The Tibetan sheep is an indigenous animal of the Tibetan plateau, and after a long period of adaptation have adapted to high-altitude hypoxia. Many physiological changes occur in Tibetan sheep as they adapt to high-altitude hypoxia, especially in the lungs. To reveal the physiological changes and their molecular mechanisms in the lungs of Tibetan sheep during adaptation to high altitudes, we selected Tibetan sheep from three altitudes (2500 m, 3500 m, and 4500 m) and measured blood-gas indicators, observed lung structures, and compared lung proteome changes. The results showed that the Tibetan sheep increased their O₂-carrying capacity by increasing the hemoglobin (Hb) concentration and Hematocrit (Hct) at an altitude of 3500 m. While at altitude of 4500 m, Tibetan sheep decreased their Hb concentration and Hct to avoid pulmonary hypertension and increased the efficiency of air-blood exchange and O₂ transfer by increasing the surface area of gas exchange and half-saturation oxygen partial pressure. Besides these, some important proteins and pathways related to gas transport, oxidative stress, and angiogenesis identified by proteome sequencing further support these physiology findings, including HBB, PRDX2, GPX1, GSTA1, COL14A1, and LTBP4, etc. In conclusion, the lungs of Tibetan sheep are adapted to different altitudes by different strategies; these findings are valuable for understanding the basis of hypoxic adaptation in Tibetan sheep.

Lymphatic enhancer factor 1 (Lef1) and distal-less homeobox 3 (Dlx3) are the transcription factors involved in regulating hair follicle development in mice, goats, and other animals. Their deletion can lead to hair follicle deficiency. In this study, hematoxylin–eosin staining (HE), real-time quantitative PCR (RT-qPCR), immunohistochemistry, and immunofluorescence were used to analyze the expression, location, and biological functions of Lef1 and Dlx3 in the lateral skin of Gansu Alpine Merino aged 1, 30, 60, and 90 days. The results revealed that the number of hair follicles decreased with age and was significantly higher at 1 day than in the other three age groups (p < 0.05). The mRNA levels of Lef1 and Dlx3 in the skin of 30-day old Gansu Alpine Merino were significantly higher than those in the other three age groups (p < 0.05). Protein expression of Lef1 and Dlx3 was lowest at 1 day (p < 0.05) and peaked at 60 days. Lef1 and Dlx3 exhibited a high density and strong positive expression in the dermal papillae; additionally, Dlx3 exhibited a high density and strong positive expression in the inner and outer root sheaths. Collectively, Lef1 and Dlx3 may facilitate the maturation of secondary hair follicles, which is mainly achieved through the dermal papillae and inner and outer root sheaths.

In our previous study, microRNA (miR)-381 was found to be the most down-regulated miRNA in skeletal muscle of Liaoning cashmere goats with higher skeletal muscle mass, but the molecular mechanism involved remains unclear. In this study, primary caprine skeletal muscle satellite cells (SMSCs) were isolated and identified. We investigated the effect of miR-381 on the viability, proliferation and differentiation of caprine SMSCs, and the target relationships of miR-381 with jagged canonical Notch ligand 2 (JAG2) and phosphatase and tensin homolog (PTEN). Cells isolated were positive for SMSC-specific marker protein Pax7. This suggests that purified SMSCs were obtained. The expression level of miR-381 achieved a peak value on day 4 after SMSC differentiation, and miR-381 also significantly increased the expression levels of myogenic differentiation marker genes: myosin heavy chain (MyHC), myogenin (MyoG) and myocyte enhancer factor 2C (MEF2C) in differentiated SMSCs, the area of MyHC-positive myotubes and the myogenic index. These findings suggest that miR-381 promoted myogenic differentiation of caprine SMSCs. The CCK8 assay and EDU staining analysis showed that miR-381 mimic both inhibited the viability of SMSCs and decreased the percentage of EDU-labeled positive SMSCs. In contrast, miR-381 inhibitor had the opposite effect with miR-381 mimic. A dual luciferase reporter assay verified that miR-381 can target JAG2 and PTEN by binding to the 3′-untranslated regions (3′-UTR) of the genes. The transfection of miR-381 mimic into caprine SMSCs resulted in decreases in expression levels of JAG2 and PTEN, while miR-381 inhibitor increased the two target genes in expression. This is the first study to reveal the biological mechanisms by which miR-381 regulates caprine SMSC activities.

MicroRNAs (miRNAs) are crucial regulatory molecules in lipid deposition and metabolism. However, the effect of miR-200b on the regulation of proliferation and adipogenesis of ovine preadipocytes is unknown in the sheep (Ovis aries). In this study, the expression profiles of miR-200b were investigated in the seven tissues of Tibetan ewes and differentiated preadipocytes. The effect of miR-200b, as well as its target genes p27 and KLF9, on the proliferation of ovine preadipocytes and adipogenesis was also investigated, using cell viability analysis, EdU staining, Oil Red O staining and reverse transcription-quantitative PCR (RT-qRCR). The miR-200b was expressed in all the tissues investigated, and it was highly expressed in lung, liver, subcutaneous adipose and spleen tissues. The expression of miR-200b continuously decreased when the differentiation of ovine preadipocytes initiated. The miR-200b mimic dramatically accelerated the proliferation but inhibited differentiation of ovine preadipocytes. The miR-200b inhibitor resulted in an opposite effect on the proliferation and differentiation of ovine preadipocytes. The dual luciferase reporter assay results showed that miR-200b mimic significantly decreased the luciferase activity of p27 and KLF9 in HEK293 cells transfected with wild-type dual luciferase reporter vectors. This suggests that p27 and KLF9 are the target genes of miR-200b. In over-expressed-p27 preadipocytes, the number of EdU-labeled preadipocytes and the expression levels of proliferation marker genes CDK2, CDK4, CCND1 and PCNA significantly decreased. In addition, the transfection of over-expressed-KLF9 vector into adipocytes remarkably increased the accumulation of lipid droplets and the expression levels of differentiation marker genes aP2, PPARγ, LPL and GLUT4. These results suggest that miR-200b accelerated the proliferation but inhibited the adipogenic differentiation of ovine preadipocytes by targeting p27 and KLF9, respectively.