be predominantly regulated by the sex steroids, progesterone (P4) and (17)estradiol (E2). Although steroid receptors share analogous structures and binding preferences, the interaction between GCs and E2/P4 in the oviduct has attracted little attention. Using an air-liquid interface culture model, porcine oviduct epithelial cells were stimulated with single (cortisol, E2, P4) or hormone mixtures (cortisol/E2, cortisol/P4) for 12 hours and 72 hours. Cultures were subsequently assessed for epithelial morphometry, bioelectrical properties, and gene expression responses (steroid hormone signaling, oviductal function, immune response, and apoptosis). Results confirmed the suppressive role of P4 in regulating oviduct epithelium characteristics, which was partially opposed by E2. Besides increasing the ratio of ciliated cells, cortisol antagonized the effect of P4 on epithelial polarity and modified sex steroid-induced changes in transepithelial electrical properties. Both sex steroids affected the glucocorticoid receptor expression, while cortisol downregulated the expression of progesterone receptor. The overall gene expression pattern suggests that sex steroid dominates the cotreatment, but cortisol contributes by altering the gene responses to sex steroids. We conclude that besides its individual action, maternal cortisol interplays with sex steroids at phenotypic and molecular levels in the oviduct epithelium, thereby influencing the microenvironment of gametes and early embryos.

DNA methylation landscapes from pig's limbic structures underline regulatory mechanisms relevant for brain plasticity.

Epigenetic dynamics are essential for reconciling stress-induced responses in neuro-endocrine routes between the limbic brain and adrenal gland. CpG methylation associates with the initiation and end of regulatory mechanisms underlying responses critical for survival, and learning. Using Reduced Representation Bisulfite Sequencing, we identified methylation changes of functional relevance for mediating tissue-specific responses in the hippocampus, amygdala, hypothalamus, and adrenal gland in pigs. We identified 4186 differentially methylated CpGs across all tissues, remarkably, enriched for promoters of transcription factors (TFs) of the homeo domain and zinc finger classes. We also detected 5190 differentially methylated regions (DMRs, 748 Mb), with about half unique to a single pairwise. Two structures, the hypothalamus and the hippocampus, displayed 860 unique brain-DMRs, with many linked to regulation of chromatin, nervous development, neurogenesis, and cell-to-cell communication. TF binding motifs for TFAP2A and TFAP2C are enriched amount DMRs on promoters of other TFs, suggesting their role as master regulators, especially for pathways essential in long-term brain plasticity, memory, and stress responses. Our results reveal sets of TF that, together with CpG methylation, may serve as regulatory switches to modulate limbic brain plasticity and brain-specific molecular genetics in pigs.

Prenatal transcript levels and metabolomics analyses reveal metabolic changes associated with intrauterine growth restriction and sex.

The metabolic changes associated with intrauterine growth restriction (IUGR) particularly affect the liver, which is a central metabolic organ and contributes significantly to the provision of energy and specific nutrients and metabolites. Therefore, our aim was to decipher and elucidate the molecular pathways of developmental processes mediated by miRNAs and mRNAs, as well as the metabolome in fetal liver tissue in IUGR compared to appropriate for gestational age groups (AGA). Discordant siblings representing the extremes in fetal weight at day 63 post conception (dpc) were selected from F2 fetuses of a cross of German Landrace and Pietrain. Most of the changes in the liver of IUGR at midgestation involved various lipid metabolic pathways, both on transcript and metabolite levels, especially in the category of sphingolipids and phospholipids. Differentially expressed miRNAs, such as miR-34a, and their differentially expressed mRNA targets were identified. Sex-specific phenomena were observed at both the transcript and metabolite levels, particularly in male. This suggests that sex-specific adaptations in the metabolic system occur in the liver during midgestation (63 dpc). Our multi-omics network analysis reveals interactions and changes in the metabolic system associated with IUGR and identified an important biosignature that differs between IUGR and AGA piglets.

Tissue-Wide Expression of Genes Related to Vitamin D Metabolism and FGF23 Signaling following Variable Phosphorus Intake in Pigs.

