Transcriptomic profiling of mare endometrium at different stages of endometrosis.

In the current study, transcriptome profiles of mare endometrium, classified into categories I, IIA, and IIB according to Kenney and Doig, were compared using RNA sequencing, analyzed, and functionally annotated using in silico analysis. In the mild stage (IIA) of endometrosis compared to category I endometrium, differentially expressed genes (DEGs) were annotated to inflammation, abnormal metabolism, wound healing, and quantity of connective tissue. In the moderate stage (IIB) of endometrosis compared to category I endometrium, DEGs were annotated to inflammation, fibrosis, cellular homeostasis, mitochondrial dysfunction, and pregnancy disorders. Ingenuity pathway analysis (IPA) identified cytokines such as transforming growth factor (TGF)-ß1, interleukin (IL)-4, IL-13, and IL-17 as upstream regulators of DEGs associated with cellular homeostasis, metabolism, and fibrosis signaling pathways. In vitro studies showed the effect of these cytokines on DEGs such as ADAMTS1, -4, -5, -9, and HK2 in endometrial fibroblasts at different stages of endometrosis. The effect of cytokines on ADAMTS members' gene transcription in fibroblasts differs according to the severity of endometrosis. The identified transcriptomic changes associated with endometrosis suggest that inflammation and metabolic changes are features of mild and moderate stages of endometrosis. The changes of ADAMTS-1, -4, -5, -9, in fibrotic endometrium as well as in endometrial fibroblast in response to TGF-ß1, IL-4, IL-13, and IL-17 suggest the important role of these factors in the development of endometrosis.

Differential expression and methylation patterns of NFATC1, NADSYN1 and JAK3 gene in equine chondrocytes expanded in monolayer culture.

Ex vivo expansion of chondrocytes in monolayer (ML) culture for therapeutic purposes is burdened with difficulties related to the loss of cartilaginous phenotype. Epigenetic mechanisms responsible for regulation of gene expression are believed to underlie chondrocyte dedifferentiation. We have inspected the relevance of DNA methylation alterations for passage-related differential expression of NFATC1 gene involved in hard connective tissue turnover and development, NADSYN1 influencing redox metabolism, and JAK3 - an important driver of inflammation. We have assessed relative amount of transcript abundance and performed DNA bisulfite sequencing of upstream located elements. It seems that anabolic-like effects of chondrogenic differentiation were observed in form of NFATC1 and NADSYN1 upregulation in chondrocytes at the earlier stages of passaging whereas JAK3 upregulation at the 11th passage was the sign of chondrocytes dedifferentiation. Summarizing the inversely correlated DNA methylation and expression patterns in NFATC1 and JAK3 locus might be relevant for cellular dedifferentiation during chondrocyte expansion in monolayer. Obtained results are supportive for further studies on the role of encoded proteins in regenerative biology of articular cartilage using in vitro expanded chondrocytes.

Short communication: Locus-specific interrelations between gene expression and DNA methylation patterns in bovine mammary gland infected by coagulase-positive and coagulase-negative staphylococci.

Pathogens are able to alter the cell cycle program and immune response of the host by changing the transcription and epigenetics of genes responsible for cell cycle control and inflammation. In this regard, we evaluated interrelations between DNA methylation and expression of autophagy, apoptosis, and lipid metabolism-related genes in a sample set of mammary gland secretory tissue sections derived from bovine mammary glands infected with coagulase-negative and coagulase-positive staphylococci. We assessed relative transcript abundance and DNA bisulfite sequencing in loci of the ATG5, IGF1R, TERT, and DGAT1 genes. Lack of DNA methylation in ATG5 and DGAT1 loci might be associated with maintenance of ATG5 and DGAT1 expression regardless of the health status of bovine mammary gland. Complete methylation of intragenic CpG regions in the IGF1R locus was apparently not related to the presence of its transcript in the investigated udder parenchyma samples. Detected hypermethylation of the TERT upstream element was associated with a small amount of TERT mRNA in bovine mammary gland, regardless of the presence, or absence, of the pathogen. A significant decrease in TERT gene expression in tissue sections of mammary gland free of bacteria and in those infected with coagulase-positive staphylococci was observed in parenchyma samples infected with coagulase-negative staphylococci. Two possible explanations are the direct involvement of the TERT gene in the etiology of bovine mastitis or the increase of TERT mRNA due to activation of the MAPK signaling pathway in response to release of exotoxins by coagulase-negative bacteria in the bovine mammary gland.

