MicroRNA expression profiling of porcine mammary epithelial cells after challenge with Escherichia coli in vitro.

BACKGROUND: Coliform mastitis is a symptom of postpartum dysgalactia syndrome (PDS), a multifactorial infectious disease of sows. Our previous study showed gene expression profile change after bacterial challenge of porcine mammary epithelial cells (PMECs). These mRNA expression changes may be regulated through microRNAs (miRNAs) which play critical roles in biological processes. Therefore, miRNA expression profile was investigated in PMECs. RESULTS: PMECs were isolated from three lactating sows and challenged with heat-inactivated potential mastitis-causing pathogen Escherichia coli (E. coli) for 3 h and 24 h, in vitro. At 3 h post-challenge with E. coli, target gene prediction identified a critical role of miRNAs in regulation of host immune responses and homeostasis of PMECs mediated by affecting pathways including cytokine binding (miR-202, miR-3277, miR-4903); IL-10/PPAR signaling (miR-3277, miR-4317, miR-548); and NF-ĸB/TNFR2 signaling (miR-202, miR-2262, miR-885-3p). Target genes of miRNAs in PMECs at 24 h were significantly enriched in pathways associated with interferon signaling (miR-210, miR-23a, miR-1736) and protein ubiquitination (miR-125, miR-128, miR-1280). CONCLUSIONS: This study provides first large-scale miRNA expression profiles and their predicted target genes in PMECs after contact with a potential mastitis-causing E. coli strain. Both, highly conserved miRNAs known from other species as well as novel miRNAs were identified in PMECs, representing candidate predictive biomarkers for PDS. Time-dependent pathogen clearance suggests an important role of PMECs in inflammatory response of the first cellular barrier of the porcine mammary gland.

Methylating micronutrient supplementation during pregnancy influences foetal hepatic gene expression and IGF signalling and increases foetal weight.

PURPOSE: Maternal diet during pregnancy impacts foetal growth and development. In particular, dietary levels of methylating micronutrients (methionine, folate, choline, vitamins B6, and B12) interfere with the availability and allocation of methyl groups for methylation reactions, thereby influencing normal transcription. However, the currently recommended methylating micronutrient supplementation regimen is haphazard and arbitrary at best. METHODS: To investigate the effects of a methylating micronutrient-rich maternal diet, pregnant Pietrain sows were fed either a standard diet (CON) or a diet supplemented with methionine, folate, choline, B6, B12, and zinc (MET). Foetal liver and muscle (M. longissimus dorsi) tissues were collected at 35, 63, and 91 days post-conception. Transcriptional responses to diet were assessed in foetal liver. Altered insulin-like growth factor (IGF) signalling in transcriptome analyses prompted investigation of IGF-2 and insulin-like growth factor binding proteins (IGFBPs) levels in muscle and liver. RESULTS: Maternal diet enriched with methylating micronutrients was associated with increased foetal weight in late gestation. Hepatic transcriptional patterns also revealed differences in vitamin B6 and folate metabolism between the two diets, suggesting that supplementation was effective. Additionally, shifts in growth-supporting metabolic routes of the lipid and energy metabolism, including IGF signalling, and of cell cycle-related pathways were found to occur in liver tissue in supplemented individuals. Weight differences and modulated IGF pathways were also reflected in the muscle content of IGF-2 (increased in MET) and IGFBP-2 (decreased in MET). CONCLUSIONS: Maternal dietary challenges provoke stage-dependent and tissue-specific transcriptomic modulations in the liver pointing to molecular routes contributing to the organismal adaptation. Subtle effects on late foetal growth are associated with changes in the IGF signalling mainly in skeletal muscle tissue that is less resilient to dietary stimuli than liver.

Analysis of non-synonymous SNPs of the porcine SERPINA6 gene as potential causal variants for a QTL affecting plasma cortisol levels on SSC7.

