Sheep Skeletal Muscle Research Articles

Regulatory Role of Nfix Gene in Sheep Skeletal Muscle Cell Development and Its Interaction Mechanism with MSTN.

Skeletal muscle development is crucial for livestock production, and understanding the molecular mechanisms involved is essential for enhancing muscle growth in sheep. This study aimed to investigate the role of Nfix, a member of the nuclear factor I (NFI) family, in regulating muscle development in sheep, filling a significant gap in the current understanding of Nfix deficiency and its impact on skeletal muscle growth, as no similar studies have been reported in this species. Bioinformatic analysis, including temporal analysis of transcriptome data, identified Nfix as a potential target gene for muscle growth regulation. The effects of Nfix overexpression and knockout on the proliferation and differentiation of sheep skeletal muscle cells were investigated. Changes in the expression of associated marker genes were assessed to explore the regulatory link between Nfix and the myostatin (MSTN) gene. Additionally, target miRNAs for Nfix and MSTN were predicted using online databases such as miRWalk, resulting in the construction of an Nfix-miRNA-MSTN interactive regulatory network. The findings revealed that Nfix promotes the proliferation and differentiation of sheep skeletal muscle cells, with further analysis indicating that Nfix may regulate muscle cell development by modulating MSTN expression. This study provides preliminary insights into the function of Nfix in sheep skeletal muscle development and its regulatory interactions, addressing a critical knowledge gap regarding Nfix deficiency and its implications for muscle growth. These findings contribute to a better understanding of muscle biology in sheep and provide a theoretical foundation for future research into the regulatory mechanisms governing muscle development.

PSI-3 Effects of a Retinoid X Receptor agonist on primary ovine satellite cells

Abstract Sheep producers are typically paid based on body weight, so understanding the mechanisms of postnatal skeletal muscle growth is vital. Satellite cells are responsible for the capacity for postnatal skeletal muscle growth. Satellite cells proliferate and then differentiate into myoblasts before fusing with existing myotubes. The retinoid X receptor (RXR) is a transcription factor that enhances myogenic differentiation 1 (MyoD) expression, which increases differentiation and fusion of myoblasts in sheep. LG 100268 is an RXR agonist, which activates the RXR. The objective of this project is to measure the effect of LG 100268 on primary ovine satellite cell (OSC) proliferation and protein synthesis rates. Primary OSC were isolated from the hind legs of 9-mo old commercial hair sheep wethers (n = 3). For proliferation, cultures were grown to approximately 70% confluency, followed by treatment with 5% fetal bovine serum (FBS) or 300 nM of LG 100268 in 5% FBS. For protein synthesis, cultures were grown to approximately 80% confluency, at which point cultures were induced to differentiate and treated 48-h later with serum free media (SFM) or 300 nM LG 100268 in SFM. Proliferation rates were measured at 24-h post-treatment, and protein synthesis rates were measured at 6-h post-treatment. Treatment with 300 nM LG 100268 increased (P = 0.004) OSC proliferation rates compared with control cultures. Preliminary protein synthesis results suggest that LG 100268 does not alter (P = 0.22) protein synthesis rates of fused OSC. However, more research is warranted to clarify the role of the RXR in sheep skeletal muscle growth.

Expression Regulation of Gluconeogenesis Related Genes in Ovine Skeletal Muscle Cells.