Structural and functional analysis of the signaling lymphocytic activation molecule family 7 (SLAMF7) gene in response to infection with coagulase-negative and coagulase-positive staphylococci.

Splice variants of the signaling lymphocytic activation molecule family 7 (SLAMF7) gene have been identified, and differences in the expression of this gene have been demonstrated at the mRNA level in the mammary glands of healthy and mastitis-infected dairy cows. At the same time, significant associations have been found between a deletion in the SLAM7 gene exon, the occurrence of different splice variants, and the occurrence of mastitis in one group of dairy cows. An expression study was conducted on 40 Polish Holstein-Friesian dairy cows of the Black and White variety (group I). Milk samples were taken for microbiological analysis 2 d before slaughter and examined for the presence of bacteria. Immediately after slaughter, mammary tissue samples were taken and divided into 3 groups according to the health status of the mammary gland: healthy (without pathogenic bacteria in milk), coagulase-negative staphylococci (CNS), and coagulase-positive staphylococci (CPS). Based on different SLAMF7 gene DNA fragments, 2 alternative variants of this gene (V1 and V2) and complete gene expression were identified. Separate analyses performed for each isoform showed that the health status of the cow was strongly associated with the expression level of individual variants. The highest expression was detected for the SLAMF7 complete amplicon in healthy cows, and in the CNS and CPS cows the expression of this variant was also higher than V1 and V2. Sanger sequencing was applied to detect the polymorphism/indel variant in the second exon of the SLAMF7 gene probably having the greatest effect on the protein structure and function of SLAMF7. Two genotypes were detected: AA (wild-type) and AB (insertion A). In healthy cows, the frequency of homozygotes AA was higher than the heterozygotes, whereas in the infected animals, the genotypic distribution was the opposite. An association analysis between the identified polymorphism and production traits-including somatic cell count, as well as lactose, protein, and casein content and yield as indicators of subclinical mastitis occurrence-was performed on the group II cows (166 Polish Holstein-Friesian dairy cows). Unfortunately, due to the low number of AB animals, no relationship was demonstrated between genotype in the second exon and the health status of cows. Additionally, the difference in the percentage of SLAMF7-targeted DNA methylation between the groups of animals was not significant, with an average of ∼66 to 68%.

Mouse testicular transcriptome after modulation of non-canonical oestrogen receptor activity.

The aims of this study were to shed light on the role of G-protein-coupled membrane oestrogen receptor (GPER) and oestrogen-related receptor (ERR) in mouse testis function at the gene expression level, as well as the involvement of GPER and ERR in cellular and molecular processes. Male mice were injected (50µg kg-1,s.c.) with the GPER antagonist G-15, the ERRα inverse agonist XCT790 or the ERRß/ERRγ agonist DY131. Next-generation sequencing (RNA-Seq) was used to evaluate gene expression. Bioinformatic analysis of read abundance revealed that 50, 86 and 171 transcripts were differentially expressed in the G-15-, XCT790- and DY131-treated groups respectively compared with the control group. Annotated genes and their protein products were categorised regarding their associated biological processes and molecular functions. In the XCT790-treated group, genes involved in immunological processes were upregulated. In the DY131-treated group, genes with increased expression were primarily engaged in protein modification (protein folding and small protein conjugation). In addition, the expression of genes recognised as oncogenes, such as BMI1 proto-oncogene, polycomb ring finger (Bmi1) and nucleophosphin 1 (Npm1), was significantly increased in all experimental groups. This study provides detailed information regarding the genetic changes in the testicular transcriptome of the mouse in response to modulation of non-canonical oestrogen receptor activity.