Recently, the SERPINA6 gene encoding corticosteroid-binding globulin (CBG) has been proposed as a candidate gene for a quantitative trait locus (QTL) affecting cortisol level on pig chromosome 7. The QTL was repeatedly detected in different lines, including a Piétrain × (German Landrace × German Large White) cross (PiF1) and purebred German Landrace (LR). In this study, we investigated whether the known non-synonymous polymorphisms c.44G>T, c.622C>T, c.770C>T, c.793G>A, c.832G>A and c.919G>A of SERPINA6 are sufficient to explain the QTL in these two populations. Our investigations revealed that SNPs c.44G>T, c.622C>T, c.793G>A and c.919G>A are associated with cortisol level in PiF1 (P < 0.01). Haplotype analysis showed that these associations are largely attributable to differences between a major haplotype carrying SNPs c.793G>A and c.919G>A and a haplotype carrying SNPs c.44G>T and c.622C>T. Furthermore, some SNPs, particularly c.44G>T and c.622C>T and the carrier haplotype, showed association with meat quality traits including pH and conductivity (P < 0.05). In LR, the non-synonymous SNPs segregate at very low frequency (<5%) and/or show only weak association with cortisol level (SNPs c.832G>A and c.919G>A; P < 0.05). These findings suggest that the non-synonymous SNPs are not sufficient to explain the QTL across different breeds. Therefore, we examined whether the expression of SERPINA6 is affected by cis-regulatory polymorphisms in liver, the major organ for CBG production. We found allelic expression imbalance of SERPINA6, which suggests that its expression is indeed affected by genetic variation in cis-acting elements. This represents candidate causal variation for future studies of the molecular background of the QTL.

Hepatic expression patterns in psychosocially high-stressed pigs suggest mechanisms following allostatic principles.

Psychosocial challenges are known to introduce cellular and humoral adaptations in various tissues and organs, including parts of the sympatho-adrenal-medullary system and hypothalamic-pituitary-adrenal axis as well as other peripheral tissue being responsive to cortisol and catecholamines. The liver is of particular interest given its vital roles in maintaining homeostasis and health as well as regulating nutrient utilization and overall metabolism. We aimed to evaluate whether and how response to psychosocial stress is reflected by physiological molecular pathways in liver tissue. A pig mixing experiment was conducted to induce psychosocial stress culminating in skin lesions which reflect the involvement in aggressive behavior and fighting. At 27 weeks of age, animals prone to psychosocially low- and high-stress were assigned to mixing groups. Skin lesions were counted before mixing and after slaughter on the carcass. Individual liver samples (n=12) were taken. The isolated RNA was hybridized on Affymetrix GeneChip porcine Genome Arrays. Relative changes of mRNA abundances were estimated via variance analyses. Molecular routes related to tRNA charging, urea cycle, acute phase response, galactose utilization, and steroid receptor signaling were found to be increased in psychosocially high-stressed animals, whereas catecholamine degradation and cholesterol biosynthesis were found to be decreased. In particular, psychosocially high-stressed animals show decreased expression of catechol-O-methyltransferase (COMT) which has been linked to molecular mechanisms regulating aggressiveness and stress response. The expression patterns of high-stressed animals revealed metabolic alterations of key genes related to energy-mobilizing processes at the expense of energy consuming processes. Thus, the coping following psychosocial challenges involves transcriptional alterations in liver tissue which may be summarized with reference to the concept of allostasis, a strategy which is critical for survival.

High- and low-protein gestation diets do not provoke common transcriptional responses representing universal target-pathways in muscle and liver of porcine progeny.

AIM: Maternal diets introduce transcriptional changes in the offspring, highlighting the concept of genetic and physiological plasticity. Our previous analyses investigated stage-dependent transcriptional responses to either maternal high or low protein/carbohydrate ratios in either muscle or liver. Foetal programming is proposed to be mediated by a small number of gatekeeper processes, such as cytoskeleton remodelling and cell-cycle regulation. Here, we conducted an overall analysis of a three-dimensional data set aiming to elucidate, whether there are universally targeted pathways of adaptive transcriptional response to different protein/carbohydrate ratios. METHODS: Microarray analyses were performed on liver and skeletal muscle tissue sampled at 94 days post-conception and 1, 28 and 188 days post-natum from offspring (n = 253) of German Landrace gilts that were fed isoenergetic diets containing low, high or adequate protein. RESULTS: Cluster analyses revealed a hierarchical influence of tissue, ontogenetic stage and diet on transcript levels. Considering results cumulatively over stages, liver showed only marginal transcriptional differences between the dietary groups, whereas considerable differences appeared in muscle. Considering results cumulatively over tissues, nutrition-responsive transcriptions were observed along ontogenesis. Pathway analyses revealed transcript differences in genes related to tissue remodelling, cell-cycle regulation and mitochondrial function. CONCLUSION: The factors tissue, stage and diet impact gene expression in a hierarchical order. Porcine liver appeared to be a tissue that was more resilient to nutritional modulation compared with skeletal muscle tissue. Differential modulation between tissues and dietary groups reveal that there are no universal target-pathways of adaptive transcriptional response to different protein diets.