Under fasting conditions, the pathway converting gluconeogenesis precursors into muscle glycogen becomes crucial due to reduced glycogen reserves. However, there is limited research on skeletal muscle gluconeogenesis and the impact of fasting on gluconeogenic gene expression. Sheep fetal skeletal muscle cells cultured in vitro were used to study the effects of varying lactic acid concentrations (0 to 30 mM) and 2.5 mM glucose on the expression of gluconeogenesis-related genes after 6 h of fasting. The effects on mRNA and protein expression of key genes involved in skeletal muscle gluconeogenesis were measured by quantitative real time polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting at 48 h. Fasting increased the expression of key gluconeogenic genes, fructose-1,6-bisphosphatase 2 (FBP2), glucose-6-phosphatase 3 (G6PC3), pyruvate kinase M (PKM), monocarboxylate transporter1 (MCTS1), glucose transporter type 4 (GLUT4), pyruvate carboxylase (PC), and lactate dehydrogenase A (LDHA). The mRNA levels of FBP2, G6PC3, and MCTS1 significantly decreased with glucose addition. Additionally, 10 mM lactic acid significantly promoted the expression of FBP2, PC, MCTS1, LDHA, GLUT4, and PKM while inhibiting phosphoenolpyruvate carboxykinase (PEPCK) expression. At the protein level, 10 mM lactic acid significantly increased FBP2 and PKM protein expression. This study shows that fasting regulates key gluconeogenic gene expression in sheep skeletal muscle cells and highlights the role of lactic acid in inducing these gene expressions.

Transcriptome analysis revealed differences in gene expression in sheep muscle tissue at different developmental stages

BackgroundThe analysis of differentially expressed genes in muscle tissues of sheep at different ages is helpful to analyze the gene expression trends during muscle development. In this study, the longissimus dorsi muscle of pure breeding Hu sheep (H), Suffolk sheep and Hu sheep hybrid F1 generation (SH) and East Friesian and Hu sheep hybrid sheep (EHH) three strains of sheep born 2 days (B2) and 8 months (M8) was used as the research object, and transcriptome sequencing technology was used to identify the differentially expressed genes of sheep longissimus dorsi muscle in these two stages. Subsequently, GO and KEGG enrichment analysis were performed on the differential genes. Nine differentially expressed genes were randomly selected and their expression levels were verified by qRT-PCR.ResultsThe results showed that 842, 1301 and 1137 differentially expressed genes were identified in H group, SH group and EHH group, respectively. Among them, 191 differential genes were enriched in these three strains, including pre-folding protein subunit 6 (PFDN6), DnaJ heat shock protein family member A4 (DNAJA4), myosin heavy chain 8 (MYH8) and so on. GO and KEGG enrichment analysis was performed on 191 differentially expressed genes shared by the three strains to determine common biological pathways. The results showed that the differentially expressed genes were significantly enriched in ribosomes, unfolded protein binding, FoxO signaling pathway, glycolysis / glycogen generation and glutathione signaling pathway that regulate muscle protein synthesis and energy metabolism. The results of qRT-PCR were consistent with transcriptome sequencing, which proved that the sequencing results were reliable.ConclusionsOverall, this study revealed the important genes and signaling pathways related to sheep skeletal muscle development, and the result laid a foundation for further understanding the mechanism of sheep skeletal muscle development.

Global DNA Methylation, miRNA, and mRNA Profiles in Sheep Skeletal Muscle Promoted by Hybridization.

With the development of high-throughput sequencing technology, several nongenetic variations, including noncoding RNAs such as miRNAs, and DNA methylation, have been found to play an important role in animal muscle development and fat metabolism. In this study, Southdown and Suffolk were selected as male parents for hybridization with Hu sheep (Southdown × Hu (NH), Suffolk × Hu (SH), and Hu × Hu (HH)). RNA sequencing, bisulfite sequencing, and small-RNA sequencing were used to study the methylation patterns and differences in miRNA and mRNA expression in the F1 sheep longissimus dorsi muscle tissue. We identified 765 differentially expressed genes (DEGs), 10,161 differentially methylated regions (DMRs), and 164 differentially expressed miRNAs, which were significantly enriched in AMPK signaling, fatty acid degradation, metabolism, and other related pathways (P < 0.05). In addition, we constructed a DNA methylation-mRNA and miRNA-mRNA coexpression network. A total of 42 common genes were identified from DMRs and DEGs. Importantly, we predicted that 33 differentially expressed miRNAs directly or indirectly targeted the SLC27A6. The data obtained in this study provide useful information and evidence to support further understanding of the miRNA and DNA methylation of key genes regulating muscle growth and fat metabolism in hybrid sheep populations.

