Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone.

Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice.

Muscle Transcriptional Profile Based on Muscle Fiber, Mitochondrial Respiratory Activity, and Metabolic Enzymes.

Skeletal muscle is a highly metabolically active tissue that both stores and consumes energy. Important biological pathways that affect energy metabolism and metabolic fiber type in muscle cells may be identified through transcriptomic profiling of the muscle, especially ante mortem. Here, gene expression was investigated in malignant hyperthermia syndrome (MHS)-negative Duroc and Pietrian (PiNN) pigs significantly differing for the muscle fiber types slow-twitch-oxidative fiber (STO) and fast-twitch-oxidative fiber (FTO) as well as mitochondrial activity (succinate-dependent state 3 respiration rate). Longissimus muscle samples were obtained 24 h before slaughter and profiled using cDNA microarrays. Differential gene expression between Duroc and PiNN muscle samples were associated with protein ubiquitination, stem cell pluripotency, amyloid processing, and 3-phosphoinositide biosynthesis and degradation pathways. In addition, weighted gene co-expression network analysis within both breeds identified several co-expression modules that were associated with the proportion of different fiber types, mitochondrial respiratory activity, and ATP metabolism. In particular, Duroc results revealed strong correlations between mitochondrion-associated co-expression modules and STO (r = 0.78), fast-twitch glycolytic fiber (r = -0.98), complex I (r=0.72) and COX activity (r = 0.86). Other pathways in the protein-kinase-activity enriched module were positively correlated with STO (r=0.93), while negatively correlated with FTO (r = -0.72). In contrast to PiNN, co-expression modules enriched in macromolecule catabolic process, actin cytoskeleton, and transcription activator activity were associated with fiber types, mitochondrial respiratory activity, and metabolic enzyme activities. Our results highlight the importance of mitochondria for the oxidative capacity of porcine muscle and for breed-dependent molecular pathways in muscle cell fibers.

MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation.

In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2.

Prostaglandin E synthase interacts with inducible heat shock protein 70 after heat stress in bovine primary dermal fibroblast cells.

Exposure to heat stress in dairy cows leads to undesired side effects that are reflected by complex alterations in endocrine parameters, such as reduced progesterone, estradiol, and thyroid hormone concentrations. These endocrine maladaptation leads to failure to resume cyclicity, a poor uterine environment and inappropriate immune responses in postpartum dairy cows. Prostaglandins (PG's) are lipid mediators, which serve as signal molecules in response to various external stimuli as well as to cell-specific internal signal molecules. A central role in PG synthesis plays prostaglandin E synthase (PGES) that catalyzes the isomerization of PGH2 to PGE2 .The present study was conducted to investigate heat stress associated PGES expression. Expression of PGES and inducible heat shock protein 70 (HSP70), as a putative chaperonic protein, was studied in bovine primary fibroblasts under different heat shock conditions. Bovine primary fibroblasts produce PGE2 at homoiothermical norm temperature (38.5°C in bovine), but reduce PGE2 production rates under extreme heat stress (at 45°C for 6 h). By contrast, PGE2 production rates are maintained after a milder heat stress (at 41.5°C for 6 h). PGE2 synthesis is abolished by application of cyclooxygenase inhibitor indomethacin, indicating de novo synthesis. Heat stress increases HSP70 but not PGES protein concentrations. HSP70 physically interacts with PGES and the PGES-HSP70 complex did not dissociate upon heat stress at 45°C even after returning the cells to 37°C. The PGE2 production negatively correlates with the portion of PGES-HSP70 complex. These results suggest a protein interaction between HSP70 and PGES in dermal fibroblast cells. Blockage of PGES protein by HSP70 seems to interfere with the regulatory processes essential for cellular adaptive protection. © 2014 International Society for Advancement of Cytometry.

Differential expression of miRNAs and their target mRNAs in endometria prior to maternal recognition of pregnancy associates with endometrial receptivity for in vivo- and in vitro-produced bovine embryos.

