Identification of genes differentially expressed during prenatal development of skeletal muscle in two pig breeds differing in muscularity

<p>Abstract</p> <p>Background</p> <p>Postnatal muscle growth is largely depending on the number and size of muscle fibers. The number of myofibers and to a large extent their metabolic and contractile properties, which also influence their size, are determined prenatally during the process of myogenesis. Hence identification of genes and their networks governing prenatal development of skeletal muscles will provide insight into the control of muscle growth and facilitate finding the source of its variation. So far most of the genes involved in myogenesis were identified by <it>in vitro </it>studies using gene targeting and transgenesis. Profiling of transcriptome changes during the myogenesis <it>in vivo </it>promises to obtain a more complete picture. In order to address this, we performed transcriptome profiling of prenatal skeletal muscle using differential display RT-PCR as on open system with the potential to detect novel transcripts. Seven key stages of myogenesis (days 14, 21, 35, 49, 63, 77 and 91 <it>post conception</it>) were studied in two breeds, Pietrain and Duroc, differing markedly in muscularity and muscle structure.</p> <p>Results</p> <p>Eighty prominent cDNA fragments were sequenced, 43 showing stage-associated and 37 showing breed-associated differences in the expression, respectively. Out of the resulting 85 unique expressed sequence tags, EST, 52 could be assigned to known genes. The most frequent functional categories represented genes encoding myofibrillar proteins (8), genes involved in cell adhesion, cell-cell signaling and extracellular matrix synthesis/remodeling (8), genes regulating gene expression (8), and metabolism genes (8). Some of the EST that showed no identity to any known transcripts in the databases are located in introns of known genes and most likely represent novel exons (e.g. <it>HMGA2</it>). Expression of thirteen transcripts along with five reference genes was further analyzed by means of real-time quantitative PCR. Nine of the target transcripts showed higher than twofold differences in the expression between the two breeds (<it>GATA3</it>, <it>HMGA2</it>, <it>NRAP</it>, <it>SMC6L1</it>, <it>SPP1</it>, <it>RAB6IP2, TJP1 </it>and two EST).</p> <p>Conclusion</p> <p>The present study revealed several genes and novel transcripts not previously associated with myogenesis and expands our knowledge of genetic factors operating during myogenesis. Genes that exhibited differences between the divergent breeds represent candidate genes for muscle growth and structure.</p

Dual effect of a single nucleotide polymorphism in the first intron of the porcine Secreted phosphoprotein 1 gene: allele-specific binding of C/EBP beta and activation of aberrant splicing

<p>Abstract</p> <p>Background</p> <p>Secreted phosphoprotein 1 (SPP1 or Osteopontin, OPN) is a multifunctional matricellular glycoprotein involved in development and regeneration of skeletal muscle. Previously, we have demonstrated that porcine <it>SPP1 </it>shows breed-related differential mRNA expression during myogenesis. With the aim to identify putative contributing <it>cis</it>-regulatory DNA variation we resequenced the 5' upstream region of the gene in the respective breeds Pietrain and Duroc. We found two single nucleotide polymorphisms (SNP; [GenBank:<ext-link ext-link-id="M84121" ext-link-type="gen">M84121</ext-link>]: g.1804C>T and g.3836A>G). We focused our investigation on the SNP g.3836A>G, because <it>in silico </it>analysis and knowledge about the regulation of <it>SPP1 </it>suggested an effect of this SNP on a CCAAT/enhancer binding protein beta (C/EBPβ) responsive transcriptional enhancer.</p> <p>Results</p> <p>Using electrophoretic mobility shift assay we demonstrated that, similar to human <it>SPP1</it>, the 3' terminal end of the first intron of porcine <it>SPP1 </it>harbors a C/EBPβ binding site and showed that this binding site is negatively affected by the mutant G allele. Genotyping of 48 fetuses per breed revealed that the G allele segregated exclusively in Duroc fetuses with a frequency of 57 percent. Using real-time quantitative PCR we showed that, consistent with its negative effect on a transcriptional enhancer element, the G allele tends to decrease mRNA abundance of <it>SPP1 </it>in the fetal <it>musculus longissimus dorsi </it>(~1.3 fold; P ≥ 0.1).</p> <p>Moreover, we showed that the SNP g.3836A>G leads to ubiquitous aberrant splicing of the first intron by generating a <it>de novo </it>and activating a cryptic splice acceptor site. Aberrantly spliced transcripts comprise about half of the <it>SPP1 </it>messages expressed by the G allele. Both aberrant splice variants differ from the native transcript by insertions in the leader sequences which do not change the reading frame of <it>SPP1</it>.</p> <p>Conclusion</p> <p>At the 3' terminal end of the first intron of the porcine <it>SPP1 </it>we identified a unique, dually functional SNP g.3836A>G. This SNP affects the function of the <it>SPP1 </it>gene at the DNA level by affecting a C/EBPβ binding site and at the RNA level by activating aberrant splicing of the first intron, and thus represents an interesting DNA-marker to study phenotypic effects of <it>SPP1 </it>DNA-variation.</p