OR29-02 Energy Deficient Osteoblasts Inefficiently Mineralize Developing Bone In Sheep With Hypophosphatasia

Abstract Disclosure: J.C. Bertels: None. M. McCord: None. L.G. White: None. S. Huggins: None. D. Landrock: None. E.D. Giles: None. L.J. Suva: None. D. Gaddy: None. Hypophosphatasia (HPP) is a rare genetic disorder characterized by low enzymatic activity of tissue nonspecific alkaline phosphatase (TNSALP), resulting in decreased bone mineralization, muscle weakness, and premature loss of deciduous teeth. All HPP phenotypes have been characterized in our CRISPR/Cas9 gene-edited sheep model of HPP. Previous studies identified small, abnormal mitochondria in HPP sheep cells, leading to the hypothesis that reduced bone mineralization may result from decreased osteoblast (OB) differentiation and cellular energetics. To test this idea, iliac crest bone biopsies and sternal bone marrow stromal cells (BMSCs) were harvested from 1 yr old WT and HPP sheep (n=3 per genotype). Histomorphometric analysis of bone biopsies from HPP sheep revealed increased unmineralized osteoid matrix compared to WT, indicative of decreased OB activity, whereas osteoblast and osteoclast number were unchanged. To determine whether BMSCs from HPP sheep were altered from WT in regard to differentiation capacity toward the adipocyte (AD) or OB lineage, 21 day cultures of BMSCs in OB or AD differentiation medium were performed. HPP BMSCs had significantly increased AD differentiation and similar OB differentiation compared to WT. Next, high resolution respirometry of undifferentiated and differentiating cells toward either the OB or AD lineage using Seahorse XF analysis to measure oxidative respiration and glycolysis on days 0, 7, 14 and 21 of both OB and AD differentiation was performed. Results demonstrated that both undifferentiated and differentiating HPP cells (to OB and AD) had significantly decreased oxygen consumption rates (OCR) and extracellular acidification rates (ECAR), reflective of diminished maximal and ATP-linked oxidative respiration and glycolysis. When measured, intracellular ATP levels in differentiating BMSCs, both undergoing osteoblastogenic and adiopogenic differentiation, showed a higher accumulation of ATP. Collectively, these data provide the first evidence that decreased bone mineralization in HPP sheep results from decreased BMSC and OB energetics through both oxidative respiration and glycolysis. In addition, the data demonstrate that reduced TNSALP activity also leads to enhanced bone marrow adipogenesis. Coupled with our previous results demonstrating reduced oxidative respiration and glycolysis in sheep skeletal muscle, these findings demonstrate a fundamental requirement for TNSALP activity in the maintenance of normal cellular respiration and purinergic metabolism in cells of the musculoskeletal system. Moreover, they provide important insight into the potential for targeting increased cellular respiration to alleviate the deleterious effects of HPP. Presentation: Sunday, June 18, 2023

Astragalus polysaccharide promotes sheep satellite cell differentiation by regulating miR-133a through the MAPK/ERK signaling pathway

Astragalus polysaccharide (APS) possesses extensive biological activities, pharmacological effects, and anti-fatigue function. MiR-133a is a specifically expressed miRNA in skeletal muscle that participates in the regulation of myoblast proliferation and differentiation. However, little is known about the role of APS in the development of sheep skeletal muscle. In this study, we aimed to investigate the underlying mechanism of APS and miR-133a on the differentiation of sheep skeletal muscle satellite cells (SMSCs) and the regulatory relationship between APS and miR-133a. The results suggested that APS plays a positive regulatory role in the proliferation and differentiation of sheep SMSCs. Moreover, miR-133a significantly promotes SMSC differentiation and the activity of the MAPK/ERK signaling pathway. Importantly, we found that APS function requires the mediation of miR-133a in the differentiation of sheep SMSCs. Taken together, our results indicate that APS accelerates SMSC differentiation by regulating miR-133a via the MAPK/ERK signaling pathway in sheep.

