Comparative proteomic analysis of Tibetan pig spermatozoa at high and low altitudes.

BACKGROUND: To illuminate the mechanisms underlying the high-altitude tolerance of Tibetan pig spermatozoa, proteomes of spermatozoa from Tibetan pigs raised in high and low altitudes were compared using a tandem mass tag (TMT)-labeled quantitative proteomics approach. RESULTS: A total of 77 differentially expressed proteins (DEPs) were identified. Gene Ontology (GO) analysis revealed DEPs that were predominantly associated with the actin cytoskeleton, the tricarboxylic acid (TCA) cycle, and adenosine triphosphate (ATP) metabolism, and were from 12 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Three subnetworks were significantly enriched and 10 centric proteins were identified by protein-protein interaction (PPI) network analysis. Relative expression levels of the proteins (ATP5H, CYCS, MYH9 and FN1) were confirmed using Western blotting. CONCLUSIONS: Our study is the first to use a tandem mass tag (TMT) approach to analyze Tibetan pig spermatozoa, and provides a foundation to understand the mechanisms underlying the reproductive adaptations of Tibetan pigs to high-altitude environments.

Correction to: Comparative proteomic analysis of Tibetan pig spermatozoa at high and low altitudes.

Following publication of the original article [1], the authors reported an error in one of the authors' names. In this Correction the incorrect and correct author name are shown. The original publication of this article has been corrected.

The association of NR1H3 gene with lipid deposition in the pig.

BACKGROUND: Nuclear receptor subfamily 1, group H, member 3 (NR1H3, an alias for Liver X receptor α, LXRα) is a member of the LXR nuclear receptor super family and is an important regulator of lipid and fatty acid accumulation in the liver, adipose and skeletal muscle. METHODS: In this study, single-nucleotide polymorphisms (SNPs) from six populations of pig (Sus scrofa) were screened by PCR-sequencing and genotyped, and its association with backfat thickness was analyzed in a population of New Huai line (NHP, n = 117). In addition, quantitative real-time PCR and western blot were used to measure expression of NR1H3 in the liver tissue, backfat and longissimus dorsi muscle of DSP (n = 10), TP (n = 10) and YY (n = 10) pigs. RESULTS: Three SNPs (exon2-C105T, exon2-G106C, and exon5-A201C) were screened and exon5-A201C was identified; the genotype frequencies were significantly different between indigenous and introduced breeds. The CC genotype was associated with higher backfat thickness than the AA and AC genotypes in the NYP. NR1H3 mRNA and protein expression were higher in the liver and longissimus dorsi of DSP and TP than in those of YY. This increased NR1H3 expression might be associated with higher lipid deposition. NR1H3 expression in the backfat of YY was not lower than that in DSP or TP, which might because NR1H3 has an alternative regulatory function for lipid metabolism in the subcutaneous fat of pigs. CONCLUSIONS: Our results suggest that allele A of the exon5-A201C in NR1H3 may promote a reduction in backfat thickness, and differences in NR1H3 expression may be associated with differences in lipid deposition capacity among pigs.

Effect of a single nucleotide polymorphism in the growth hormone secretagogue receptor (GHSR) gene on growth rate in pigs.

The growth hormone secretagogue receptor (GHSR) gene controls growth hormone (GH) release by inducing a strong stimulatory effect on the endogenous ligand, ghrelin. In this study, we examined the possible role of GHSR in the growth traits of four pig breeds, namely Tibetan pigs (n=45), Diannan small-eared pigs (n=40), Yorkshire pigs (n=45), and New Huai pigs (n=122). Single nucleotide polymorphisms (SNPs) in these pigs were identified by polymerase chain reaction (PCR) sequencing and genotyping was performed using PCR-restriction fragment length polymorphisms (PCR-RFLPs). A SNP (C/A) named C-1595A (the "C" allele), which is located 1595bp upstream of the initiation codon of the GHSR gene, was found at a higher frequency in the fast-growing Yorkshire pigs than in the slow-growing Tibetan and Diannan small-eared pigs. In preliminary assays, the C-1595A genotype was found to be associated with growth traits in New Huai pigs. Quantitative real-time PCR and western blotting assays were used to measure the levels of GHSR1a, a functionally active form of the GHSR protein, in the tissues of the growth axis. The estimated levels of mRNA and protein in pituitary and liver tissues were significantly higher in Yorkshire pigs than in Diannan small-eared or Tibetan pigs (P<0.05). The results indicated that GHSR had a positive influence on the growth rate of pigs and suggested that the C-1595A SNP could be of value as a molecular marker for improving the production performance of pig breeds.

Gene expression of vascular endothelial growth factor A and hypoxic adaptation in Tibetan pig.