Endometrial receptivity is a prerequisite for successful embryo implantation and pregnancy. Receptivity involves complex processes promoted by many transcripts that are key components of molecular pathways that depend on ovarian hormones and that contribute to shaping structural, metabolic, and communication properties of endometrial cells toward reception of embryos. MicroRNAs (miRNAs) are important regulators of the expression of these transcripts encoding effector molecules. We acquired miRNA and mRNA signatures, miRNA-mRNA pairs, and regulatory networks linked with the emergence and maintenance of postimplantation pregnancy. Endometrial tissue samples were obtained at Days 3 and 7 of the estrous cycle of cows that did or did not become pregnant after transfer of either in vivo-produced (IVV) or in vitro-produced (IVT) embryos in the next cycle following the biopsy. We report a list of endometrial miRNAs that were differentially expressed between Day 3 and Day 7 of the bovine estrous cycle (including miR-1290, miR-3437, miR-1246, miR-486, miR-3107, and miR-382), that differed with high or low endometrial receptivity (miR-3902-3p, miR-1825, miR-H14-3p, miR-885-3p, miR-504-3p, and miR-186), or that differed among the IVT and IVV transfers (miR-449a/b/c, miR-138, miR-874, miR-4342, miR-2231, and miR-2751). Moreover, mRNA transcripts were also analyzed, and pairs of negatively correlated miRNAs and mRNAs were predicted in silico. The miRNA-mRNA target pairs had roles in response to hormonal stimuli and oxidative stress, chromatin organization, miRNA-mediated epigenetic histone changes, cell proliferation, p53 signaling, and apoptosis. Overall, we identified significant miRNAs, miRNA-mRNA pairs, and functional networks that are associated with the state of pregnancy at Day 28 as a parameter of endometrial receptivity and that are affected by estrous cycle and embryo culture systems.

Gene expression and DNA-methylation of bovine pretransfer endometrium depending on its receptivity after in vitro-produced embryo transfer.

Embryonic implantation to establish a pregnancy is a complex process that requires appropriate communication between the embryo and the maternal endometrium. Inadequate uterine receptivity may contribute to the majority of implantation failures. To provide a comprehensive inventory of genes and functional networks that represent the maternal input of the embryo-maternal cross-talk, a longitudinal, holistic study of the endometrial transcriptome in relation to the days of estrous and to the receptivity of the endometrium was performed in bovine. At day 3 of estrous, genes related to cell communication and mitochondrial energy metabolism were differentially expressed among high- and low-receptive endometria (HR, LR); at day 7, transcripts functioning in immune and inflammatory pathways, oxidative stress, and angiogenesis had different abundances. Additionally, temporal transcriptional changes between days 3 and 7 differed considerably among HR and LR. Further, several transcription factors were predicted as relevant for receptivity because they were either differentially expressed among HR and LR animals or are known to be associated with genes we detected to have differential expression. Finally, global DNA methylation varied according to the interaction of receptivity group and day of estrous, and a divergent trend, which correlated with abundance of DNMT1 transcript, was observed in LR and HR along the estrous cycle days. The study revealed that, even in early estrous, transcripts related to cell communication and response to exogenous stimuli, vascularization, and energy supply show divergent expression and longitudinal temporal regulation in HR and LR. Key components of these molecular pathways are known to be dependent on ovarian hormones that promote uterine receptivity.

Maternal dietary protein restriction and excess affects offspring gene expression and methylation of non-SMC subunits of condensin I in liver and skeletal muscle.