The identification and validation of target genes of IGFBP3 protein in sheep skeletal muscle cells

This study aimed to identify the target genes of IGFBP3（insulin growth factor binding protein）protein and to investigate its target genes effects on the proliferation and differentiation of Hu sheep skeletal muscle cells. IGFBP3 was an RNA-binding protein that regulates mRNA stability. Previous studies have reported that IGFBP3 promotes the proliferation of Hu sheep skeletal muscle cells and inhibits differentiation, but the downstream genes that bind to it have not been reported yet. We predicted the target genes of IGFBP3 through RNAct and sequencing data, and verified by qPCR and RIP（RNA Immunoprecipitation）experiments, and demonstrated GNAI2（G protein subunit alpha i2）as one of the target gene of IGFBP3. After interference with siRNA, we carried out qPCR, CCK8, EdU, and immunofluorescence experiments, and found that GNAI2 can promote the proliferation and inhibit differentiation of Hu sheep skeletal muscle cells. This study revealed the effects of GNAI2 and provided one of the regulatory mechanisms of IGFBP3 protein underlying sheep muscle development.

Genome-Wide Analysis of Circular RNAs Reveals circCHRNG Regulates Sheep Myoblast Proliferation via miR-133/SRF and MEF2A Axis.

As relatively new members of the non-coding RNA family, circRNAs play important roles in a variety of biological processes. However, the temporal expression pattern and the function of circRNAs during sheep skeletal muscle development remains unclear. This study aimed to identify circRNAs related to sheep skeletal muscle development and explore their roles in myoblast proliferation. The circRNA expression profiles of longissimus dorsi of sheep from F90, L30, and A3Y were obtained by the RNA-seq method. The function and mechanisms of the novel circCHRNG in muscle satellite cell proliferation were explored using CCK-8 assay, Western blot, qPCR, and dual-luciferase reporter assay. We identified 12,375 circRNAs, including 476, 133, and 233 DEcircRNAs found among three comparative groups. KEGG results showed that DEcircRNAs were enriched in muscle contraction, the regulation of cell proliferation, and the AMPK, insulin, and PI3K-Akt signaling pathways. Notably, a novel circRNA, termed circRNA CHRNG, acts as a miR-133 sponge to promote skeletal muscle satellite cell proliferation. Our study provides a systematic description of circRNAs of ovine skeletal muscle across fetal, lamb, and adult stages. GO and KEGG analyses showed that DEcircRNAs were enriched in multiple pathways associated with muscle development, such as the PI3K-Akt and AMPK signaling pathways. In addition, we propose that circCHRNG acts as a miR-133 sponge to upregulate the expression levels of SRF and MEF2A, thereby promoting myoblast proliferation.

Isolation, culture and identification of sheep skeletal muscle satellite cells

Skeletal muscle satellite cells (SMSCs) play an important role in muscle growth, regeneration and maintenance. This study aimed to isolate SMSCs from sheep, establish a system for isolation, culture and identification of SMSCs in vitro, and provide seed cells for subsequent studies. SMSCs were isolated and purified from newborn 2-day-old healthy sheep by collagenase type I and trypsin two-step digestion and pre-plating method. The results indicated that the isolated and purified SMSCs showed full spindle shape and strong refractive index. The cell growth curve detected by CCK-8 kit was typical “S” type. Immunofluorescence analysis showed that the isolated cells expressed SMSC marker proteins Pax7 and MyoD1. After induction of myogenic differentiation, the cells fused with each other to form multinucleated myotubes and expressed the myoblast specific marker MHC. RT-PCR results showed that the cells expressed SMSC marker gene Pax 7. This experiment established an in vitro isolation, purification and identification system for sheep skeletal muscle satellite cells, which provided a good cell model for studying the biological mechanism of sheep skeletal muscle cells, optimizing sheep breeds, and cell transplantation repair.

