Comparative study of equine bone marrow and adipose tissue-derived mesenchymal stromal cells.

REASONS FOR PERFORMING STUDY: Mesenchymal stromal cells (MSCs) represent an attractive source for regenerative medicine. However, prior to their application, fundamental questions regarding molecular characterisation, growth and differentiation of MSCs must be resolved. OBJECTIVES: To compare and better understand the behaviour of equine MSCs obtained from bone marrow (BM) and adipose tissue (AT) in culture. METHODS: Five horses were included in this study. Proliferation rate was measured using MTT assay and cell viability; apoptosis, necrosis and late apoptosis and necrosis were evaluated by flow cytometry. The mRNA expression levels of 7 surface marker genes were quantified using RT-qPCR and CD90 was also analysed by flow cytometry. Differentiation was evaluated using specific staining, measurement of alkaline phosphatase activity and analysis of the mRNA expression. RESULTS: High interindividual differences were observed in proliferation in both cell types, particularly during the final days. Statistically significant differences in viability and early apoptosis of cultured AT- and BM-MSCs were found. The highest values of early apoptosis were observed during the first days of culture, while the highest percentage of necrosis and late apoptosis and lowest viability was observed in the last days. Surface marker expression pattern observed is in accordance to other studies in horse and other species. Osteogenic differentiation was evident after 7 days, with an increasing of ALP activity and mRNA expression of osteogenic markers. Adipogenic differentiation was achieved in BM-MSCs from 2 donors with one of the 16 media tested. Chondrogenic differentiation was also observed. CONCLUSIONS: Proliferation ability is different in AT-MSCs and BM-MSCs. Differences in viability and early apoptosis were observed between both sources and CD34 was only found in AT-MSCs. Differences in their osteogenic and adipogenic potential were detected by staining and quantification of specific tissue markers. POTENTIAL RELEVANCE: To provide data to better understand AT-MSCs and BM-MSCs behaviour in vitro.

A single nucleotide polymorphism in the coding region of bovine transferrin is associated with milk fat yield.

The bovine transferrin gene (TF) is located at 125 cM on bovine chromosome 1 (BTA1); it codes for transferrin, a glycoprotein that is highly conserved in many species and that is responsible for iron transport. The TF gene has been located in several QTL regions, and some transferrin classes have been associated with fat and milk yields. We analyzed by means of allele-specific oligonucleotide real-time PCR the c.1455A>G SNP in exon 12 of the TF cDNA sequence (accession number U02564), which induces an Asp/Gly substitution at position 469 of the peptide. The c.1455A>G SNP was assayed in eight Spanish cattle breeds, as well as in two groups of Holstein-Friesian animals that had the highest and lowest estimated breeding values for milk fat yield. Analysis of the cSNP showed balanced frequencies in all breeds, with a mean of 0.44. Evaluation of a potential association between the cSNP and the groups of Holstein-Friesian animals selected for milk fat yield showed a significant association (P < 0.0006); the G allele was associated with high fat production. Significant differences in genotypic frequencies between the groups were also detected (P < 0.0028). These results lead us to suggest that the TF gene has an effect on milk fat yield.

The bovine annexin 9 gene (ANXA9) is significantly associated with milk-fat yield in a Spanish Holstein-Friesian population.

On the basis of QTL studies for milk-fat yield trait on BTA3, annexin 9 protein (ANXA9), fatty acid transport protein type 3 (SLC27A3) and diacylglycerol O-acyltransferase 1 (DGAT1) were selected as candidate genes. Three different single nucleotide polymorphisms (SNPs) of bovine ANXA9, SLC27A3 and DGAT1 genes have been tested in a selective genotyping design for milk-fat yield. Significant allele frequency differences were found for ANXA9 (p=0.02), in Holstein-Friesian animals with high and low breeding values for milk-fat yield. Regression analysis also showed a significant effect (p=0.0207) between estimated breeding values (EBVs) for fat milk content and ANXA9 polymorphism. So ANXA9 gene falls into a significant quantitative trait loci interval for milk-fat yield that was previously reported on bovine chromosome 3 in other dairy populations. Our results suggest that the ANXA9 gene polymorphism or a linked segregating QTL contributes to variation in milk-fat yield.

