Tibetan pigs that inhabit the Tibetan Plateau exhibit striking phenotypic and physiological differences from lowland pigs, and have adapted well to extreme conditions. However, the mechanisms involved in regulating gene expression at high altitude in these animals are not fully understood. In this study, we obtained transcriptomic and proteomic data from the heart tissues of Tibetan and Yorkshire pigs raised in the highlands (TH and YH) and lowlands (TL and YL) via RNA-seq and iTRAQ (isobaric tags for relative and absolute quantitation) analyses, respectively. Comparative analyses of TH vs. YH, TH vs.TL, TL vs. YL, and YH vs. YL yielded 299, 169, 242, and 368 differentially expressed genes (DEGs), and 473, 297, 394, and 297 differentially expressed proteins (DEPs), respectively. By functional annotation of these DEGs and DEPs, genes that were enriched in the HIF-1 signaling pathway (NPPA, ERK2, ENO3, and EGLN3), VEGF signaling pathway (ERK2, A2M, FGF1, CTGF, and DPP4), and hypoxia-related processes (CRYAB, EGLN3, TGFB2, DPP4, and ACE) were identified as important candidate genes for high-altitude adaptation in the Tibetan pig. This study enhances our understanding of the molecular mechanisms involved in hypoxic adaptation in pigs, and furthers our understanding of human hypoxic diseases.

Ovarian follicle selection largely depends on the transition of granulosa cells from an undifferentiated to a fully differentiated state, which is accompanied by morphological and functional changes in follicles. The processes and transcriptional regulation of follicles during follicle selection are unclear; we thus used follicles from the prehierarchal to the hierarchal stage to investigate histology, reproductive endocrinology, and transcription. The morphology of follicles changed markedly during follicle selection. The numbers of large white, small yellow, and large yellow follicles (LWF, SYF, and LYF, respectively) were 11.83 ± 2.79, 6.83 ± 2.23, and 1.00, respectively, per ovary. LYF showed thicker granulosa cell layers than those of other prehierarchal follicles. Progesterone concentrations were significantly higher in LYF than that in LWF and SYF. In total, 16,823 genes were positively expressed in LWF, SYF, and LYF. Among follicle types, 1290 differentially expressed genes were enriched regarding cell differentiation, blood vessel morphogenesis, and response to steroid hormones. Candidate genes associated with follicle selection participated in the Wnt signaling pathway, steroid hormone biosynthesis, and the TGF-β signaling pathway. We produced insights into crucial morphological characteristics of transcriptional regulation in follicle development. Our results provide an important basis for revealing the mechanism of follicle selection and potential impact on the poultry industry.

Subcutaneous fat and intramuscular fat (IMF) deposition are closely related to meat production and pork quality. Dingyuan pig, as a native pig breed in China, low selection leads to obvious genetic and phenotypic differences in the population. Individuals with extreme fat content in the population are ideal models for studying the mechanism of fat deposition. In this study, we used RNA-Seq and tandem mass tags-based (TMT) proteomics to analyze the key pathways and genes that specifically regulate subcutaneous fat and IMF deposition in Dingyuan pigs. We identified 191 differentially expressed genes (DEGs) and 61 differentially abundant proteins (DAPs) in the high and low back fat thickness (HBF, LBF) groups, 85 DEGs and 12 DAPs were obtained in the high and low intramuscular fat (HIMF, LIMF) groups. The functional analysis showed that the DEGs and DAPs in the backfat groups were mainly involved in carbohydrates, amino acids, and fatty acids metabolism, whereas the IMF groups were involved in the insulin pathway, longevity, and some disease-related pathways. We found 40 candidate genes that might tissue-specifically lipids deposition for subcutaneous and intramuscular fat. Our research provides theoretical reference materials for the improvement of fat deposition traits of local pig breeds in my country.

Proliferation, apoptosis, and steroid hormone secretion by granulosa cells (GCs) and theca cells (TCs) are essential for maintaining the fate of chicken follicles. Our previous study showed that the Wnt inhibitor factor 1 (WIF1) plays a role in follicle selection. However, the significance of WIF1 in GC- and TC-associated follicular development was not explicitly investigated. This study found that WIF1 expression was strongly downregulated during follicle selection (p < 0.05) and was significantly higher in GCs than in TCs (p < 0.05). WIF1 inhibits proliferation and promotes apoptosis in GCs. Additionally, it promotes progesterone secretion in prehierarchal GCs (pre-GCs, 1.16 ± 0.05 ng/mg vs. 1.58 ng/mg ± 0.12, p < 0.05) and hierarchal GCs (hie-GCs, 395.00 ng/mg ± 34.73 vs. 527.77 ng/mg ± 27.19, p < 0.05) with the participation of the follicle-stimulating hormone (FSH). WIF1 affected canonical Wnt pathways and phosphorylated β-catenin expression in GCs. Furthermore, 604 upregulated differentially expressed genes (DEGs) and 360 downregulated DEGs in WIF1-overexpressed GCs were found through RNA-seq analysis (criteria: |${\log }_2\left(\mathrm{F}\mathrm{o}\mathrm{l}\mathrm{d}\mathrm{C}\mathrm{h}\mathrm{a}\mathrm{n}\mathrm{g}\mathrm{e}\right)$| > 1 and p_adj < 0.05). Cytokine–cytokine receptor interaction and the steroid hormone biosynthesis pathway were identified. In addition, the transcript of estrogen receptor 2 (ESR2) increased significantly (${\log }_2\left(\mathrm{F}\mathrm{o}\mathrm{l}\mathrm{d}\mathrm{C}\mathrm{h}\mathrm{a}\mathrm{n}\mathrm{g}\mathrm{e}\right)$ = 1.27, p_adj < 0.05). Furthermore, we found that WIF1 regulated progesterone synthesis by upregulating ESR2 expression in GCs. Additionally, WIF1 suppressed proliferation and promoted apoptosis in TCs. Taken together, these results reveal that WIF1 stimulates follicle development by promoting GC differentiation and progesterone synthesis, which provides an insight into the molecular mechanism of follicle selection and egg-laying performance in poultry.

Tibetan pigs, which inhabit the Tibetan Plateau, exhibit distinct phenotypic and physiological characteristics from those of lowland pigs and have adapted well to the extreme conditions at high altitude. However, the genetic and epigenetic mechanisms of hypoxic adaptation in animals remain unclear. Whole-genome DNA methylation data were generated for heart tissues of Tibetan pigs grown in the highland (TH, n = 4) and lowland (TL, n = 4), as well as Yorkshire pigs grown in the highland (YH, n = 4) and lowland (YL, n = 4), using methylated DNA immunoprecipitation sequencing. We obtained 480 million reads and detected 280679, 287224, 259066, and 332078 methylation enrichment peaks in TH, YH, TL, and YL, respectively. Pairwise TH vs. YH, TL vs. YL, TH vs. TL, and YH vs. YL comparisons revealed 6829, 11997, 2828, and 1286 differentially methylated regions (DMRs), respectively. These DMRs contained 384, 619, 192, and 92 differentially methylated genes (DMGs), respectively. DMGs that were enriched in the hypoxia-inducible factor 1 signaling pathway and pathways involved in cancer and hypoxia-related processes were considered to be important candidate genes for high-altitude adaptation in Tibetan pigs. This study elucidates the molecular and epigenetic mechanisms involved in hypoxic adaptation in pigs and may help further understand human hypoxia-related diseases.

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. 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. 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. 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.