Calcium (Ca) and phosphorus (P) homeostasis is maintained by several regulators, including vitamin D and fibroblast growth factor 23 (FGF23), and their tissue-specific activation and signaling cascades. In this study, the tissue-wide expression of key genes linked to vitamin D metabolism (CYP2R1, CYP27A1, CYP27B1, CYP24A1, GC, VDR) and FGF23 signaling (FGF23, FGFR1-4, KL) were investigated in pigs fed conventional (trial 1) and divergent P diets (trial 2). The tissue set comprised kidney, liver, bone, lung, aorta, and gastrointestinal tract sections. Expression patterns revealed that non-renal tissues and cells (NRTC) express genes to form active vitamin D [1,25(OH)2D3] according to site-specific requirements. A low P diet resulted in higher serum calcitriol and increased CYP24A1 expression in the small intestine, indicating local suppression of vitamin D signaling. A high P diet prompted increased mRNA abundances of CYP27B1 for local vitamin D synthesis, specifically in bone. For FGF23 signaling, analyses revealed ubiquitous expression of FGFR1-4, whereas KL was expressed in a tissue-specific manner. Dietary P supply did not affect skeletal FGF23; however, FGFR4 and KL showed increased expression in bone at high P supply, suggesting regulation to balance mineralization. Specific NRTC responses influence vitamin D metabolism and P homeostasis, which should be considered for a thrifty but healthy P supply.

Genetic regulation and variation of expression of miRNA and mRNA transcripts in fetal muscle tissue in the context of sex, dam and variable fetal weight.

BACKGROUND: Impaired skeletal muscle growth in utero can result in reduced birth weight and pathogenesis of intrauterine growth restriction. Fetal and placental growth is influenced by many factors including genetic, epigenetic and environmental factors. In fact, the sex and genotype of the fetus itself, as well as the mother providing it with a suitable environment, influence the growth of the fetus. Hence, our goal was to decipher and elucidate the molecular pathways of developmental processes mediated by miRNAs and mRNAs in fetal muscle tissue in the context of sex, dam, and fetal weight. Therefore, we analyse the variation of miRNA and mRNA expression in relation to these factors. In addition, the coincidence of genetic regulation of these mRNAs and miRNAs, as revealed by expression quantitative trait loci (eQTL) analyses, with sex-, mother- and weight-associated expression was investigated. METHODS: A three-generation pig F2 population (n = 118) based on reciprocal crossing of German Landrace (DL) and Pietrain (Pi) was used. Genotype information and transcriptomic data (mRNA and miRNA) from longissimus dorsi muscle (LDM) of pig fetuses sampled at 63 days post-conception (dpc) were used for eQTL analyses. RESULTS: The transcript abundances of 13, 853, and 275 probe-sets were influenced by sex, dam and fetal weight at 63 dpc, respectively (FDR < 5%). Most of significant transcripts affected by sex were located on the sex chromosomes including KDM6A and ANOS1 or autosomes including ANKS1B, LOC100155138 and miR-153. The fetal muscle transcripts associated with fetal weight indicated clearer metabolic directions than maternally influenced fetal muscle transcripts. Moreover, coincidence of genetic regulation (eQTL) and variation in transcript abundance due to sex, dam and fetal weight were identified. CONCLUSIONS: Integrating information on eQTL, sex-, dam- and weight-associated differential expression and QTL for fetal weight allowed us to identify molecular pathways and shed light on the basic biological processes associated with differential muscle development in males and females, with implications for adaptive fetal programming.

Brain Transcriptome Responses to Dexamethasone Depending on Dose and Sex Reveal Factors Contributing to Sex-Specific Vulnerability to Stress-Induced Disorders.