Células madre satélite musculares en el modelo de ELA murino hSOD-1G93A / No disponible

No disponible

Structural and functional characterization of the bovine solute carrier family 27 member 1 (SLC27A1) gene.

The Solute Carrier Family 27 Member 1 (SLC27A1) is an evolutionarily conserved protein involved in regulating the long chain fatty acid uptake into cells. It has been shown to be expressed in tissues undergoing rapid fatty acid metabolism such as heart, skeletal muscle and adipose tissues, but no expression is detected in liver. Here we report the molecular characterization of the bovine SLC27A1 gene and draw a comparison with orthologous genes of some monogastric species. The bovine SLC27A1 gene is organized in 13 exons and extends over more than 40 kb of genomic DNA. It codes for a protein of 646 amino acids with a predicted molecular weight of 71 kDa which has 92%, 88% and 88% similarity with the human, mouse and rat SLC27A1 proteins respectively. The bovine SLC27A1 RNA expression was high in heart, testis, nervous tissue and muscle and very low in liver. Surprisingly, adipose tissues showed very low RNA expression levels contrary to the results described for both human and mouse genes. On the other hand, discordances observed between the bovine SLC27A1 RNA and protein expression patterns suggest that complex regulation mechanisms may be involved in determining the final SLC27A1 protein levels in each tissue. Finally, we have identified an alternative transcript generated by exon skipping of exon 3 to 7 which could encode a cytosolic SLC27A1 isoform of approximately 37 kDa.

Association of polymorphisms in the bovine FASN gene with milk-fat content.

Fatty acid synthase (FASN) is a multifunctional protein that carries out the synthesis of fatty acids so it plays a central role in de novo lipogenesis in mammals. Previously, we defined the genetic structure and expression of the bovine FASN gene. Our mapping studies placed FASN on BTA19 (19q22) where several quantitative trait loci (QTL) affecting milk-fat content and related traits have been described. This study was conducted to identify polymorphisms in the bovine FASN gene and to study their association with milk-fat content. The bovine FASN gene was screened for polymorphisms in two cattle breeds. Sequence analysis revealed several single nucleotide polymorphisms (SNPs), and two of them were analysed: a G>C substitution in the untranslated exon 1 (g.763G>C), altering a potential Sp1 transcription factor-binding site, and an A>G substitution in exon 34 (g.16009A>G), which determines a non-conservative substitution of threonine by alanine. Allele-specific amplification of the SNPs in FASN revealed significant frequency differences for both polymorphisms in Holsteins with high and low breeding values for milk-fat content. The intragenic haplotypes comprising exon 1 (alleles G and C) and exon 34 (alleles A and G) polymorphisms were studied, and the existence of linkage disequilibrium between these SNPs was found (D(CG) = 0.048, P < 0.001). Our results suggest that the FASN gene polymorphisms contribute to variation in milk-fat content. We propose that the bovine FASN gene is a candidate gene for a milk-fat content QTL.

Genomic structure and an alternative transcript of bovine mitochondrial glycerol-3-phosphate acyltransferase gene (GPAM).

GPAM maps in BTA26q22, where several QTLs affecting milk production, milk fat and protein content have been mapped. On the basis of the QTL location, the GPAM gene could be considered a good candidate gene for the mentioned traits. Glycerol-3-phosphate acyltransferase mitochondrial (GPAM) is the enzyme that catalyses the initial and committed step of glycerolipid synthesis and, therefore, it is a potential site for triacylglycerol synthesis regulation. In this study, the structure of the cDNA and the genomic DNA of the bovine GPAM gene were determined and the expression of its mRNA was studied. The cDNA of the gene was cloned by RT-PCR, 5' and 3' rapid amplification of cDNA ends. The GPAM mRNA sequence contains a 2,475-bp coding region and a 3,689-bp 3' UTR. Its ORF encoded for an 825-amino acid protein and has an 89% homology with the coding regions of previously characterized mouse and human GPAM genes. The predicted amino acid sequence had an 89 and 93% similarity with mouse and human GPAM proteins, respectively. Using a 5' RACE strategy, two different 5' UTRs were cloned. Northern blot analysis confirmed the presence of two different transcripts. Adipose tissues and lung had the highest levels of GPAM mRNA expression, whereas it was barely detectable in liver. This expression pattern differs with those of non-ruminant animals where liver is one of the tissues with higher GPAM mRNA expression level.