Recent evidence indicates that maternal nutrition during pregnancy influences gene expression in offspring through epigenetic alterations. In the present study we evaluated the effect of protein excess and deficiency during porcine pregnancy on offspring hepatic and skeletal muscular expression patterns of key genes of methionine metabolism (DNMT1, DNMT3a, DNMT3b, BHMT, MAT2B and AHCYL1), condensin I subunit genes (NCAPD2, NCAPG and NCAPH), important for chromosome condensation and segregation, global DNA methylation and gene-specific DNA methylation. German Landrace sows were randomly assigned to control (CO), high protein (HP) and low protein (LP) diet groups. Tissue samples of offspring were collected from fetal (dpc95), newborn (dpn1), weanling (dpn28) and finisher pigs (dpn188). Gene expression of DNMT1, DNMT3a and DNMT3b was influenced by both HP and LP diets, indicating an involvement of DNA methylation in fetal programming by maternal protein supply. Moreover, hepatic global methylation was significantly affected by protein restriction at dpc95 (p = 0.004) and by protein excess at dpn188 (p = 0.034). Gene expression in fetal liver was significantly different between CO and LP for NCAPD2 (p = 0.0005), NCAPG (p = 0.0009) and NCAPH (p < 0.0001). In skeletal muscle, LP fetuses had significantly altered gene expression of NCAPD2 (p = 0.020) and NCAPH (p = 0.001), compared with CO. Furthermore, NCAPG was differentially methylated among LP, HP and CO; indeed, a significant positive correlation was detected with transcript amount in fetal pigs (r = 0.47, p = 0.002). These data demonstrate that both restriction and excess dietary protein during pregnancy alters the offspring's epigenetic marks and influences gene expression.

Comparative expression profiling of E. coli and S. aureus inoculated primary mammary gland cells sampled from cows with different genetic predispositions for somatic cell score.

BACKGROUND: During the past ten years many quantitative trait loci (QTL) affecting mastitis incidence and mastitis related traits like somatic cell score (SCS) were identified in cattle. However, little is known about the molecular architecture of QTL affecting mastitis susceptibility and the underlying physiological mechanisms and genes causing mastitis susceptibility. Here, a genome-wide expression analysis was conducted to analyze molecular mechanisms of mastitis susceptibility that are affected by a specific QTL for SCS on Bos taurus autosome 18 (BTA18). Thereby, some first insights were sought into the genetically determined mechanisms of mammary gland epithelial cells influencing the course of infection. METHODS: Primary bovine mammary gland epithelial cells (pbMEC) were sampled from the udder parenchyma of cows selected for high and low mastitis susceptibility by applying a marker-assisted selection strategy considering QTL and molecular marker information of a confirmed QTL for SCS in the telomeric region of BTA18. The cells were cultured and subsequently inoculated with heat-inactivated mastitis pathogens Escherichia coli and Staphylococcus aureus, respectively. After 1, 6 and 24 h, the cells were harvested and analyzed using the microarray expression chip technology to identify differences in mRNA expression profiles attributed to genetic predisposition, inoculation and cell culture. RESULTS: Comparative analysis of co-expression profiles clearly showed a faster and stronger response after pathogen challenge in pbMEC from less susceptible animals that inherited the favorable QTL allele 'Q' than in pbMEC from more susceptible animals that inherited the unfavorable QTL allele 'q'. Furthermore, the results highlighted RELB as a functional and positional candidate gene and related non-canonical Nf-kappaB signaling as a functional mechanism affected by the QTL. However, in both groups, inoculation resulted in up-regulation of genes associated with the Ingenuity pathways 'dendritic cell maturation' and 'acute phase response signaling', whereas cell culture affected biological processes involved in 'cellular development'. CONCLUSIONS: The results indicate that the complex expression profiling of pathogen challenged pbMEC sampled from cows inheriting alternative QTL alleles is suitable to study genetically determined molecular mechanisms of mastitis susceptibility in mammary epithelial cells in vitro and to highlight the most likely functional pathways and candidate genes underlying the QTL effect.

Temporary consumption of diet with unbalanced amino acid pattern affects long-lasting growth retardation correlated with oxidative stress response associated gene expression in juvenile pigs.