Host-Microbiota Interactions in Ileum and Caecum of Pigs Divergent in Feed Efficiency Contribute to Nutrient Utilization

peer-reviewedThe composition of the intestinal microbiota plays an important role in the digestion and utilization of nutrients and for gut health. Low-fiber diets stimulate digestion and absorption processes, predominantly in the upper region of the gastrointestinal tract, thereby increasing the conversion of feed into body weight. As a consequence, the chemical composition of digesta after duodenal and jejunal absorption processes and passage has a limited complexity affecting colonization and molecular profiles of enterocytes in the hind gut. To decipher ileal and caecal microbial ecosystems and host transcriptional profiles that are beneficial for effective use of the remaining nutrients, pigs differing in feeding efficiency were studied. Biological functions that were consistently enriched at both the gene and microbiota levels comprise immunity-related processes, which ensure the integrity of the gastrointestinal tract. In addition, the differential abundance of certain genera, including Rothia, Subdoligranulu, Leeia and Cellulosilyticum, reflects the establishment of a microbial profile that supports the digestion of endogenously indigestible dietary components in highly feed-efficient pigs. Overall, the results indicate the potential to promote these beneficial functions and further improve feed efficiency through manipulation of dietary and probiotic strategies

Association of HPA axis-related genetic variation with stress reactivity and aggressive behaviour in pigs

<p>Abstract</p> <p>Background</p> <p>Stress, elicited for example by aggressive interactions, has negative effects on various biological functions including immune defence, reproduction, growth, and, in livestock, on product quality. Stress response and aggressiveness are mutually interrelated and show large interindividual variation, partly attributable to genetic factors. In the pig little is known about the molecular-genetic background of the variation in stress responsiveness and aggressiveness. To identify candidate genes we analyzed association of DNA markers in each of ten genes (<it>CRH </it>g.233C>T, <it>CRHR1 </it>c.*866_867insA, <it>CRHBP </it>c.51G>A, <it>POMC </it>c.293_298del, <it>MC2R </it>c.306T>G, <it>NR3C1 </it>c.*2122A>G, <it>AVP </it>c.207A>G, <it>AVPR1B </it>c.1084A>G, <it>UCN </it>g.1329T>C, <it>CRHR2 </it>c.*13T>C) related to the hypothalamic-pituitary-adrenocortical (HPA) axis, one of the main stress-response systems, with various stress- and aggression-related parameters at slaughter. These parameters were: physiological measures of the stress response (plasma concentrations of cortisol, creatine kinase, glucose, and lactate), adrenal weight (which is a parameter reflecting activity of the central branch of the HPA axis over time) and aggressive behaviour (measured by means of lesion scoring) in the context of psychosocial stress of mixing individuals with different aggressive temperament.</p> <p>Results</p> <p>The SNP <it>NR3C1 </it>c.*2122A>G showed association with cortisol concentration (p = 0.024), adrenal weight (p = 0.003) and aggressive behaviour (front lesion score, p = 0.012; total lesion score p = 0.045). The SNP <it>AVPR1B </it>c.1084A>G showed a highly significant association with aggressive behaviour (middle lesion score, p = 0.007; total lesion score p = 0.003). The SNP <it>UCN </it>g.1329T>C showed association with adrenal weight (p = 0.019) and aggressive behaviour (front lesion score, p = 0.029). The SNP <it>CRH </it>g.233C>T showed a significant association with glucose concentration (p = 0.002), and the polymorphisms <it>POMC </it>c.293_298del and <it>MC2R </it>c.306T>G with adrenal weight (p = 0.027 and p < 0.0001 respectively).</p> <p>Conclusions</p> <p>The multiple and consistent associations shown by SNP in <it>NR3C1 </it>and <it>AVPR1B </it>provide convincing evidence for genuine effects of their DNA sequence variation on stress responsiveness and aggressive behaviour. Identification of the causal functional molecular polymorphisms would not only provide markers useful for pig breeding but also insight into the molecular bases of the stress response and aggressive behaviour in general.</p

