Defining ovine dermal papilla cell markers and identifying key signaling pathways regulating its intrinsic properties

Dermal papilla cell (DPC), one of the key cell types during hair follicle development and regeneration, specifies hair size, shape and cycling. It is also an important in vitro screening model for hair growth. Although some characteristics of DPCs, such as agglutinative growth and marker genes, have been studied in mice and humans, the intrinsic properties of ovine DPCs and the regulatory mechanism of the intrinsic properties during continued culture in vitro remained unknown. In this study, based on our previous single-cell transcriptome sequencing on sheep lambskin, we verified SOX18 and PDGFRA as the novel marker genes of ovine DPCs through immunofluorescence staining on skin sections and cultured DPCs. Using continued cell culture and alkaline phosphatase staining, we found that different from mice and humans, ovine DPCs exhibit particularly robust and stable aggregation with unbated alkaline phosphatase activity till 30 passages during continued culture in vitro. Also, we found that the expression of some marker genes and the activity of Wnt/β-catenin signaling differ between early passaged DPCs and multiple passaged DPCs. Further, using Wnt/β-catenin agonist and antagonist, we demonstrated that Wnt/β-catenin signaling could regulate cell aggregation and alkaline phosphatase activity of ovine DPCs through regulating FGF and IGF signaling. This study provides the basis for isolating ovine DPCs and defines their intrinsic properties, which contribute to improving wool performance and medicine of hair regeneration

MicroRNA-181a Regulates the Proliferation and Differentiation of Hu Sheep Skeletal Muscle Satellite Cells and Targets the YAP1 Gene

MicroRNA (miRNA) is of great importance to muscle growth and development, including the regulation of the proliferation and differentiation of skeletal muscle satellite cells (SMSCs). In our research group’s previous study, we found that miR-181a is differentially expressed in the longissimus dorsi muscle of Hu sheep at different stages. We speculated that miR-181a may participate in the growth and development process of Hu sheep. To understand the mechanism of miR-181a regulating the growth and development of Hu sheep skeletal muscle, we extracted skeletal muscle satellite cells from the longissimus dorsi muscle of 3-month-old Hu sheep fetuses and performed a series of experiments. Our results showed that miR-181a suppressed SMSCs’ proliferation using QRT-PCR, Western blot, CCK-8, EDU, and Flow cytometry cycle tests. In addition, QRT-PCR, Western blot, and immunofluorescence indicated that miR-181a facilitated the differentiation of SMSCs. Then, we used dual-luciferase reporter gene detection, QRT-PCR, and Western blot to find that the Yes1-related transcription regulator (YAP1) is the target gene of miR-181a. Our study supplies a research basis for understanding the regulation mechanism of miR-181a on the growth of Hu sheep skeletal muscle

Association of Polymorphisms in Candidate Genes with the Litter Size in Two Sheep Breeds

Hu sheep and Small-tailed Han sheep are the most widely raised and most famous maternal sheep breeds in China, which are known for precocious puberty, perennial oestrus and high fecundity (1–6 lambs each parity). Therefore, it is crucial to increase litter size of these two breeds for intensive sheep industry. The objective of this study was to identify potential genetic markers linked with sheep litter size located at ten genes. This study collected blood sample of 537 Hu sheep and 420 Small-tailed Han sheep with litter size of first parity. The average litter sizes in Hu sheep and Small-tailed Han sheep were 2.21 and 1.93. DNA-pooling sequencing method was used for detecting the potential single nucleotide polymorphisms (SNPs) in ten genes related to follicle development and female reproduction. SNPscan® was used for individually genotyping. As a result, a total of 78 putative SNPs in nine out of ten candidate genes (except NOG) were identified. In total, 50 SNPs were successfully genotyped in Hu sheep and Small-tailed Han sheep. After quality control, a total of 42 SNPs in Hu sheep and 44 SNPs in Small-tailed Han sheep were finally used for further analysis. Association analysis revealed that nine SNPs within six genes (KIT: g.70199073A>G, KITLG: g.124520653G>C, ADAMTS1: g.127753565T>C, ADAMTS1: g.127754640G>T, NCOA1: g.31928165C>T, NCOA1: g.32140565G>A, LIFR: g.35862868C>T, LIFR: g.35862947G>T and NGF: g.91795933T>C) were significantly associated with litter size in Hu sheep or Small-tailed Han sheep. A combined haplotypes analysis of the two loci (LIFR: g.35862868C>T and LIFR: g.35862947G>T) revealed that H2H3 (CTTT) combined haplotypes had the largest litter size than the rest combined haplotypes and more than those with either mutation alone in Small-tailed Han sheep. Taken together, our study suggests that nine significant SNPs in six genes can be served as useful genetic markers for MAS in sheep