Methylation Marks of Blood Leukocytes of Native Hucul Mares Differentiated in Age.

Horses are one of the longest-living species of farm animals. Advanced age is often associated with a decrease in body condition, dysfunction of immune system, and late-onset disorders. Due to this, the search for new solutions in the prevention and treatment of pathological conditions of the advanced age of horses is desirable. That is why the identification of aging-related changes in the horse genome is interesting in this respect. In the recent years, the research on aging includes studies of age-related epigenetic effects observed on the DNA methylation level. We applied reduced representation bisulfite sequencing (RRBS) to uncover a range of age DMR sites in genomes of blood leukocytes derived from juvenile and aged horses of native Hucul breed. Genes colocated with age-related differentially methylated regions (age DMRs) are the members of pathways involved in cellular signal transduction, immune response, neurogenesis, differentiation, development, and cancer progression. A positive correlation was found between methylation states and gene expression in particular loci from our data set. Some of described age DMR-linked genes were also reported elsewhere. Obtained results contribute to the knowledge about the molecular basis of aging of equine blood cells.

Chondrogenic expression and DNA methylation patterns in prolonged passages of chondrocyte cell lines of the horse.

We investigated the activity of chondrogenic markers and variation of methylation patterns in equine cartilaginous cells cultivated in monolayer. The transcriptional and epigenetic effect of the long-term culture of chondrocytes has been evaluated using several passages of chondrocyte cell-lines derived from equine articular cartilage. Using 3 genes as endogenous control we tested the expression of 7 genes important for different stages of chondrocyte differentiation and maturation. CpG islands in RUNX3 locus were inspected for the evaluation of differential methylation state of passaged cell-lines. The general decline of transcript abundance of marker loci was detected in passage 11 which is the sign of dedifferentiation of cultivated chondrocytes in prolonged monolayer culture. Passages 13 and 14 were characterized by the upregulation of a number of genes, possibly due to the heterogeneity of developed cell lines at this stage of the culture. Instead, gradual increase of methylation percent at particular CpG sites of RUNX3 locus was associated with the growing number of passage. This finding led us to the conclusion that epigenetic alterations better describe the stage of cultivated chondrocytes.

Characterisation of genome-wide structural aberrations in canine mammary tumours using single nucleotide polymorphism (SNP) genotyping assay.

Characterisation of copy number variation (CNV) and loss of heterozygosity (LOH) has pro- vided evidence for the relationship of this type of genetic variation with the occurrence of a broad spectrum of diseases, including cancer lesions. The role of CNVs and germinal or somatic LOHs in canine mammary tumours is still unknown. Therefore, the aim of this study was to identify CNVs and LOHs in canine mammary tumours. Forty-eight samples obtained from normal (n=24) and tumour (n=24) tissues of dogs were analysed. In the study, we used CanineHD BeadChip assay (Illumina) and OncoSNP software to identify copy number alternations in genomes of dif- ferent dog breeds and in different mammary cancer types occurring in this species. The analyses revealed that, in the case of CNV, the amplification-type variants were longer and more frequent than deletions. Based on the analysis of the frequency of different types of aberrations in the in- dividual parts of the genome, regions that are particularly susceptible to structural aberrations were indicated. The fraction of genes identified within these regions was associated with major processes of neoplastic transformation. Association analysis of such traits as tumour grading as well as the size and age of dogs demonstrated that structural aberrations were more frequent in dogs diagnosed with tumour malignancy grade II and III, in dogs with a larger body size, and in large dogs aged 7-8. The promising results of these pioneering investigations prompt continuation thereof to analyse other types of cancer.

Diversifying selection signatures among divergently selected subpopulations of Polish Red cattle.