Deoxynivalenol affects the composition of the basement membrane proteins and influences en route the migration of CD16(+) cells into the intestinal epithelium.

The numerous pores in the basement membrane (BM) of the intestinal villi are essential for the communication of enterocytes with cells in the lamina propria, an important mechanism for the induction of intestinal immune responses. The intestinal epithelial barrier is affected by the mycotoxin deoxynivalenol (DON) from both the apical (luminal) and basolateral (serosal) side. The pig is the most susceptible species to the anorectic and immune-modulating effects of DON, which is most prevalent in crops. We analysed in pigs the effect of DON-contaminated feed on the composition and perforation of the BM and the presence of CD16(+) cells or their dendrites in the epithelium. In addition to in vivo experiments, in vitro studies were carried out. Using microarray analyses, the effects of DON on IPEC-J2 cells were studied with the focus on the BM. Our in vivo results showed in the control pigs: (1) a significant increased pore number (p ≤ 0.001) in the jejunum in comparison to ileum, (2) no difference in the pore size, and (3) comparable frequency of intraepithelial CD16(+) cells/dendrites in the jejunum and ileum. There was a marked trend that DON feeding increases: (1) the pore number in jejunum, and (2) the number of CD16(+) cells/dendrites in the epithelium (Tukey-Kramer; p = 0.055 and p = 0.067, respectively). The in vivo results were extended with microarray analyses of epithelial cell (IPEC-J2 cells). The down-regulation of genes like syndecan, fibulin 6 and BM-40 was observed. These proteins are important factors in the BM composition and in formation of pores. Our results provide evidence that already low basolateral concentrations of DON (50 ng/mL) influence the production of the BM protein laminin by epithelial cells. Thus, DON affects the composition of the BM.

Transcriptional profiling and miRNA-dependent regulatory network analysis of longissimus dorsi muscle during prenatal and adult stages in two distinct pig breeds.

MicroRNAs (miRNAs) and mRNAs establish a complex regulatory network influencing diverse biological pathways including muscle development and growth. Elucidating miRNA-dependent regulatory networks involved in muscle development could provide additional insights into muscle traits largely predefined during prenatal development. The present study aimed to determine differentially expressed transcripts and functional miRNA-mRNA relationships associated with different stages of skeletal muscle development in two pig breeds, German Landrace and Pietrain, distinct in muscle characteristics. A comparative transcriptional profiling of longissimus dorsi muscle tissues from fetuses at 35, 63 and 91 days post-conception as well as adult pigs (180 days postnatum) was performed using the Affymetrix GeneChip porcine genome microarray. Differential expression patterns were identified to be associated with muscularly developmental stages and breed types. The integration of miRNA expression data and ingenuity pathways analysis (ipa) pathway analysis revealed several miRNA-dependent regulatory networks related to muscle growth and development. The present results provide insights into muscle biology for further improvement of porcine meat quality.

QTL region-specific microarrays reveal differential expression of positional candidate genes of signaling pathways associated with the liability for the inverted teat defect.

