Comparative analysis of alternative splicing events in skeletal muscle of different sheep.

This paper aims to investigate the relationship between genes with alternative splicing (AS) events and breed-specific differences in muscle development in two breeds of sheep. RNA-seq was utilized to identify genes with AS between Small-tailed Han sheep and Dorset sheep. The gene lists of differentially spliced genes were identified, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were conducted on these genes. In this study, 299 genes with 356 AS indicated significant differences between two diffrerent breeds. There are differences in 31 genes with 35 AS. Cassette, alt5' and alt3' exhibited the highest levels of enrichment across various significant levels. GO and KEGG enrichment analysis demonstrated a significant correlation between Wnt, TGF-beta, Notch and MAPK signaling pathways and the development of muscle in sheep. These findings indicate that genes with AS are linked to variations in muscle development in sheep. These results offer significant scientific and practical implications for improving the quality of sheep meat.

Comparison of alternative splicing (AS) events in adipose tissue of polled dorset versus small tail han sheep.

Background: During the alternative splicing (AS), the exons of primary transcripts are spliced in various arrangements, resulting in structurally and functionally distinct mRNAs and proteins. This study aimed to examine genes with AS events from Small Tail Han sheep and Dorset sheep to explore the mechanism of adipose developments. Methods: This study identified the genes with AS events in adipose tissues of two different sheep with next-generation sequencing. In this paper, genes with significantly different AS events were performed gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Results: 364 genes with 411 A S events showed significant differences in adipose tissues between the two breeds; 108 genes with 120 A S events were extremely significant differences between the two breeds. We identified several novel genes that are related with adipose growth and development. The results of KEGG and GO analysis indicated that oocyte meiosis, mitogen-activated protein kinase (Wnt), mitogen-activated protein kinase (MAPK) signaling pathway, etc. Were closely related to the adipose tissue developments. Conclusions: This paper revealed that the genes with AS events are important for adipose tissues in sheep, exploring the mechanisms of AS events associated with adipose tissue developments in sheep of different breeds.

Impact of FecB Mutation on Ovarian DNA Methylome in Small-Tail Han Sheep.

Booroola fecundity (FecB) gene, a mutant of bone morphogenetic protein 1B (BMPR-1B) that was discovered in Booroola Merino, was the first prolificacy gene identified in sheep related to increased ovulation rate and litter size. The mechanism of FecB impact on reproduction is unclear. METHODS: In this study, adult Han ewes with homozygous FecB(B)/FecB(B) mutations (Han BB group) and ewes with FecB(+)/FecB(+) wildtype (Han ++ group) were selected. Methylated DNA immunoprecipitation and high-throughput sequencing (MeDIP-seq) was used to identify differences in methylated genes in ovary tissue. RESULTS: We examined differences in DNA methylation patterns between HanBB and Han ++ sheep. In both sheep, methylated reads were mainly distributed at the gene body regions, CpG islands and introns. The differentially methylated genes were enriched in neurotrophy in signaling pathway, Gonadotropin Releasing Hormone (GnRH) signaling pathway, Wnt signaling pathway, oocyte meiosis, vascular endothelial growth factor (VEGF) signaling pathway, etc. Differentially-methylated genes were co-analyzed with differentially-expressed mRNAs. Several genes which could be associated with female reproduction were identified, such as FOXP3 (forkhead box P3), TMEFF2 (Transmembrane Protein with EGF Like and Two Follistatin Like Domains 2) and ADAT2 (Adenosine Deaminase TRNA Specific 2). CONCLUSIONS: We constructed a MeDIP-seq based methylomic study to investigate the ovarian DNA methylation differences between Small-Tail Han sheep with homozygous FecB mutant and wildtype, and successfully identified FecB gene-associated differentially-methylated genes. This study has provided information with which to understand the mechanisms of FecB gene-induced hyperprolificacy in sheep.

Comparative DNA methylome analysis of estrus ewes reveals the complex regulatory pathways of sheep fecundity.

BACKGROUND/AIMS: Sheep are important livestock with variant ovulation rate and fertility. Dorset sheep is a typical breed with low prolificacy, whereas Small Tail Han sheep with FecB mutation (HanBB) have hyperprolificacy. Our previous studies have revealed the gene expression difference between the ovaries from Dorset and HanBB sheep contributes to the difference of fecundity, however, what leads to these gene expression difference remains unclear. DNA methylation, an important epigenetic process, plays a crucial role in gene expression regulation. METHODS: In the present study, we constructed a methylated DNA immunoprecipitation combined with high throughput sequencing (MeDIP-seq) strategy to investigate the differentially methylated genes between the Dorset and HanBB ovaries. RESULTS: Our findings suggest the genes involved in immune response, branched-chain amino acid metabolism, cell growth and cell junction were differentially methylated in or around the gene body regions. CONCLUSIONS: These findings provide prospective insights on the epigenetic basis of sheep fecundity.