BACKGROUND: Glucocorticoid (GC) receptor (GR) signaling in the hypothalamus (Hyp) and in the superordinate limbic structures, such as the hippocampus (Hip), conveys feedback regulation of the neuroendocrine stress response and acts upon other neurobiological functions that ultimately influence mental health. These responses are strongly influenced by sex, but the molecular causes are still largely unexplored. METHODS: To investigate GR targets and their GC sensitivity in the Hyp and Hip, we treated juvenile male and female piglets with 10 (D10) or 60 (D60) µg/kg dexamethasone (DEX), a selective GR agonist, and analyzed transcriptome responses compared to a saline control group using RNA sequencing. RESULTS: Both doses influenced similar biological functions, including cellular response to lipid and immune cell-related functions, but the transcriptional response to D10 was considerably weaker, particularly in the Hip. Weighted Gene Co-expression Network Analysis revealed a network of genes coordinately regulated by DEX in both structures, among which the alpha-arrestin ARRDC2 takes a central position. Distinct functional groups of genes were differentially regulated by DEX between sexes depending on the dose; at D10, these included particularly mitochondrial genes, whereas at D60 interferon signaling and lipid homeostasis genes were enriched. The general and sex-specific transcriptional responses to DEX highlight microglia as the prominent target. Several key marker genes of disease-associated microglia were regulated by DEX depending on sex, such as TREM2 and LPL. CONCLUSION: The discovered expression signatures suggest that DEX induced a dysfunctional state of microglia in males, while in females microglia were primed, which could entail predisposition for different mental disorders.

Jejunal transcriptomic profiling of two layer strains throughout the entire production period.

The jejunum plays crucial roles for the digestion and absorption of nutrients and minerals and for barrier functions that are essential for a healthy, productive life cycle of farm animals, including laying hens. Accordingly, knowledge of the molecular pathways that emerge in the intestine during development, and particularly at the beginning of laying activity, will help to derive strategies for improving nutrient efficiency in laying hens. In this study, jejunal samples were obtained from two high-yielding layer strains at five developmental stages (weeks 10, 16, 24, 30 and 60 of life) for RNA-sequencing, alongside the profiling of blood plasma parameters to approximate the dynamics of mineral homeostasis. The results reflected a marked distinction between the pre-laying and laying phase as inferred from levels of parathyroid hormone, triiodothyronine, estradiol, vitamin D, and calcium. Moreover, the expression patterns of the intestinal mucosa responded directly to the changing metabolic and nutritional profiles at the beginning of the laying phase in maturing high-yielding strains of laying hens. These comprise signaling events namely RANK/RANKL signaling and cellular senescence. Taken together, the timing of sexual maturity of laying hens demands closer examination to unravel metabolic requirements and associated endogenous mechanisms.

Erratum: Ali et al. In Utero Fetal Weight in Pigs Is Regulated by microRNAs and Their Target Genes. Genes 2021, 12, 1264.

The authors wish to make the following correction to their paper published in Genes [...].

Multi-Transcript Level Profiling Revealed Distinct mRNA, miRNA, and tRNA-Derived Fragment Bio-Signatures for Coping Behavior Linked Haplotypes in HPA Axis and Limbic System.

The molecular basis of porcine coping behavior (CB) relies on a sophisticated interplay of genetic and epigenetic features. Deep sequencing technologies allowed the identification of a plethora of new regulatory small non-coding RNA (sncRNA). We characterized mRNA and sncRNA profiles of central parts of the physiological stress response system including amygdala, hippocampus, hypothalamus and adrenal gland using systems biology for integration. Therefore, ten each of high- (HR) and low- (LR) reactive pigs (n = 20) carrying a CB associated haplotype in a prominent QTL-region on SSC12 were selected for mRNA and sncRNA expression profiling. The molecular markers related to the LR group included ATP1B2, MPDU1, miR-19b-5p, let-7g-5p, and 5'-tiRNA Leu in the adrenal gland, miR-194a-5p, miR-125a-5p, miR-7-1-5p, and miR-107-5p in the hippocampus and CBL and PVRL1 in the hypothalamus. Interestingly, amygdalae of the LR group showed 5'-tiRNA and 5'-tRF (5'-tRF Lys , 5'-tiRNA Lys , 5'-tiRNA Cys , and 5'-tiRNA Gln ) enrichment. Contrarily, molecular markers associated with the HR group encompassed miR-26b-5p, tRNA Arg , tRNA GlyiF in the adrenal gland, IGF1 and APOD in the amygdala and PBX1, TOB1, and C18orf1 in the hippocampus and miR-24 in the hypothalamus. In addition, hypothalami of the HR group were characterized by 3'-tiRNA enrichment (3'-tiRNAGln, 3'-tiRNA Asn , 3'-tiRNA Val , 3'-tRF Pro , 3'-tiRNA Cys , and 3'-tiRNA Ala ) and 3'-tRFs enrichment (3'-tRF Asn , 3'-tRF Glu , and 3'-tRF Val ). These evidence suggest that tRNA-derived fragments and their cleavage activity are a specific marker for coping behavior. Data integration revealed new bio-signatures of important molecular interactions on a multi-transcript level in HPA axis and limbic system of pigs carrying a CB-associated haplotype.