Effects of Intronic and Exonic Polymorphisms of Paraoxonase 1 (PON1) Gene on Serum PON1 Activity in a Korean Population

Paraoxonase 1 (PON1) hydrolyzes a number of toxic organophosphorous compounds and reduces lipid peroxide accumulation, and PON1 genetic polymorphisms in the coding region modulate serum PON1 activity. In this study, we investigated the association between 3 polymorphisms of PON1 located in intron 5 (17899insdelTT and 17974CT) and exon 6 (192QR) and serum PON1 activity. The genetic polymorphisms and serum activity of PON1 were analyzed in 153 healthy Koreans by using a direct sequencing assay and spectrophotometric method, respectively. A significant linkage disequilibrium (LD) was observed between all tested single nucleotide polymorphisms, with the strongest LD observed between 17899insdelTT and 192QR (D' = 0.984). The 17899insdelTT, 17974CT and 192QR genetic polymorphisms were associated with significant differences in serum paraoxonase activity. In multiple regression analyses, smoking, triglyceride level, high-density lipoprotein (HDL) level, and the 17899insdelTT and 192QR genetic polymorphisms were significant determinants of serum paraoxonase activity, while age, smoking, triglyceride level, HDL level, and the 192QR genetic polymorphism were significant determinants of serum arylesterase activity. These results suggest that although the 192QR genetic polymorphism in the coding region of PON1 is primarily associated with serum PON1 activity, the intronic polymorphisms are also involved in serum PON1 activity, and this association may be mediated by LD

Human Skeletal Muscle Possesses an Epigenetic Memory of Hypertrophy

It is unknown if adult human skeletal muscle has an epigenetic memory of earlier encounters with growth. We report, for the first time in humans, genome-wide DNA methylation (850,000 CpGs) and gene expression analysis after muscle hypertrophy (loading), return of muscle mass to baseline (unloading), followed by later hypertrophy (reloading). We discovered increased frequency of hypomethylation across the genome after reloading (18,816 CpGs) versus earlier loading (9,153 CpG sites). We also identified AXIN1, GRIK2, CAMK4, TRAF1 as hypomethylated genes with enhanced expression after loading that maintained their hypomethylated status even during unloading where muscle mass returned to control levels, indicating a memory of these genes methylation signatures following earlier hypertrophy. Further, UBR5, RPL35a, HEG1, PLA2G16, SETD3 displayed hypomethylation and enhanced gene expression following loading, and demonstrated the largest increases in hypomethylation, gene expression and muscle mass after later reloading, indicating an epigenetic memory in these genes. Finally, genes; GRIK2, TRAF1, BICC1, STAG1 were epigenetically sensitive to acute exercise demonstrating hypomethylation after a single bout of resistance exercise that was maintained 22 weeks later with the largest increase in gene expression and muscle mass after reloading. Overall, we identify an important epigenetic role for a number of largely unstudied genes in muscle hypertrophy/memory

Causes, consequences and biomarkers of stress in swine: an update

BACKGROUND: In recent decades there has been a growing concern about animal stress on intensive pig farms due to the undesirable consequences that stress produces in the normal physiology of pigs and its effects on their welfare and general productive performance. This review analyses the most important types of stress (social, environmental, metabolic, immunological and due to human handling), and their biological consequences for pigs. The physio-pathological changes associated with stress are described, as well as the negative effects of stress on pig production. In addition an update of the different biomarkers used for the evaluation of stress is provided. These biomarkers can be classified into four groups according to the physiological system or axis evaluated: sympathetic nervous system, hypothalamic-pituitary-adrenal axis, hypothalamic-pituitary-gonadal axis and immune system. CONCLUSIONS: Stress it is a process with multifactorial causes and produces an organic response that generates negative effects on animal health and production. Ideally, a panel of various biomarkers should be used to assess and evaluate the stress resulting from diverse causes and the different physiological systems involved in the stress response. We hope that this review will increase the understanding of the stress process, contribute to a better control and reduction of potential stressful stimuli in pigs and, finally, encourage future studies and developments to better monitor, detect and manage stress on pig farms