The inverted teat defect is the most common disorder of the mammary complex in pigs. It is characterized by the failure of teats to protrude from the udder surface, preventing normal milk flow and thus limiting the rearing capacity and increasing the risk of mastitis. The inverted teat defect is a liability trait with a complex mode of inheritance. We previously identified QTL for inverted teats. As a complementary approach that integrates map-based efforts to identify candidate genes for the inverted teat defect with function-driven expression analysis, application-specific microarrays were constructed that cover 1525 transcripts mapping in QTL regions on pig chromosomes 2, 3, 4, 6 and 11. About 950 transcripts were expressed in epithelial and mesenchymal teat tissue. The expression of three categories of teats was compared: normal teats of both non-affected and affected animals and inverted teats of affected animals. In epithelium and mesenchyme, 62 and 24 genes respectively were significantly differentially expressed (DE). The majority of biofunctions to which a significant number of DE genes were assigned are related to the following: (1) cell maintenance, proliferation, differentiation and replacement; (2) organismal, organ and tissue development; or (3) genetic information and nucleic acid processing. Moreover, the DE genes belong almost exclusively to canonical pathways related to signaling rather than metabolic pathways. This is in line with findings obtained by genome-wide catalogue microarrays. This study adds another piece to the puzzle of the etiology of inverted teats by indicating that causal genetic variation leading to the disorder is likely among the genes encoding for members of the signaling cascades of growth factors.

Association of TLR5 sequence variants and mRNA level with cytokine transcription in pigs.

The Toll-like receptor 5 (TLR5) plays a crucial role in host defense against flagellated bacteria by recognizing flagellin. Accumulating evidence suggests that single nucleotide polymorphisms (SNPs) in TLR5 have an effect on flagellin recognition and are associated with susceptibility/resistance to disease. In this study, we analyzed association of SNPs, including c.834T>G, c.1065T>C, c.1205C>T, c.1246A>T, c.1269G>A, and c.1398C>T, as well as mRNA level of TLR5 with the abundance of transcripts of cytokines in pigs. SNPs c.1246A>T and c.1269G>A were significantly associated with the transcript abundance of interleukin (IL)-2, and SNPs c.834T>G and c.1398C>T with IL-10 (P < 0.05); the haplotypes showed a tendency to affect the transcript abundance of IL-10 (P = 0.0660) and significantly associated with the transcription of TLR5 (P < 0.01); the abundance of transcripts of TLR5 and IL-10 were strongly correlated (P < 0.01). The results indicated that the SNPs, associated with the transcript abundance of cytokines, were related to immune responsiveness mediated by cytokine, which, in turn, would have a role in pig breeding for disease resistance. Furthermore, the positive correlation between the abundance of TLR5 and IL10 suggest a link between TLR5 activation and IL-10 expression in porcine.

Association of TLR4 polymorphism with cytokine expression level and pulmonary lesion score in pigs.

The toll-like receptor 4 (TLR4), recognizing lipopolysaccharide of gram-negative bacteria, plays an essential role in immune responses. Variation in TLR4 alters host immune responses to pathogen and is associated with resistance/susceptibility to infectious diseases, as suggested by studies in humans and agricultural species, including cattle and chicken. In this study, we analyzed association of single nucleotide polymorphisms (SNPs) of TLR4 with cytokine expression level and pulmonary lesion score in swine. The SNP c.611 T>A showed significant association with the transcription levels of IFNG, TNFA, and IL-6 (P < 0.05); the SNP c.962 G>A showed significant association with the transcription of IFNG, IL-2, and IL-4 (P < 0.05); the SNP c.1,027 C>A showed significant association with the transcription of IFNG and IL-6 (P < 0.05); the haplotypes showed significant association with the transcription of IFNG, IL-2, IL-4, IL-6, and TNFA (P < 0.05). Both SNPs c.611 T>A and c.962 G>A showed significant association with pulmonary lesion scores (P < 0.01); and the combination genotypes of 3 polymorphic sites were also significantly associated with pulmonary lesion scores (P < 0.01). The observed relationship between TLR4 polymorphism and the transcription levels of cytokines indicate that these SNPs are related to the modulation of the cytokine mediated immune response.

Genes with expression levels correlating to drip loss prove association of their polymorphism with water holding capacity of pork.