Polish Red cattle is one of the few indigenous breeds of European red cattle which is characterized by several desired features, such as high disease resistance, good health, longevity, good fertility, and high nutritional value of milk. Currently, Polish Red cattle population is a subject of two independent breeding programs: (i) improvement program and (ii) genetic resources conservation program. The aim of the improvement program is the genetic progress in terms of milk production and body conformation traits, while the conservation program mainly focuses on protection of the genetic resources of Polish Red cattle and preservation of the existing, original gene pool. By the analysis of FST genetic distances across genome-wide SNP panel, we detected diversifying selection signatures among these two subpopulations and indicated (among others) the significance of DGAT1 and FGF2 genes for milk production traits in these cattle. We also found that among genes being presumably under selection in terms of milk production, there are genes responsible, for example, for mammary gland development (e.g., SOSTDC1, PYGO2, MED1, and CCND1) and immune system response (e.g., IL10RA, IL12B, and IL21). The most important finding of this study is that the most pronounced genetic differences between the analyzed populations were associated with ß-defensin genes (e.g., DEFB1, DEFB4A, DEFB5, DEFB7, DEFB10, DEFB13, EBD, BNBD-6, and LAP) located within so-called bovine cluster D on BTA27. The ß-defensins are expressed mainly in the mammary gland and are antimicrobial peptides against the Gram-negative and Gram-positive bacteria, viruses, and other unicellular parasites. This suggests that antimicrobial resistance of mammary gland is of high importance during selection towards increased milk production and that genes responsible for this process are selected together with increasing levels of productivity.

Sequence analysis and expression profiling of the equine ACTN3 gene during exercise in Arabian horses.

The ACTN3 gene codes for α-actinin-3, a protein localized in the Z-line in the skeletal muscle. Actinin-3 is critical in anchoring the myofibrillar actin filaments and plays a key role in muscle contraction. ACTN3 (α-actinin-3) cross-links glycogen phosphorylase (GP), which is the key enzyme catalysing glycogen metabolism. The aim of present study was to establish the expression level of the ACTN3 gene (for both isoforms separately and together in the gene expression analysis) in the gluteus medius muscle in order to verify if the α-actinin-3 gene can be related to training intensity in Arabian horses. A structural analysis of the ACTN3 gene was performed simultaneously to identify polymorphisms that can be related to racing performance traits. Our results showed the significant decrease (p < 0.05) of ACTN3 expression in the skeletal muscle of Arabian horses during the training periods preparing for flat-racing, and this decrease differed by the intensity of the exercises. The highest mRNA abundance measured for all ACTN3 genes was detected in the muscle of untrained horses, while the lowest expression was identified at the end of the racing season when horses had fully adapted to the physical effort. This gene expression profile was confirmed for both ACTN3 isoforms. The analysis of the ACTN3 sequence allowed us to identify 14 polymorphisms, which were localized in the promoter region, the 5'UTR (7 SNPs), exons (2 SNPs) and introns (5 SNPs). Two of them, a novel c.2334C>T - splice variant and the g.1104G>A polymorphism in the promoter region, were proposed as the causative mutations that might affect gene expression. The presented gene expression analyses indicated the significant role of the ACTN3 gene in adaptation to physiological effort in horses. Due to previous reports and our findings, further studies should be conducted to verify the usage of the ACTN3 gene as a potential genetic marker for determining exercise performance in Arabian horses and other horse breeds.

Molecular characterization of the apoptosis-related SH3RF1 and SH3RF2 genes and their association with exercise performance in Arabian horses.