The regulation of LncRNA GTL2 expression by DNA methylation during sheep skeletal muscle development

DNA methylation has crucial roles in regulating the expression of genes involved in skeletal muscle development. However, the DNA methylation pattern of lncRNA during sheep skeletal muscle development remains unclear. This study investigated previous WGBS and LncRNA data in skeletal muscle of sheep (fetus and adult). We then focused on LncRNA GTL2, which is differentially expressed in skeletal muscle and has multiple DMRs. We found that the expression level of GTL2 decreased with age. GTL2 DMRs methylation levels were significantly higher in adult muscle than in fetal muscle. After 5AZA treatment, GTL2 expression was significantly increased in a dose-dependent manner.The dCas9-DNMT3A-sgRNA significantly reduced the expression level of GTL2 in cells, but increased GTL2 DMR methylation levels. The above studies indicate that dCas9-DNMT3A can effectively increase the methylation level in the DMR region of GTL2, the expression level of GTL2 is regulated by DNA methylation during muscle development.

Effects of YAP1 on proliferation and differentiation of Hu sheep skeletal muscle satellite cells in vitro

This study aimed to understand the expression level of YAP1 in the skeletal muscle of Hu sheep and to reveal the regulatory mechanism of YAP1 on Hu sheep skeletal muscle satellite cells (SMSCs). Previous research by our group has found that YAP1 may affect the growth and development of Hu sheep skeletal muscle. In the present study, we found the expression of YAP1 in the skeletal muscle is higher than in other tissues of Hu sheep. Then, we detected the effect of YAP1 on proliferation and differentiation in Hu sheep SMSCs. According to the results of qPCR, CCK-8, EDU, and Western blot, compared to the group of negative control, overexpression of YAP1 promoted the proliferation and inhibited the differentiation of SMSCs according to the results of qPCR, CCK-8, EDU, Western blot, while the interference of YAP1 was on the contrary. Overall, our study suggests that YAP1 is an important functional molecule in the growth and development of skeletal muscle by regulating the proliferation and differentiation of SMSCs. These findings are of great use for understanding the roles of YAP1 in the skeletal muscle of Hu sheep.

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.

MiR-22-3p Inhibits Proliferation and Promotes Differentiation of Skeletal Muscle Cells by Targeting IGFBP3 in Hu Sheep.

Simple SummaryThe meat production of Hu sheep affects the mutton supply, and how to increase the meat production is the primary focus of genetic breeding. Therefore, in order to explore the key factors affecting the proliferation and differentiation of Hu sheep skeletal muscle cells, we performed functional verification of miRNA at the cellular level. Our findings are helpful to clarify the molecular regulation mechanism of proliferation and differentiation of skeletal muscle cells, which will benefit the molecular breeding of Hu sheep.The growth and development of skeletal muscle require a series of regulatory factors. MiRNA is a non-coding RNA with a length of about 22 nt, which can inhibit the expression of mRNA and plays an important role in the growth and development of muscle cells. The role of miR-22-3p in C2C12 cells and porcine skeletal muscle has been reported, but it has not been verified in Hu sheep skeletal muscle. Through qPCR, CCK-8, EdU and cell cycle studies, we found that overexpression of miR-22-3p inhibited proliferation of skeletal muscle cells (p < 0.01). The results of qPCR and immunofluorescence showed that overexpression of miR-22-3p promoted differentiation of skeletal muscle cells (p < 0.01), while the results of inhibiting the expression of miR-22-3p were the opposite. These results suggested that miR-22-3p functions in growth and development of sheep skeletal muscle cells. Bioinformatic analysis with mirDIP, miRTargets, and RNAhybrid software suggested IGFBP3 was the target of miR-22-3p, which was confirmed by dual-luciferase reporter system assay. IGFBP3 is highly expressed in sheep skeletal muscle cells. Overexpression of IGFBP3 was found to promote proliferation of skeletal muscle cells indicated by qPCR, CCK-8, EdU, and cell cycle studies (p < 0.01). The results of qPCR and immunofluorescence experiments proved that overexpression of IGFBP3 inhibited differentiation of skeletal muscle cells (p < 0.01), while the results of interfering IGFBP3 with siRNA were the opposite. These results indicate that miR-22-3p is involved in proliferation and differentiation of skeletal muscle cells by targeting IGFBP3.