Expression of DAZL Gene in Selected Tissues and Association of Its Polymorphisms with Testicular Size in Hu Sheep

The deleted in azoospermia-like (DAZL) gene encoding an RNA binding protein is pivotal in gametogenesis in lots of species and also acts as a pre-meiosis marker. The current study was conducted to detect expression profiles and single nucleotide polymorphisms (SNPs) of DAZL in sheep using qPCR, DNA-pooled sequencing, improved multiplex ligase detection reaction (iMLDR®) and restriction fragment length polymorphism (RFLP) methods. The results confirmed that ovine DAZL showed the highest expression level at six-months of age across five developmental stage. At six-month stage, DAZL expressed primarily in testis across seven tissues analyzed. The abundance of DAZL in the large-testis group is higher than that in the small-testis group although it is not significant. In addition, six SNPs (SNP1-SNP6) were identified in DAZL. Of those, SNP1 (p < 0.05) and SNP6 (p < 0.01) were significantly correlated with the variation coefficient between left and right epididymis weight (VCTW). The current study implies DAZL may play important roles in testicular development and its SNPs are associated with testicular parameters, which supply important indicators for ram selection at early stage

Integrative analysis of Iso-Seq and RNA-seq data reveals transcriptome complexity and differentially expressed transcripts in sheep tail fat

Background Nowadays, both customers and producers prefer thin-tailed fat sheep. To effectively breed for this phenotype, it is important to identify candidate genes and uncover the genetic mechanism related to tail fat deposition in sheep. Accumulating evidence suggesting that post-transcriptional modification events of precursor-messenger RNA (pre-mRNA), including alternative splicing (AS) and alternative polyadenylation (APA), may regulate tail fat deposition in sheep. Differentially expressed transcripts (DETs) analysis is a way to identify candidate genes related to tail fat deposition. However, due to the technological limitation, post-transcriptional modification events in the tail fat of sheep and DETs between thin-tailed and fat-tailed sheep remains unclear. Methods In the present study, we applied pooled PacBio isoform sequencing (Iso-Seq) to generate transcriptomic data of tail fat tissue from six sheep (three thin-tailed sheep and three fat-tailed sheep). By comparing with reference genome, potential gene loci and novel transcripts were identified. Post-transcriptional modification events, including AS and APA, and lncRNA in sheep tail fat were uncovered using pooled Iso-Seq data. Combining Iso-Seq data with six RNA-sequencing (RNA-Seq) data, DETs between thin- and fat-tailed sheep were identified. Protein protein interaction (PPI) network, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were implemented to investigate the potential functions of DETs. Results In the present study, we revealed the transcriptomic complexity of the tail fat of sheep, result in 9,001 potential novel gene loci, 17,834 AS events, 5,791 APA events, and 3,764 lncRNAs. Combining Iso-Seq data with RNA-Seq data, we identified hundreds of DETs between thin- and fat-tailed sheep. Among them, 21 differentially expressed lncRNAs, such as ENSOART00020036299, ENSOART00020033641, ENSOART00020024562, ENSOART00020003848 and 9.53.1 may regulate tail fat deposition. Many novel transcripts were identified as DETs, including 15.527.13 (DGAT2), 13.624.23 (ACSS2), 11.689.28 (ACLY), 11.689.18 (ACLY), 11.689.14 (ACLY), 11.660.12 (ACLY), 22.289.6 (SCD), 22.289.3 (SCD) and 22.289.14 (SCD). Most of the identified DETs have been enriched in GO and KEGG pathways related to extracellular matrix (ECM). Our result revealed the transcriptome complexity and identified many candidate transcripts in tail fat, which could enhance the understanding of molecular mechanisms behind tail fat deposition