BACKGROUND & AIMS: The present study was conducted to study whether oxidative stress is the trigger of long-term physiological effects of temporary consumption of a soy protein isolate (SPI) based diet with an imbalanced amino acid pattern. MATERIAL AND METHODS: Hepatic expression of 19 genes that are involved in and co-regulated with oxidative stress response and showing diet-associated expression after chronic SPI feeding using quantitative RT-PCR, growth and liver composition were investigated in a model of protein-underfeeding juvenile pigs, which were fed a casein (CAS) based diet for four weeks subsequent to a four week consumption of an SPI diet in comparison with chronically CAS fed animals. RESULTS: Temporary feeding of SPI diet resulted in prolonged up-regulation of genes involved in oxidative/cellular stress response (glutathione-S-transferase, peptide methionine sulfoxide reductase, calnexin, organic anion transport polypeptide 2). Cluster analysis of gene expression data indicated persistent SPI-related co-regulation of the genes involved in stress response with genes involved in the regulation of protein biosynthesis and in neuronal signalling for at least four weeks after replacement of SPI by CAS. Gene expression data are negatively correlated with body weight and liver protein content. CONCLUSION: Significant association of oxidative stress responsiveness with growth retardation and liver composition underline the possible impact of diet-affected oxidative stress for long-lasting deleteriously metabolic consequences.

Stress-related gene expression in brain and adrenal gland of porcine fetuses and neonates.

This study was conducted to examine stress-induced effects on gene expression of specific markers for HPA axis and neuronal activity in fetuses and neonatal pigs. Brain, pituitary gland, and adrenal gland were obtained to determine the mRNA levels for corticotropin-releasing hormone (CRH), CRH receptor 1 (CRHR1), pro-opiomelanocortin (POMC), ACTH receptor (MC2R), c-jun and c-fos. The suitability of these molecular markers was determined in neonatal pigs which were maternally deprived for two hours. It was found that maternal deprivation caused significantly higher transcript levels of c-fos and CRH in brain accompanied by a down-regulation of CRHR1 mRNA and an up-regulation of c-jun in the pituitary gland. To determine the effect of elevated maternal cortisol levels on gene expression of these molecular markers in fetuses, pregnant sows were treated with 100 IU ACTH (Synacthen Depot) s.c. every two days between Day 49 and Day 75 of gestation (normal gestation length 114 days). Animals were killed 48 hours after the last ACTH administration and fetuses of each sow were isolated. The ACTH treatment of sows significantly increased mRNA expression of c-fos but not of CRH in the fetal brain, and significantly decreased MC2R mRNA expression in the adrenal gland. However, HPA axis seems not to be fully developed in Day 77-fetuses because fetal pituitary CRHR1 and POMC mRNA expression was low in most of the fetuses. Although the expression of endocrine regulatory factors was partially incomplete in fetuses at the beginning of the third-trimester, ACTH dependent activation of c-fos mRNA in brain indicates a stress-related increase of neuronal activity. Based on these results it is assumed that prenatal stress in pigs may also have effects on the activity of the HPA axis in the offspring.

Application of disease-associated differentially expressed genes--mining for functional candidate genes for mastitis resistance in cattle.

In this study the mRNA differential display method was applied to identify mastitis-associated expressed DNA sequences based on different expression patterns in mammary gland samples of non-infected and infected udder quarters of a cow. In total, 704 different cDNA bands were displayed in both udder samples. Five hundred-and-thirty two bands, (75.6%) were differentially displayed. Ninety prominent cDNA bands were isolated, re-amplified, cloned and sequenced resulting in 87 different sequences. Amongst the 19 expressed sequence tags showing a similarity with previously described genes, the majority of these sequences exhibited homology to protein kinase encoding genes (26.3%), to genes involved in the regulation of gene expression (26.3%), to growth and differentiation factor encoding genes (21.0%) and to immune response or inflammation marker encoding genes (21.0%). These sequences were shown to have mastitis-associated expression in the udder samples of animals with and without clinical mastitis by quantitative RT-PCR. They were mapped physically using a bovine-hamster somatic cell hybrid panel and a 5000 rad bovine whole genome radiation hybrid panel. According to their localization in QTL regions based on an established integrated marker/gene-map and their disease-associated expression, four genes (AHCY, PRKDC, HNRPU, OSTF1) were suggested as potentially involved in mastitis defense.