RNA-sequencing reveals the metabolism regulation mechanism of sheep skeletal muscle under nutrition deprivation stress

Although the skeletal muscle is one of the main sites of metabolism, little is known about the molecular mechanisms involving its response to nutrition stress. The aim of the study was to screen the transcriptome of sheep muscle to identify the metabolism-related genes under nutrition deprivation stress. Ten healthy adult female Small-tailed Han sheep with similar age and weight were randomly divided into a normal group and fasted group. After 3 days, three sheep were randomly selected from each group and the semitendinosus samples were subjected to RNA-sequencing (RNA-seq) and a series of analyses and function annotations. Compared with the normal group, 391 differentially expressed genes (DEGs) were identified in the fasted group that had obvious weight loss, including 278 down-regulated and 113 up-regulated genes. Gene Ontology enrichment annotation classified 228 DEGs in the metabolic process, 11 of which were new genes and only Sheep_newGene_4578 had been annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. The results of Clusters of Orthologous Groups annotation indicated that 11, 9, and 4 DEGs were respectively classified in lipid transport and metabolism, amino acid transport and metabolism, and carbohydrate transport and metabolism. In addition, KEGG enrichment analysis showed that there were not only pathways which were directly related to metabolisms such as protein digestion and absorption pathway, fatty acid metabolism pathway, and biosynthesis pathway of unsaturated fatty acids, but also PI3K-AKT pathway, AMPK pathway, MAPK pathway, and FoxO pathway which were important to metabolism among the top 20 pathways with the lowest significant Q value. The MCODE analysis of protein–protein interaction revealed that two identified subnetworks with top score were closely associated with metabolism. The correlation analysis showed that the mRNA levels of most of DEGs that might be related in the two subnetworks were significantly correlated respectively, and the mRNA levels of most of 10 metabolism-related DEGs including Sheep_newGene_4578 were significantly correlated. Finally, 16 random and 10 metabolism-related DEGs were chosen for confirmation by quantitative real-time PCR, demonstrating the same expression change as determined by RNA-seq. In conclusion, multiple interrelated metabolism-related DEGs in skeletal muscle contributed to the response of sheep to nutritional deprivation stress.

Ubiquitin-specific peptidase 8 regulates proliferation and early differentiation of sheep skeletal muscle satellite cells

Ubiquitin-specific protease 8 (USP8), a member of the ubiquitin-specific protease (USP) family, was originally identified as playing a role in the regulation of growth and cell cycle. However, its functional role in myogenesis is unknown. In this study, we investigated the role of USP8 in proliferation and differentiation of sheep skeletal muscle satellite cells. The results showed that the expression level of USP8 was significantly increased on days 2 and 3 following the induction of the differentiation process. Furthermore, knocking down USP8 resulted in a significant increase in myogenin-positive cells, and promoted early differentiation of satellite cells by regulating the expression level of paired box 7 (PAX7). Additionally, knocking down USP8 suppressed muscle satellite cell proliferation, possibly explaining that the relative mRNA level of USP8 was linearly related to muscle fibre density of Hu sheep. Overall, our research demonstrates that USP8 plays a role in proliferation and early differentiation of skeletal muscle satellite cells.

Analysis of dynamic and widespread lncRNA and miRNA expression in fetal sheep skeletal muscle.