Whole-Genome Resequencing Reveals Selection Signal Related to Sheep Wool Fineness

Wool fineness affects the quality of wool, and some studies have identified about forty candidate genes that affect sheep wool fineness, but these genes often reveal only a certain proportion of the variation in wool thickness. We further explore additional genes associated with the fineness of sheep wool. Whole-genome resequencing of eight sheep breeds was performed to reveal selection signals associated with wool fineness, including four coarse wool and four fine/semi-fine wool sheep breeds. Multiple methods to reveal selection signals (Fst and θπ Ratio and XP-EHH) were applied for sheep wool fineness traits. In total, 269 and 319 genes were annotated in the fine wool (F vs. C) group and the coarse wool (C vs. F) group, such as LGR4, PIK3CA, and SEMA3C and NFIB, OPHN1, and THADA. In F vs. C, 269 genes were enriched in 15 significant GO Terms (p p p p < 0.05), such as negative regulation of focal adhesion assembly (GO: 0051895) and Axon guidance (oas 04360). Our study has uncovered genomic information pertaining to significant traits in sheep and has identified valuable candidate genes. This will pave the way for subsequent investigations into related traits

Whole-genome resequencing of Dorper and Hu sheep to reveal selection signatures associated with important traits

Dorper and Hu sheep exhibit different characteristics in terms of reproduction, growth, and meat quality. Comparison of the genomes of two breeds help to reveal important genomic information. In this study, whole genome resequencing of 30 individuals (Dorper, DB and Hu sheep, HY) identified 15,108,125 single nucleotide polymorphisms (SNPs). Population differentiation (Fst) and cross population extended haplotype homozygosity (XP-EHH) were performed for selective signal analysis. In total, 106 and 515 overlapped genes were present in both the Fst results and XP-EHH results in HY vs DB and in DB vs HY, respectively. In HY vs DB, 106 genes were enriched in 12 GO terms and 83 KEGG pathways, such as ATP binding (GO:0005524) and PI3K-Akt signaling pathway (oas04151). In DB vs HY, 515 genes were enriched in 109 GO terms and 215 KEGG pathways, such as skeletal muscle cell differentiation (GO:0035914) and MAPK signaling pathway (oas04010). According to the annotation results, we identified a series of candidate genes associated with reproduction (UNC5C, BMPR1B, and GLIS1), meat quality (MECOM, MEF2C, and MYF6), and immunity (GMDS, GALK1, and ITGB4). Our investigation has uncovered genomic information for important traits in sheep and provided a basis for subsequent studies of related traits

Rumen Bacteria Communities and Performances of Fattening Lambs with a Lower or Greater Subacute Ruminal Acidosis Risk