Dietary protein modifies hepatic gene expression associated with oxidative stress responsiveness in growing pigs.

Understanding the basis for differences in nutrient requirements and for nutrient effects on health and performance requires an appreciation of the links between nutrition and gene expression. We developed and applied molecular probes to characterize diet-associated postabsorptive hepatic gene expression in growing pigs chronically fed protein-restricted diets based on either casein (CAS) or soy protein isolate (SPI). Eighty-eight expressed sequence tags (ESTs) were identified on the basis of diet-related changes in expression, by using an mRNA differential display method. Expression profiling based on transcription analysis by real-time reverse transcriptase-polymerase chain reaction showed that the SPI diet significantly changed the pattern of gene expression as compared with the CAS diet and allowed identification of coregulated genes. The expression of six genes involved in the metabolism of stress response (glutathione S-transferase, peptide methionine sulfoxide reductase, apolipoprotein A-I, organic anion transport polypeptide 2, calnexin, heat shock transcription factor 1) exhibited significant changes in the transcription level and indicated an increased oxidative stress response in pigs fed the SPI diet. Hierarchical clustering of gene expression data of all 33 ESTs analyzed across 14 pigs fed the two different diets resulted in clustering of genes related to the oxidative stress response with genes related to the regulation of gene expression and neuronal signaling.

Targeted construction of a high-resolution, integrated, comprehensive, and comparative map for a region specific to bovine chromosome 6 based on radiation hybrid mapping.

To resolve a candidate chromosome region on the middle part of bovine chromosome 6 (BTA6) containing several different quantitative trait locus (QTL) intervals, we constructed a high-resolution, integrated, comprehensive, and comparative map using a 12,000-rad, whole-genome, cattle-hamster radiation hybrid (RH) panel. The RH map includes a total of 71 loci either selected from bovine and comparative maps or targeted directly from a microdissection library specific for the BTA6 region. All loci typed were placed in one linkage group at a lod score threshold of 4.0. The length of the comprehensive RH map, which is the first high-resolution RH map in cattle, spans 2568.8 cR(12,000). The order of markers obtained principally agrees with the order on published bovine genetic maps. Our RH map integrates markers as well as genes and ESTs available from several physical and genetic maps of BTA6 and the orthologous ovine chromosome 6, human chromosome 4, and mouse chromosomes 5/3. Comparative analysis confirms and refines current knowledge about conservation and rearrangements in corresponding chromosomal regions on BTA6. We identified and localized two new breakpoints for intrachromosomal rearrangements between human chromosome 4 and BTA6. This RH map is a powerful tool in all aspects of genetic, physical, transcript, and comparative mapping. Due to its links to the gene-dense maps of human and mouse, it can serve as a prerequisite to identify possible candidate genes for quantitative trait loci localized in the targeted BTA6 region.

A comparative radiation hybrid map of bovine chromosome 18 and homologous chromosomes in human and mice.

A comprehensive radiation hybrid (RH) map and a high resolution comparative map of Bos taurus (BTA) chromosome 18 were constructed, composed of 103 markers and 76 markers, respectively, by using a cattle-hamster somatic hybrid cell panel and a 5,000 rad whole-genome radiation hybrid (WGRH) panel. These maps include 65 new assignments (56 genes, 3 expressed-sequence tags, 6 microsatellites) and integrate 38 markers from the first generation WGRH(5,000) map of BTA18. Fifty-nine assignments of coding sequences were supported by somatic hybrid cell mapping to markers on BTA18. The total length of the comprehensive map was 1666 cR(5,000). Break-point positions within the chromosome were refined and a new telomeric RH linkage group was established. Conserved synteny between cattle, human, and mouse was found for 76 genes of BTA18 and human chromosomes (HSA) 16 and 19 and for 34 cattle genes and mouse chromosomes (MMU) 7 and 8. The new RH map is potentially useful for the identification of candidate genes for economically important traits, contributes to the expansion of the existing BTA18 gene map, and provides new information about the chromosome evolution in cattle, humans, and mice.