Six genes that were known to exhibit expression levels that are correlated to drip loss BVES, SLC3A2, ZDHHC5, CS, COQ9, and EGFR have been for candidate gene analysis. Based on in silico analysis SNPs were detected, confirmed by sequencing, and used for genotyping. The SNPs were genotyped in about 1,800 animals from six pig populations including commercial herds of Pietrain (PI) and German Landrace (DL), different commercial herds of Pietrain×(German Large White×German Landrace) (PIF1(a/b/c)), and one experimental F2-population Duroc×Pietrain (DUPI). Comparative and genetic mapping established the location of BVES on SSC1, of SLC3A2 and ZDHHC5 on SSC2, of CS on SSC5, of COQ9 on SSC6 and of EGFR on SSC9, respectively, coinciding with QTL regions for carcass and meat quality traits. BVES, SLC3A2, and CS revealed association at least with drip loss and with several other measures of water holding capacity (WHC). Moreover, COQ9 and EGFR were associated with several meat quality traits such as meat color and/or thawing loss. This study reveals statistic evidence in addition to the functional relationship of these genes to WHC previously evidenced by expression analysis. This study reveals positional and genetic statistical evidence for a link of genetic variation at these loci or close to them and promotes those six candidate genes as functional and/or positional candidate genes for meat quality traits.

Microarray analysis reveals genes and functional networks relevant to the predisposition to inverted teats in pigs.

The inverted teat defect is characterized by the failure of teats to protrude from the udder surface and has a negative effect on the economic efficiency of pig production. The inverted teat defect is influenced by genetic factors, but the number and identity of relevant genes are unknown. In this study, we compared the mRNA expression of teat tissues from unaffected pigs and affected pigs by using microarrays. Simultaneously, 24,123 probe sets were screened, of which some 15,000 had present calls and were analyzed for differential expression between mesenchymal and epithelial tissue of 3 categories of teats (i.e., normal teats of unaffected and affected animals, and inverted teats of the latter). Differential expression was more pronounced in epithelial than in mesenchymal tissue, and the comparisons among the 3 categories of teats showed that local processes at the side of the affected area as well as processes taking place at the level of the organ contribute to the development of inverted teats. Genes related to biofunctions of cell maintenance, proliferation, differentiation, and replacement; organismal, organ, and tissue development; genetic information and nucleic acid processing; and cell-to-cell signaling and interaction were differentially expressed, depending on the teat phenotype and the status of the animal as affected or unaffected. In particular, genes encoding members of canonical pathways of growth factor signaling were highlighted. Complementary to previous real-time quantitative reverse-transcription PCR experiments showing upregulation of growth factors (epidermal growth factor, fibroblast growth factor, hepatocyte growth factor, platelet-derived growth factor, vascular endothelial growth factor) and their receptors in the inverted teat, here it is shown that the abundance of transcripts encoding subordinated proteins (acid phosphatase 1, soluble; activating transcription factor 2; casein kinase 2, α 1 polypeptide; casein kinase 2, α prime polypeptide; actinin, α 2; and Homo sapiens growth factor receptor-bound protein 2) within the growth factor signaling pathways are also affected. Tuning of the expression of genes of these pathways balances the differentiation and proliferation of epithelial and mesenchymal teat tissue and finally affects the shape and structure of the teats.

Association of PPARGC1A and CAPNS1 gene polymorphisms and expression with meat quality traits in pigs.

This study aimed to investigate the genes PPARGC1A (peroxisome proliferator-activated receptor gamma-coactivator 1A) and CAPNS1 (calpain small subunit 1) as candidate genes affecting meat quality traits in pigs. Four polymorphisms were identified in PPARCG1A and three in CAPNS1. The PPARGC1A polymorphism c.1288T>A was associated with pH and cooking loss in a F2 Duroc×Pietrain experimental cross (DuPi, n=313) and with pH values in Italian Large White (ILW, n=380) and Italian Landrace (ILA, n=158) populations (P<0.05). The CAPNS1 polymorphism c.429A>C was associated with pH and conductivity in DuPi and with meat color in ILA (P<0.05). PPARGC1A mRNA expression associated with drip loss (P<0.01) and the same tendency was found for CAPNS1 (P=0.06). The promoter methylation profiling suggested that methylation is not involved in CAPNS1 expression regulation. In conclusion, porcine PPARGC1A and CAPNS1 genes may affect meat quality traits, with breed-specific differences, and they could be used as markers for the improvement of meat quality in pigs.

Polymorphism and expression of the porcine Tenascin C gene associated with meat and carcass quality.