BACKGROUND: Apoptosis plays an important role in the regulation of healthy tissue growth and development as well as in controlling the maintenance of homeostasis in exercising muscles. During an intensive physical effort, the regulation of cell death by apoptosis results in the replacement of unaccustomed muscle cells by new cells that are better suited to exercise. The aim of this study was to determine the expression of two genes (SH3FR1 and SH3RF2) that control apoptosis in muscle tissues during training periods characterized by different intensities. The gene expression levels were estimated using real-time PCR method in skeletal muscle biopsies collected from 15 Arabian horses (untrained, after an intense gallop phase, and at the end of the racing season). An association study was performed on 250 Arabian horses to assess the effect of the SH3RF2:c.796 T > C (p.Ser266Pro) variant on race performance traits in flat gallop-racing. RESULTS: A gene expression analysis confirmed a significant decrease (p < 0.01) in the anti-apoptotic SH3RF2 (POSHER) gene during training periods that differed in intensity. The highest SH3RF2 expression level was detected in the muscles of untrained horses, whereas the lowest expression was identified at the end of the racing season in horses that were fully adapted to the exercise. A non-significant decrease in SH3RF1 gene expression following the training periods was observed. Moreover, a serine substitution by proline at amino acid position 266 (CC genotype) was negatively associated with the probability of winning races, the number of races in which a horse occurred and the financial value of the prizes. Horses with the TT genotype achieved the highest financial benefits, both for total winnings and for winnings per race in which the horses participated. CONCLUSIONS: The present study showed the supposed regulation mechanism of exercise-induced apoptosis in horses at the molecular level. The identified SH3RF2: c.796 T > C missense variant was associated with selected racing performance traits, which is important information during the evaluation of horses' exercise predisposition. The association results and frequencies of the CT and TT genotypes suggest the possibility of using SH3RF2 variant in selection to improve the racing performance of Arabian horses.

Comparative analysis of DNA methylation patterns of equine sarcoid and healthy skin samples.

OBJECTIVE: In this study, for the first time we report the genome-wide DNA methylation profile of skin tumour in horses and describe differentially methylated genomic regions (DMRs) with respect to healthy skin. MATERIALS & METHODS: The comparative analysis of DNA methylation patterns detected using Reduced Representation Bisulfite Sequencing (RRBS) technique, allowed identification of 136 regions showing differential methylation between sarcoid and normal skin tissue. RESULTS: Most of the identified DMRs were short fragments, less than 1 kb in size, located in the intergenic regions. Among identified DMRs there were also regions located within genes directly or indirectly related with oncogenesis. We additionally validated 9 CpG sites showing hypomethylation and 9 CpG sites that were hypermethylated in lesional sample, confirming the identified changes in the DNA methylation. CONCLUSION: Knowledge on the changes taking place in the process of DNA methylation may provide a basis for the development of new alternative diagnostic or therapeutic approaches to equine sarcoids.

The use of runs of homozygosity for estimation of recent inbreeding in Holstein cattle.

Controlling inbreeding in livestock populations is of great importance because excess relatedness among animals leads to a rapid loss of genetic variation and to adverse phenotypical effects associated with an inbreeding depression. Recent advances in genotyping technology have made it possible to study inbreeding at a molecular level by the analysis of genome-wide single nucleotide polymorphism panels. In this study, we used BovineSNP50 assay (Illumina) to estimate genomic inbreeding coefficient in 298 Holstein cattle by the analysis of the genome portion in runs of homozygosity (FROH) or using genomic relationship matrix (FGRM), and compared this data with conventional pedigree-based inbreeding coefficients (FPED). Weak or moderate Spearman's rank correlations were observed between FROH and FPED which depended on the ROH length categories used for calculations and inclusion of animals with different number of complete generations registered in pedigrees. The highest correlations were observed when using ROH with lengths over 8 Mb (0.334). The correlations tended to increase as pedigree depth increased, and were the highest for animals with seven complete generations of pedigree data. FGRM correlated poorly with pedigree-based estimates, which suggests that ROH-based inbreeding coefficients better reflect recent relatedness among animals.

Age-related methylation profiles of equine blood leukocytes in the RNASEL locus.