Ovarian transcriptomic analysis reveals the alternative splicing events associated with fecundity in different sheep breeds.

Alternative splicing (AS) is one of the most common mechanisms that accounts for the greater macromolecular and cellular complexity of higher eukaryotic organisms. This study focused on the splicing events in the ovaries of different sheep breeds, namely the Han and Dorset breeds. Of the groups studied, cassette splicing events accounted for the maximum number of the AS events with significant differences, whereas the splicing events that were mutually exclusive with introns accounted for the smallest proportion of splicing events. Greater than 1000 AS events with significant differences were identified between the Han BB and Dorset sheep. The number of AS events with significant differences between Han ++ and Dorset sheep, however, was fewer than that of the comparison of Han BB and Dorset sheep. Seven randomly selected genes with AS events were detected in this study and were validated by reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, there are many genes which were common to the two genotype groups (Han BB and Dorset sheep, as well as the Han ++ and Dorset sheep). In addition, genes detected in the present study were involved in different pathways, including the pathways related with fertility or fecundity. The present study could provide the detailed understanding on the mechanisms of alternative splicing events associated with fecundity in different sheep breeds.

An Integrated Analysis of miRNAs and Methylated Genes Encoding mRNAs and lncRNAs in Sheep Breeds with Different Fecundity.

In our previous study, we investigated the regulatory relationship between lncRNAs, miRNA, and mRNAs in an effort to shed light onto the regulatory mechanisms involved in sheep fecundity. As an extension of this study, here, we aimed to identify potential regulators of sheep fecundity using a genome-wide analysis of miRNAs and the methylated genes encoding mRNAs and lncRNAs in the ovaries of Dorset sheep (low fecundity) and Small Tail Han ewes (high fecundity) with the genotype BB (Han BB) and the genotype ++ (Han ++) by performing RNA-Seq and MeDIP-Seq analyses. Methylated coding-non-coding gene co-expression networks for Han and Dorset sheep were constructed using the methylated genes encoding the differentially expressed mRNAs and lncRNAs identified in this study. In the Han BB vs. Dorset comparison, the lncRNAs TTC26 and MYH15 had the largest degree. Similarly, the lncRNA NYAP1 had the largest degree in the Han ++ vs. Dorset comparison. None of the methylated genes encoding lncRNAs were co-expressed with the methylated genes encoding mRNAs in the Han BB vs. Han ++ comparison. The methylated genes encoding lncRNAs identified here may play a vital regulatory role in sheep breeding. Our results suggest that miRNAs might play a key role in sheep prolificacy by regulating target genes related to thyroid hormone synthesis, and methylated genes encoding lncRNAs associated with tight junctions might contribute to the high breeding rate in Han sheep. These findings may contribute to a deeper understanding of sheep prolificacy.

Co-expression analysis and identification of fecundity-related long non-coding RNAs in sheep ovaries.

Small Tail Han sheep, including the FecBBFecBB (Han BB) and FecB+ FecB+ (Han++) genotypes, and Dorset sheep exhibit different fecundities. To identify novel long non-coding RNAs (lncRNAs) associated with sheep fecundity to better understand their molecular mechanisms, a genome-wide analysis of mRNAs and lncRNAs from Han BB, Han++ and Dorset sheep was performed. After the identification of differentially expressed mRNAs and lncRNAs, 16 significant modules were explored by using weighted gene coexpression network analysis (WGCNA) followed by functional enrichment analysis of the genes and lncRNAs in significant modules. Among these selected modules, the yellow and brown modules were significantly related to sheep fecundity. lncRNAs (e.g., NR0B1, XLOC_041882, and MYH15) in the yellow module were mainly involved in the TGF-ß signalling pathway, and NYAP1 and BCORL1 were significantly associated with the oxytocin signalling pathway, which regulates several genes in the coexpression network of the brown module. Overall, we identified several gene modules associated with sheep fecundity, as well as networks consisting of hub genes and lncRNAs that may contribute to sheep prolificacy by regulating the target mRNAs related to the TGF-ß and oxytocin signalling pathways. This study provides an alternative strategy for the identification of potential candidate regulatory lncRNAs.

