High-altitude stress threatens the survival rate of Tibetan sheep and reduces their fertility. However, the molecular basis of this phenomenon remains elusive. Here, we used RNA-seq to elucidate the transcriptome dynamics of high-altitude stress in Tibetan sheep ovaries. In total, 104 genes were characterized as high-altitude stress-related differentially expressed genes (DEGs). In addition, 36 DEGs contributed to ovarian follicle development, and 28 of them were downregulated under high-altitude stress. In particular, high-altitude stress significantly suppressed the expression of two ovarian lymphatic system marker genes: LYVE1 and ADAMTS-1. Network analysis revealed that luteinizing hormone (LH)/follicle-stimulating hormone (FSH) signaling-related genes, such as EGR1, FKBP5, DUSP1, and FOS, were central regulators in the DEG network, and these genes were also suppressed under high-altitude stress. As a post-transcriptional regulation mechanism, alternative splicing (AS) is ubiquitous in Tibetan sheep. High-altitude stress induced 917 differentially alternative splicing (DAS) events. High-altitude stress modulated DAS in an AS-type-specific manner: suppressing skipped exon events but increasing retained intron events. C₂H₂-type zinc finger transcription factors and RNA processing factors were mainly enriched in DAS. These findings revealed high-altitude stress repressed ovarian development by suppressing the gene expression of LH/FSH hormone signaling genes and inducing intron retention of C₂H₂-type zinc finger transcription factors.

MicroRNAs (miRNAs) have been found to be involved in lipid deposition and metabolism. However, there have been no reports on the roles of miR-148a in the proliferation and adipogenesis of preadipocytes in sheep. In this study, the expression of miR-148a was profiled in the eight tissues of Tibetan ewes and differentiated preadipocytes, and the role of miR-148a in differentiation and proliferation of ovine preadipocytes was investigated using Oil Red O staining, CCK-8, EdU staining, cell cycle detection, and RT-qPCR. The effect of PTEN on the differentiation of ovine preadipocytes was also investigated. The miR-148a was widely expressed in the eight tissues investigated and had significantly increased expression in liver, spleen and subcutaneous adipose tissues, and the heart. The expression of miR-148a continued to increase with the differentiation of ovine preadipocytes. The over-expression of miR-148a significantly promoted differentiation but inhibited the proliferation of ovine preadipocytes. The inhibition of miR-148a had the opposite effect on the differentiation and proliferation of ovine preadipocytes with over-expressed miR-148a. The results from the dual luciferase reporter assays showed that miR-148a mimic significantly decreased the luciferase activity of PTEN-3′UTR dual luciferase reporter vector, suggesting that PTEN is a target gene of miR-148a. In over-expressed-PTEN preadipocytes, the number of lipid droplets remarkably decreased, and the expression levels of adipogenesis marker genes PPARγ, FASN, FATP4, GLUT4, C/EBPβ and LPL were also significantly down-regulated. These results suggest that miR-148a accelerated the adipogenic differentiation of ovine preadipocytes by inhibiting PTEN expression, and also inhibited the proliferation of ovine preadipocytes.

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.

Yaks are tough animals living in Tibet’s hypoxic stress environment. However, the metabolite composition of yak milk and its role in hypoxic stress tolerance remains largely unexplored. The similarities and differences between yak and human milk in hypoxic stress tolerance are also unclear. This study explored yak colostrum (YC) and yak mature milk (YMM) using GC–MS, and 354 metabolites were identified in yak milk. A comparative metabolomic analysis of yak and human milk metabolites showed that over 70% of metabolites were species-specific. Yak milk relies mainly on essential amino acids- arginine and essential branched-chain amino acids (BCAAs): L-isoleucine, L-leucine, and L-valine tolerate hypoxic stress. To slow hypoxic stress, human breast milk relies primarily on the neuroprotective effects of non-essential amino acids or derivates, such as citrulline, sarcosine, and creatine. In addition, metabolites related to hypoxic stress were significantly enriched in YC than in YMM. These results reveal the unique metabolite composition of yak and human milk and provide practical information for applying yak and human milk to hypoxic stress tolerance.

Circular RNAs (circRNAs) are a class of non-coding RNAs that play important roles in preadipocyte differentiation and adipogenesis. However, little is known about genome-wide identification, expression profile, and function of circRNAs in sheep. To investigate the role of circRNAs during ovine adipogenic differentiation, the subcutaneous adipose tissue of Tibetan rams was collected in June 2022. Subsequently, the preadipocytes were immediately isolated from collected adipose tissue and then induced to begin differentiation. The adipocytes samples cultured on day 0, day 2 and day 8 of preadipocytes differentiation were used to perform RNA sequencing (RNA-seq) analysis to construct the expression profiles of circRNAs. Subsequently, the function of differentially expressed circRNAs was investigated by performing the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of their parent genes. Finally, a circRNAs-miRNAs-mRNAs network involved in adipogenic differentiation was been analyzed. As a result, a total of 6,449 candidate circRNAs were identified in ovine preadipocytes. Of these circRNAs identified, 63 candidate circRNAs were differentially expressed among the three differentiation stages and their parent genes were mainly enriched in acetyl-CoA metabolic process, positive regulation of lipid biosynthetic process, positive regulation of steroid biosynthetic process, and focal adhesion pathway (P < 0.05). Based on a circRNAs-miRNAs-mRNAs regulatory network constructed, circ_004977, circ_006132 and circ_003788 were found to function as competing endogenous RNAs (ceRNAs) to regulate ovine preadipocyte differentiation and lipid metabolism. The results provide an improved understanding of functions and molecular mechanisms of circRNAs underlying ovine adipogenesis in sheep.

KRTAP24-1 belongs to the high-sulphur KAP family and has been associated with cashmere fibre diameter in goats, but its role in ovine wool traits remains unclear. This study assessed KRTAP24-1 tissue expression by RT-qPCR and investigated genetic variation and associations with wool traits in 277 Tibetan sheep. Polymorphisms in the coding region were identified by PCR amplification and Sanger sequencing, and genotyping was performed using PARMS. A linear mixed model (LMM) incorporating a genomic relationship matrix (GRM) was used to evaluate associations between SNPs, haplotypes, and 12 wool traits. Bioinformatic analyses were restricted to the five haplotypes observed in the study population and were used as preliminary in silico assessments. Three missense SNPs were identified: c.191C>T (p.L64P), c.527G>A (p.G176D), and c.656C>T (p.A219V). The c.191C>T variant was associated with mean fibre length (MFL), single fibre tenacity (SFT), and scoured yield (SY), whereas c.656C>T was associated with lock length (LL) and clean fleece yield (CFY). Several haplotype combinations were also associated with LL, elongation at break (EB), and CFY. KRTAP24-1 showed high expression in skin. The observed haplotypes showed only minor differences in predicted mRNA secondary structure and mainly local changes in predicted protein features. These findings suggest that KRTAP24-1 may provide a preliminary basis for marker-assisted selection in Tibetan sheep breeding, but its functional role requires further validation in independent populations and experimental systems.