Methylation profiles across three CpG islands of the RNASEL gene were determined in blood leukocyte samples of Anglo-Arabian and Hucul horses. Bisulfite sequencing revealed hypomethylated state of the RNASEL promoter coinciding with methylated CpG island placed inside the gene. Several CpG sites were identified for which the methylation state was influenced by DNA polymorphism. Two of them showed monoallelic methylation. One of the CpG sites revealed functional polymorphism. A number of partially methylated CpG sites have been observed in the promoter area of RNASEL, which were used for the comparison of breed- and age-related effects. Clone bisulfite sequencing of blood leukocyte samples collected at different ages from particular individuals of AA and HC breeds and, also, BSPCR sequencing of 50 samples of juvenile and old AA and HC horses revealed increased methylation in particular CpG sites during aging. The age-related heterogeneity of white blood cells was hypothesized as being one of the potential causes of observed variability of methylation profiles in the RNASEL promoter.

Identification of genome-wide selection signatures in the Limousin beef cattle breed.

The study is aimed at identifying selection footprints within the genome of Limousin cattle. With the use of Extended Haplotype Homozygosity test, supplemented with correction for variation in recombination rates across the genome, we created map of selection footprints and detected 173 significant (p < 0.01) core haplotypes being potentially under positive selection. Within these regions, a number of candidate genes associated inter alia with skeletal muscle growth (GDF15, BMP7, BMP4 and TGFB3) or postmortem proteolysis and meat maturation (CAPN1 and CAPN5) were annotated. Noticeable clusters of selection footprints were detected on chromosomes 1, 4, 8 and 14, which are known to carry several quantitative trait loci for growth traits and meat quality. The study provides information about the genes and metabolic pathways potentially modified under the influence of directional selection, aimed at improving beef production characteristics in Limousin cattle.

Genome-wide characteristics of copy number variation in Polish Holstein and Polish Red cattle using SNP genotyping assay.

Copy number variation (CNV), which results from deletions or amplifications of large fragments of genomic DNA, is widespread in mammalian genomes and apart from its potential pathogenic effect it is considered as a source of natural genetic diversity. In cattle populations, this kind of genetic variability remains still insufficiently elucidated and studies focusing on the detection of new structural genomic variants in different cattle populations may contribute to a better understanding of cattle breeds' diversity and genetic basis of production traits. In this study, by using BovineSNP50 assay and cnvPartition algorithm we identified CNVs in two different cattle breeds: Holstein (859 animals) and Polish Red (301). In Holstein cattle we found 648 CNVs which could be reduced to 91 non-redundant variable genomic regions (CNVRs) covering in total 168.6 Mb of the genomic sequence. In Polish Red cattle we detected 62 CNVs, localized in 37 variable regions encompassing 22.3 Mb of the sequence, corresponding to 0.89 % of the autosomal genome. Within the regions we identified 1,192 unique RefSeq genes which are engaged in a variety of biological processes. High concordance of the regions' distribution was found between the studied breeds, however copy number variants seemed to be more common in Holstein cattle. About 26 % of the regions described in this study could be classified as newly identified. The results of this study will broaden the knowledge of CNVs in genomes of cattle of different breeds and will provide foundations for further research aiming to identify a relationship between this type of genetic variation and phenotypic traits.

Association of calpastatin gene polymorphisms and meat quality traits in pig.

Calpastatin is associated with the rate of post mortem degradation of structural proteins due to the regulation of calpain activity. In the present research, the associations between polymorphisms within 6th intron of porcine CAST gene and several meat quality traits were analyzed. The CAST gene polymorphisms affected meat colour, pH, water holding-capacity (WHC) and texture parameters (toughness, firmness, cohesiveness, chewiness, and resilience) measured in longissimus dorsi and semimembranosus muscles. The analysis performed on the most numerous breeds maintained in Poland, suggested that the most interesting polymorphisms were CAST/HpaII and CAST/RsaI, which had the greatest effect on WHC regardless of the breed analyzed and had an effect on meat pH, firmness and toughness for most breeds. Interestingly, for almost all breeds, the significant effect of both mutations on intramuscular fat content (IMF) was detected. The provided data confirmed the use of CAST gene as a genetic marker in breeding programmes which allows performing a selection focussed on improving the quality of pork.