The research aimed to screen for polymorphism, expression of Tenascin C (TNC) and association with meat and carcass quality traits. Three single nucleotide polymorphisms were detected. In a Duroc×Pietrain F2 cross (DuPi) population, g.44488C>T was associated with meat color and ham weight; g.68794A>G was associated with pH at 24h post mortem in ham (pH24(H)) and muscle area but g.68841C>T was not statistically associated. Genotyping in a commercial Pietrain (Pi) population showed that g.44488C>T was associated with pH24(H), whereas g.68794A>G was associated with conductivity at 45 min post mortem in loin and backfat thickness. Diplotypes showed significant effects on pH24(H) in both populations. The expression was associated with pH at 45 min post mortem in loin and cooking loss. TNC was significantly higher in animals with higher muscle pH. Linkage analysis revealed four trans-regulated eQTL on four autosomes. These results suggest that TNC could be a potential candidate gene for meat quality traits in pigs.

Investigation on interferon alpha-inducible protein 6 (IFI6) gene as a candidate for meat and carcass quality in pig.

The aim of this research was to screen for polymorphism and to perform an association study of IFI6 with meat and carcass quality traits. A SNP (g.370A>G) was detected which was associated (P<0.05) with meat colour, pH 24h post mortem (p.m.) in ham, conductivity 45 min p.m. in loin and conductivity 24 h p.m. in ham, drip loss and carcass length in Duroc x Pietrain and with meat colour, muscle area and ham percentage in the Pietrain population. Highest expression of IFI6 mRNA was detected in skeletal muscle (longissimus dorsi) by qRT-PCR comparing different tissues. Both qRT-PCR and western blot revealed that the IFI6 gene and protein expressions were significantly (P<0.05) higher in skeletal muscle with low drip loss compared to that of high drip loss. IFI6 protein was localized in the myocytes membrane. Results suggested that IFI6 might play roles in meat and carcass quality and is a potential positional, physiological and functional candidate gene for improving meat quality traits in pigs.

Integrating expression profiling and whole-genome association for dissection of fat traits in a porcine model.

Traits related to fatness, important as economic factors in pork production, are associated with serious diseases in humans. Genetical genomics is a useful approach for studying the effects of genetic variation at the molecular level in biological systems. Here we applied a whole-genome association analysis to hepatic gene expression traits, focusing on transcripts with expression levels that correlated with fatness traits in a porcine model. A total of 150 crossbred pigs [Pietrain × (German Large White × German Landrace)] were studied for transcript levels in the liver. The 24K Affymetrix expression microarrays and 60K Illumina single nucleotide polymorphism (SNP) chips were used for genotyping. A total of 663 genes, whose expression significantly correlated with the trait "fat area," were analyzed for enrichment of functional annotation groups as defined in the Ingenuity Pathways Knowledge Base (IPKB). Genes involved in metabolism of various macromolecules and nutrients as well as functions related to dynamic cellular processes correlated with fatness traits. Regions affecting the transcription levels of these genes were mapped and revealed 4,727 expression quantitative trait loci (eQTL) at P < 10⁻5, including 448 cis-eQTL. In this study, genome-wide association analysis of trait-correlated expression was successfully used in a porcine model to display molecular networks and list genes relevant to fatness traits.

Association of ZYX polymorphisms with carcass and meat quality traits in commercial pigs.

Zyxin (ZYX) is one of the proteins in focal adhesions along the actin fibers playing a role in actin organization and signal transduction. By radiation hybrid and genetic mapping we assigned ZYX to porcine chromosome 18 in the area of quantitative traits loci for carcass and meat quality and muscle fiber traits and hence considered ZYX a functional positional candidate gene. Analysis of a newly detected SNPs (c.+279 C>T, c.+399 A>G, c.+522 A>G) in pigs from different commercial breeds (Pietrain [Pi], German Landrace [LR], German Large White x German Landrace [F1] and PiF1) revealed a significant association with carcass traits (including: side- and backfat thickness, loin weight and carcass lean content) and meat quality traits (including: pH, color and drip loss). However, the lack of consistent association across all pig populations in this study indicates that the association of the SNPs may be depending on causal mutations in linkage disequilibrium and/or interactions with other loci.

Expression quantitative trait loci analysis of genes in porcine muscle by quantitative real-time RT-PCR compared to microarray data.