A 192 bp ERV fragment insertion in the first intron of porcine TLR6 may act as an enhancer associated with the increased expressions of TLR6 and TLR1.

BACKGROUND: Toll-like receptors (TLRs) play important roles in building innate immune and inducing adaptive immune responses. Associations of the TLR genes polymorphisms with disease susceptibility, which are the basis of molecular breeding for disease resistant animals, have been reported extensively. Retrotransposon insertion polymorphisms (RIPs), as a new type of molecular markers developed recently, have great potential in population genetics and quantitative trait locus mapping. In this study, bioinformatic prediction combined with PCR-based amplification was employed to screen for RIPs in porcine TLR genes. Their population distribution was examined, and for one RIP the impact on gene activity and phenotype was further evaluated. RESULTS: Five RIPs, located at the 3' flank of TLR3, 5' flank of TLR5, intron 1 of TLR6, intron 1 of TLR7, and 3' flank of TLR8 respectively, were identified. These RIPs were detected in different breeds with an uneven distribution among them. By using the dual luciferase activity assay a 192 bp endogenous retrovirus (ERV) in the intron 1 of TLR6 was shown to act as an enhancer increasing the activities of TLR6 putative promoter and two mini-promoters. Furthermore, real-time quantitative polymerase chain reaction (qPCR) analysis revealed significant association (p < 0.05) of the ERV insertion with increased mRNA expression of TLR6, the neighboring gene TLR1, and genes downstream in the TLR signaling pathway such as MyD88 (Myeloid differentiation factor 88), Rac1 (Rac family small GTPase 1), TIRAP (TIR domain containing adaptor protein), Tollip (Toll interacting protein) as well as the inflammatory factors IL6 (Interleukin 6), IL8 (Interleukin 8), and TNFα (Tumor necrosis factor alpha) in tissues of 30 day-old piglet. In addition, serum IL6 and TNFα concentrations were also significantly upregulated by the ERV insertion (p < 0.05). CONCLUSIONS: A total of five RIPs were identified in five different TLR loci. The 192 bp ERV insertion in the first intron of TLR6 was associated with higher expression of TLR6, TLR1, and several genes downstream in the signaling cascade. Thus, the ERV insertion may act as an enhancer affecting regulation of the TLR signaling pathways, and can be potentially applied in breeding of disease resistant animals.

Genome-Wide Analysis for Early Growth-Related Traits of the Locally Adapted Egyptian Barki Sheep.

Sheep play a critical role in the agricultural and livestock sector in Egypt. For sheep meat production, growth traits such as birth and weaning weights are very important and determine the supply and income of local farmers. The Barki sheep originates from the northeastern coastal zone of Africa, and due to its good adaptation to the harsh environmental conditions, it contributes significantly to the meat production in these semi-arid regions. This study aimed to use a genome-wide SNP panel to identify genomic regions that are diversified between groups of individuals of Egyptian Barki sheep with high and low growth performance traits. In this context, from a phenotyped population of 140 lambs of Barki sheep, 69 lambs were considered for a genome-wide scan with the Illumina OvineSNP50 V2 BeadChip. The selected lambs were grouped into divergent subsets with significantly different performance for birth weight and weaning weight. After quality control, 63 animals and 40,383 SNPs were used for analysis. The fixation index (FST) for each SNP was calculated between the groups. The results verified genomic regions harboring some previously proposed candidate genes for traits related to body growth, i.e., EYA2, GDF2, GDF10, MEF2B, SLC16A7, TBX15, TFAP2B, and TNNC2. Moreover, novel candidate genes were proposed with known functional implications on growth processes such as CPXM2 and LRIG3. Subsequent association analysis showed significant effects of the considered SNPs on birth and weaning weights. Results highlight the genetic diversity associated with performance traits and thus the potential to improve growth traits in the Barki sheep breed.

In Utero Fetal Weight in Pigs Is Regulated by microRNAs and Their Target Genes.