The sheep is an economically important animal, and there is currently a major focus on improving its meat quality through breeding. There are variations in the growth regulation mechanisms of different sheep breeds, making fundamental research on skeletal muscle growth essential in understanding the regulation of (thus far) unknown genes. Skeletal muscle development is a complex biological process regulated by numerous genes and non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). In this study, we used deep sequencing data from sheep longissimus dorsi (LD) muscles sampled at day 60, 90, and 120 of gestation, as well as at day 0 and 360 following birth, to identify and examine the lncRNA and miRNA temporal expression profiles that regulate sheep skeletal myogenesis. We stained LD muscles using histological sections to analyse the area and circumference of muscle fibers from the embryonic to postnatal development stages. Our results showed that embryonic skeletal muscle growth can be characterized by time. We obtained a total of 694 different lncRNAs and compared the differential expression between the E60 vs. E90, E90 vs. E120, E120 vs. D0, and D0 vs. D360 lncRNA and gene samples. Of the total 701 known sheep miRNAs we detected, the following showed a wide range of expression during the embryonic stage: miR-2387, miR-105, miR-767, miR-432, and miR-433. We propose that the detected lncRNA expression was time-specific during the gestational and postnatal stages. GO and KEGG analyses of the genes targeted by different miRNAs and lncRNAs revealed that these significantly enriched processes and pathways were consistent with skeletal muscle development over time across all sampled stages. We found four visual lncRNA–gene regulatory networks that can be used to explore the function of lncRNAs in sheep and may be valuable in helping improve muscle growth. This study also describes the function of several lncRNAs that interact with miRNAs to regulate myogenic differentiation.

A multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep.

Sex is an important biological variable as many physiological as well as disease processes differ between females and males. The fundamental biological distinction between females and males starts with chromosomal sex, and the establishment of XX and XY cells and tissues. Polymerase Chain Reaction (PCR) is a simple and effective method to easily determine chromosomal or genetic sex of cells and tissues. The goal of this study was to develop a simple multiplex PCR genotyping assay to distinguish XX and XY tissues in sheep.Primers were designed to amplify a fragment of the autosomal gene myogenin (MYOG) and sex determining region on the Y chromosome (SRY). PCR analysis was performed on a variety of genomic DNA samples isolated from fetal sheep skeletal muscle, brain, liver, and placenta, and revealed a single 259bp band for MYOG in XX females, and a 259bp band for MYOG and a 167bp band for SRY in XY males. Amplicons were clearly distinguishable by gel electrophoresis, and their sequences revealed 100% identity to the known ovine MYOG and SRY sequence.The reported multiplex PCR genotyping assay provides a rapid means to distinguish XX and XY sheep tissues using low volume samples.

Analysis of DNA methylation profiles during sheep skeletal muscle development using whole-genome bisulfite sequencing

BackgroundDNA methylation is an epigenetic regulatory form that plays an important role in regulating the gene expression and the tissues development.. However, DNA methylation regulators involved in sheep muscle development remain unclear. To explore the functional importance of genome-scale DNA methylation during sheep muscle growth, this study systematically investigated the genome-wide DNA methylation profiles at key stages of Hu sheep developmental (fetus and adult) using deep whole-genome bisulfite sequencing (WGBS).ResultsOur study found that the expression levels of DNA methyltransferase (DNMT)-related genes were lower in fetal muscle than in the muscle of adults. The methylation levels in the CG context were higher than those in the CHG and CHH contexts, and methylation levels were highest in introns, followed by exons and downstream regions. Subsequently, we identified 48,491, 17, and 135 differentially methylated regions (DMRs) in the CG, CHG, and CHH sequence contexts and 11,522 differentially methylated genes (DMGs). The results of bisulfite sequencing PCR (BSP) correlated well with the WGBS-Seq data. Moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation analysis revealed that some DMGs were involved in regulating skeletal muscle development and fatty acid metabolism. By combining the WGBS-Seq and previous RNA-Seq data, a total of 159 overlap genes were obtained between differentially expressed genes (DEGs) and DMGs (FPKM > 10 and fold change > 4). Finally, we found that 9 DMGs were likely to be involved in muscle growth and metabolism of Hu sheep.ConclusionsWe systemically studied the global DNA methylation patterns of fetal and adult muscle development in Hu sheep, which provided new insights into a better understanding of the epigenetic regulation of sheep muscle development.

Isolation and Biological Characterization of Muscle-Derived Stem Cells from Sheep Skeletal Muscle

Isolation and Biological Characterization of Muscle-Derived Stem Cells from Sheep Skeletal Muscle