Genetic analysis of transcriptional profiling is a promising approach for identifying biological pathways and dissecting the genetics of complex traits. Here, we report on expression quantitative trait loci (eQTL) that were estimated from the quantitative real-time RT-PCR data of 276 F(2) animals and compared with eQTL identified using 74 microarrays. In total, 13 genes were selected that showed trait-dependent expression in microarray experiments and exhibited 21 eQTL. Real-time RT-PCR and microarray data revealed seven cis eQTL in total, of which one was only detected by real-time RT-PCR, one was only detected by microarray analysis, three were consistently found in overlapping intervals and two were in neighbouring intervals on the same chromosome; whereas no trans eQTL was confirmed. We demonstrate that cis regulation is a stable characteristic of individual transcripts. Consequently, a global microarray eQTL analysis of a limited number of samples can be used for exploring functional and regulatory gene networks and scanning for cis eQTL, whereas the subsequent analysis of a subset of likely cis-regulated genes by real-time RT-PCR in a larger number of samples is relevant to narrow down a QTL region by targeting these positional candidate genes. In fact, when modelling SNPs of six genes as fixed effects in the eQTL analysis, eQTL peaks were shifted downwards, experimentally confirming the impact of the respective polymorphic genes, although these SNPs were not located in the regulatory sequence and these shifts occur as a result of linkage disequilibrium in the F(2) population.

Differential expression of growth factors and their receptors indicates their involvement in the inverted teat defect in pigs.

In this study 8 genes of growth factors and their receptors were investigated that are known to play a significant role in signaling pathways involved in the ontogenetic, but also tumorigenic, development of breast and mammary glands. Differential expression of fibroblast growth factor receptor 2 (FGFR2), GH receptor (GHR), hepatocyte growth factor (HGF), hepatocyte growth factor receptor (HGFR), platelet-derived growth factor alpha (PDGFA), platelet-derived growth factor receptor alpha (PDGFRA), platelet-derived growth factor beta (PDGFB), and vascular endothelial growth factor (VEGF) was analyzed in mesenchymal and epithelial teat tissue of peripubertal pigs affected and nonaffected by the inverted teat defect. Comparisons were made at the level where pigs were affected between samples derived from nonaffected animals and affected animals, including specimens of normal and inverted teats. In addition, comparisons were made at the level of the teat phenotype with normal teats of nonaffected animals vs. either the normal or the inverted teat of affected animals. All genes tested, except HGFR, showed significant differential expression at P < 0.05 in the mesenchymal or the epithelial teat tissue or both. In general, we observed more pronounced differences when comparing samples obtained from inverted tissues vs. samples from normal ones. Therefore, results of our study suggest that gene expression of the growth factors and their receptors associates directly with the teat phenotype rather than with the affection status of the investigated animals, suggesting that local processes and tissue-specific compensation by means of differential expression of growth factors and their receptors are responsible for the development of impaired teat phenotypes.

Association of parathyroid hormone-like hormone (PTHLH) and its receptor (PTHR1) with the number of functional and inverted teats in pigs.

Parathyroid hormone-like hormone gene (PTHLH) and its receptor, parathyroid hormone/ parathyroid hormone-like hormone receptor 1 (PTHR1), play a role in epithelial mesenchymal interactions during growth and differentiation of different tissues and anatomic structures, including teats. Therefore, PTHLH and PTHR1 were evaluated as functional candidate genes for their effects on number and shape of teats in pigs. In particular, focus was on the occurrence and number of inverted teats, the most frequent and economically relevant teat developmental defect in pigs. For this purpose, association and linkage of the PTHLH gene and the PTHR1 gene with inverted teat defect and the total number of teats and inverted teats were studied in an experimental Duroc and Berlin Miniature pig (DUMI) population. Polymorphism C1819T of PTHR1 was significantly associated with inverted teat phenotype (p = 0.014), total number of teats (p = 0.047) and was close to significance with the number of inverted teats (p = 0.078). Polymorphism C375T of PTHLH was close to significance with the inverted teat phenotype (p = 0.122) and showed no significant association with the total number of teats (p = 0.621) and the number of inverted teats (p = 0.256) in the DUMI population. Association analyses were also performed for combined effects of PTHLH and PTHR1 in order to address potential interaction, however, revealed no indication of effects of interaction. The function, position and the association shown here promote PTHR1 as a candidate gene for number of teats and in particular for affection by and number of inverted teats.