Several ruminal cellulolytic bacteria species are sensitive to pH and could therefore be used as biomarkers to determine the risk of sub-acute ruminal acidosis (SARA) in finishing lambs. This study compared a 2–4 h post feeding ruminal pH measurement to abundances of the ruminal pH-sensitive bacteria to evaluate the risk of SARA in a herd of 120 finishing lambs. The lambs were reared in individual units for 50 days. Ruminal fluid was collected by use of an orogastric tube on day 51 2-4 h after feeding. Although the lambs were fed an identical diet, they responded differently in the abundances of four ruminal pH sensitive cellulolytic bacteria (Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes and Butyrivibrio fibrisolvens). Lambs with the most or the least cellulolytic bacteria were then classified as either lower SARA risk (LSR, n = 10) or higher SARA risk group (HSR, n = 10), respectively. Data showed that the ruminal pH and VFA profiles were uncorrelated with the number of cellulolytic bacteria (P &gt; 0.050). Lambs with the HSR showed lower ruminal pH (P = 0.013) and acetate to propionate ratio (P = 0.018), higher concentrations of lactate (P = 0.035) and proportion of propionate (P = 0.033) compared to those with the LSR. The DMI and ADG did not differ in LSR and HSR lambs (P &gt; 0.050). A diversity analysis revealed significantly lower diversity in HSR lambs than in LSR (Simpson index, P = 0.004). The relative abundances of the phyla Bacteroidetes, Fibrobacteres, Verruomicrobia, and Proteobacteria were higher in LSR lambs than in HSR (P &lt; 0.050). The abundances of several phyla including Firmicutes, Tenericutes and Actinobacteria were higher in the HSR than in the LSR group (P &lt; 0.050). The bacterial communities of the LSR and HSR clustered separately in rumen based on the Unifrac distances, indicating distinct bacteria communities at OTU level between the LSR and HSR lambs. Overall, there was no correlation between 2 and 4 h post-feeding ruminal pH and the abundance of pH-sensitive bacteria and the amount of these bacteria could be used as a potential biomarker of SARA in lamb herd

Expression profile of <i>FASN</i> gene and association of its polymorphisms with intramuscular fat content in Hu sheep

The content of intramuscular fat (IMF) is one of the most important factors that has a large impact on meat quality, and it is an effective way to improve IMF according to marker-assisted selection (MAS). Fatty-acid synthase (FASN) is a key gene in meat lipid deposition and fatty acid composition. Thus, this study was conducted to investigate the expression profile of FASN in mRNA and protein levels using real-time quantitative PCR (RT-qPCR) and western-blot methods. In addition, single nucleotide polymorphisms (SNPs) within FASN in 921 Hu rams with IMF content records were investigated using DNA-pooling sequencing and improved multiple ligase detection reaction (iMLDR) methods. Consequently, the highest mRNA expression level of FASN was observed in the perinephric fat, and the lowest in the liver among the 11 tissues analyzed, while no significant difference was found in mRNA and protein expression levels in longissimus dorsi among individuals with different IMF contents. A total of 10 putative SNPs were identified within FASN, and 9 of them can be genotyped by iMLDR method. Notably, two SNPs were significantly associated with IMF content, including NC_040262.1: g.5157 A > G in intron 5 (p = 0.046) and NC_040262.1: g.9413 T > C in intron 16 (p = 0.041), which supply molecular markers for improving meat quality in sheep breeding.</p

Integrated Hair Follicle Profiles of microRNAs and mRNAs to Reveal the Pattern Formation of Hu Sheep Lambskin

Hair follicle development is closely associated with wool curvature. Current studies reveal the crucial role of microRNAs (miRNAs) in hair follicle growth and development. However, few studies are known regarding their role in wool curvature. To reveal the potential roles of miRNAs in Hu sheep lambskin with different patterns, a total of 37 differentially expressed (DE) miRNAs were identified in hair follicles between small waves (SM) and straight wool (ST) groups using RNA-seq. Through functional enrichment and miRNA-mRNA co-expression analysis, some key miRNAs (oar-miR-143, oar-miR-200b, oar-miR-10a, oar-miR-181a, oar-miR-10b, oar-miR-125b, etc.) and miRNA-mRNA pairs (miR-125b target CD34, miR-181a target FGF12, LMO3, miR-200b target ZNF536, etc.) were identified. Though direct or indirect ways affecting hair follicle development, these miRNAs and mRNAs may have possible effects on wool curvature, and this study thus provides valuable insight on potential pattern formation