Impaired skeletal muscle growth in utero can result in reduced birth weight and poor carcass quality in pigs. Recently, we showed the role of microRNAs (miRNAs) and their target genes in prenatal skeletal muscle development and pathogenesis of intrauterine growth restriction (IUGR). In this study, we performed an integrative miRNA-mRNA transcriptomic analysis in longissimus dorsi muscle (LDM) of pig fetuses at 63 days post conception (dpc) to identify miRNAs and genes correlated to fetal weight. We found 13 miRNAs in LDM significantly correlated to fetal weight, including miR-140, miR-186, miR-101, miR-15, miR-24, miR-29, miR-449, miR-27, miR-142, miR-99, miR-181, miR-199, and miR-210. The expression of these miRNAs decreased with an increase in fetal weight. We also identified 1315 genes significantly correlated to fetal weight at 63 dpc, of which 135 genes were negatively correlated as well as identified as potential targets of the above-listed 13 miRNAs. These miRNAs and their target genes enriched pathways and biological processes important for fetal growth, development, and metabolism. These results indicate that the transcriptomic profile of skeletal muscle can be used to predict fetal weight, and miRNAs correlated to fetal weight can serve as potential biomarkers of prenatal fetal health and growth.

SINE Insertion in the Intron of Pig GHR May Decrease Its Expression by Acting as a Repressor.

The genetic diversity of the GH/IGF axis genes and their association with the variation of gene expression and phenotypic traits, principally represented by SNPs, have been extensively reported. Nevertheless, the impact of retrotransposon insertion polymorphisms (RIPs) on the GH/IGF axis gene activity has not been reported. In the present study, bioinformatic prediction and PCR verification were performed to screen RIPs in four GH/IGF axis genes (GH, GHR, IGF1 and IGF1R). In total, five RIPs, including one SINE RIP in intron 3 of IGF1, one L1 RIP in intron 7 of GHR, and three SINE RIPs in intron 1, intron 5 and intron 9 of GHR, were confirmed by PCR, displaying polymorphisms in diverse breeds. Dual luciferase reporter assay revealed that the SINE insertion in intron 1 of GHR significantly repressed the GHR promoter activity in PK15, Hela, C2C12 and 3T3-L1 cells. Furthermore, qPCR results confirmed that this SINE insertion was associated with a decreased expression of GHR in the leg muscle and longissimus dorsi, indicating that it may act as a repressor involved in the regulation of GHR expression. In summary, our data revealed that RIPs contribute to the genetic variation of GH/IGF axis genes, whereby one SINE RIP in the intron 1 of GHR may decrease the expression of GHR by acting as a repressor.

Transcriptome analysis of porcine PBMCs reveals lipopolysaccharide-induced immunomodulatory responses and crosstalk of immune and glucocorticoid receptor signaling.

The current level of knowledge on transcriptome responses triggered by endotoxins and glucocorticoids in immune cells in pigs is limited. Therefore, in the present study, we treated porcine peripheral blood mononuclear cells (PBMCs) with lipopolysaccharide (LPS) and dexamethasone (DEX) separately or combined for 2 hours. The resultant transcriptional responses were examined by mRNA sequencing. We found that the LPS treatment triggered pronounced inflammatory responses as evidenced by upregulation of pro-inflammatory cytokines, chemokines, and related signaling pathways like NF-κB. Concurrently, a series of downregulated pro-inflammatory and upregulated anti-inflammatory molecules were identified. These are involved in the inhibition of TLR, NF-κB, and MAPK cascades and activation of signaling mediated by Tregs and STAT3, respectively. These findings suggested that LPS initiated also an anti-inflammatory process to prevent an overwhelming inflammatory response. The transcriptome responses further revealed substantial crosstalk of immune responses and glucocorticoid receptor (GR) signaling. This was apparent in four aspects: constitutive inhibition of T cell signaling by DEX through a subset of genes showing no response to LPS; inhibition of LPS-induced inflammatory genes by DEX; attenuation of DEX action by LPS paralleled by the regulation of genes implicated in cytokine and calcium signaling; and DEX-induced changes in genes associated with the activation of pro-inflammatory TLR, NF-κB, iNOS, and IL-1 signaling. Consequently, our study provides novel insights into inflammatory and GR signaling in pigs, as well as an understanding of the application of glucocorticoid drugs for the treatment of inflammatory disorders.