BACKGROUND: Vascular endothelial growth factor A (VEGFA) can induce endothelial cell proliferation, promote cell migration, and inhibit apoptosis. These processes play key roles in physiological blood vessel formation and pathological angiogenesis. METHODS: In this study, we examined VEGFA gene expression in the heart, liver, and kidney of Tibetan pigs (TP), Yorkshire pigs that migrated to high altitudes (YH), and Yorkshire pigs that lived at low altitudes (YL). We used PCR and Sanger sequencing to screen for single nucleotide polymorphisms (SNPs) in 5'-flanking DNA and exons of the VEGFA gene. Quantitative real-time PCR and western blots were used to measure expression levels and PCR products were sequenced. RESULTS: Results showed that the VEGFA mRNA and protein expression in heart, liver and kidney of TP was higher than that in YH and YL. In addition, the mRNA sequence of the pig VEGFA gene was conserved among pig breeds, and only five SNPs were found in the 5'-flanking region of the VEGFA gene, the allele frequency distributions of the 5 SNPs were not significantly different between the TP, Yorkshire (YL), and Diannan small-ear (DN) pig populations. CONCLUSION: In conclusion, the Tibetan pig showed high levels of VEGFA gene expression in several hypoxic tissues, which suggests that the VEGFA gene may play a major functional role in hypoxic adaptation.

Genetic variation within exon 2 of the MHC B-LB // gene in Tibetan chicken.

Genetic variation within exon 2 of chicken major histocompatibility complex B-LB // genes was investigated by PCR amplification, cloning and sequencing of a 374 bp fragment of the indigenous Tibetan chicken genomic DNA. Fifteen novel B-LB // alleles were found. Alignment and comparison of 18 allelic sequences from the individuals sampled revealed a total of 62 variable sites (total of 80 mutations) in exon 2, of which 41 were parsimony informative sites. The nucleotide diversity (pi) within the sequence of exon 2 was calculated to be 0.0718. Analysis of nucleotide variation confirmed a lower level of divergence (0.056 +/- 0.008) as estimated by average pairwise distance within the Tibetan chicken population than the five exotic breeds detected. The relative frequencies of synonymous and non-synonymous nucleotide substitutions within the region were 3.25 +/- 0.94% and 15.61 +/- 2.69% , respectively. These results indicated that the genetic variation within exon 2 seemed to have arisen largely by gene recombination and balancing selection. Alignment of the deduced amino acid sequences of beta1 domain coded by exon 2 revealed 11 synonymous mutations and 27 non-synonymous substitutions at the 38 separate sites. Fifty percent (12/24) of the proposed peptide-binding sites were variable within beta1 domain of chicken MHC B-LB // molecules, of which 11 were unique non-synonymous amino acid substitutions. These particular non-synonymous substitutions are considered to be associated with immunological specificity of MHC B-LB // molecule in Tibetan chicken, and they can provide a molecular biological basis for the study of disease resistance in chicken.

A Comprehensive MicroRNA Expression Profile Related to Hypoxia Adaptation in the Tibetan Pig.

Tibetan pigs live between 2500 and 4300 m above sea level on the Tibetan Plateau, and are better adapted to hypoxia than lowland pigs. MicroRNAs (miRNAs) are involved in a wide variety of cellular processes; however, their regulatory role in hypoxia adaptation remains unclear. In this study, miRNA-seq was used to identify differentially expressed miRNAs (DE miRNAs) in the cardiac muscle of Tibetan and Yorkshire pigs, which were both raised in high elevation environments. We obtained 108 M clean reads and 372 unique miRNAs, which included 210 known porcine miRNAs, 136 conserved in other mammals, and 26 novel pre-miRNAs. In addition, 20 DE miRNAs, including 10 up-regulated and 10 down-regulated miRNAs, were also found after comparison between Tibetan and Yorkshire pigs. We predicted miRNA targets based on differential expression and abundance in the two populations. Furthermore, the results of a Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that DE miRNAs in Tibetan and Yorkshire pigs are involved in hypoxia-related signaling pathways such as the mitogen-activated protein kinase, which is the mechanistic target of rapamycin, and the vascular endothelial growth factor, as well as cancer-related signaling pathways. Five DE miRNAs were randomly selected to validate the results of miRNA-seq using real-time polymerase chain reaction, and the results corresponded to those from the miRNA-seq, confirming that deep-sequencing methods are feasible and efficient. In our study, we identified various previously unknown hypoxia-related miRNAs in pigs, and the data obtained suggest that hypoxia-related miRNA expression patterns are significantly altered in the Tibetan pig compared to other species. Therefore, DE miRNAs may play an important role in organisms that have adapted to hypoxic environments.