Genome-wide analysis of miRNAs in the ovaries of Jining Grey and Laiwu Black goats to explore the regulation of fecundity.

Goat fecundity is important for agriculture and varies depending on the genetic background of the goat. Two excellent domestic breeds in China, the Jining Grey and Laiwu Black goats, have different fecundity and prolificacies. To explore the potential miRNAs that regulate the expression of the genes involved in these prolific differences and to potentially discover new miRNAs, we performed a genome-wide analysis of the miRNAs in the ovaries from these two goats using RNA-Seq technology. Thirty miRNAs were differentially expressed between the Jining Grey and Laiwu Black goats. Gene Ontology and KEGG pathway analyses revealed that the target genes of the differentially expressed miRNAs were significantly enriched in several biological processes and pathways. A protein-protein interaction analysis indicated that the miRNAs and their target genes were related to the reproduction complex regulation network. The differential miRNA expression profiles found in the ovaries between the two distinctive breeds of goats studied here provide a unique resource for addressing fecundity differences in goats.

Ovarian transcriptomic study reveals the differential regulation of miRNAs and lncRNAs related to fecundity in different sheep.

miRNAs and lncRNAs, which represent one of the most highly expressed classes of ncRNAs in development, are attracting increasing interest. A variety of regulators is considered to be implicated in sheep species with different fecundity. However, interactions between miRNAs and lncRNAs and changes in the expression of regulatory lncRNAs in sheep fecundity have not yet been reported. To characterize the important roles of miRNAs and lncRNAs and elucidate their regulating networks in sheep prolificacy, a genome-wide analysis of miRNAs and lncRNAs from Small Tail Han sheep of genotypes FecBBFecBB (Han BB) and FecB+ FecB+ (Han++) and from Dorset sheep (Dorset) was performed. An integrated analysis of miRNAs and lncRNAs was performed to study the regulatory function of miRNAs and lncRNAs in fecundity, revealing significantly correlated patterns of expression. Dramatic changes of miRNAs and lncRNAs suggest their critical roles in sheep fecundity. In conclusion, this is the first study performing thorough investigations of regulatory relationships among lncRNAs, miRNA and mRNAs, which will provide a novel view of the regulatory mechanisms involved in sheep fecundity. These results may provide further insight into sheep fecundity and help us to improve sheep prolificacy.

Ovarian proteomic study reveals the possible molecular mechanism for hyperprolificacy of Small Tail Han sheep.

Small Tail Han sheep is a widely bred farm animal in China which has attracted lots of attention due to their high prolificacy and year-round estrus. However, the molecular mechanism of its fecundity remains unrevealed. The FecB gene polymorphism has been found to be associated with the ovulation rate and litter size of sheep. In the present study, we constructed an iTRAQ-based quantitative proteomics analysis to compare the ovarian proteomes of FecB+FecB+ genotype Small Tail Han sheep ewes (Han ++), FecB(B)FecB(B) Han ewes (Han BB) and Dorset ewes (Dorset). Hundreds of differentially expressed proteins between each two groups were identified; GO and KEGG pathway analysis indicated that the expressions of those proteins involved in ribosome assembly, protein translation and mTOR pathway between Dorset and both Han groups were highly different. Between Han ++ and Han BB groups, higher level of protein expressions were related to mitochondrial oxidation functions such as oxidoreductase activity, cytochrome-c oxidase activity and electron carrier activity. This was identified in Han BB group, which may contribute to the elevated ovulation rate of Han BB ewes. In conclusion, our work provided a prospective understanding of the molecular mechanism for high prolificacy of Small Tail Han sheep.

Genome-wide transcriptome analysis in the ovaries of two goats identifies differentially expressed genes related to fecundity.

The goats are widely kept as livestock throughout the world. Two excellent domestic breeds in China, the Laiwu Black and Jining Grey goats, have different fecundities and prolificacies. Although the goat genome sequences have been resolved recently, little is known about the gene regulations at the transcriptional level in goat. To understand the molecular and genetic mechanisms related to the fecundities and prolificacies, we performed genome-wide sequencing of the mRNAs from two breeds of goat using the next-generation RNA-Seq technology and used functional annotation to identify pathways of interest. Digital gene expression analysis showed 338 genes were up-regulated in the Jining Grey goats and 404 were up-regulated in the Laiwu Black goats. Quantitative real-time PCR verified the reliability of the RNA-Seq data. This study suggests that multiple genes responsible for various biological functions and signaling pathways are differentially expressed in the two different goat breeds, and these genes might be involved in the regulation of goat fecundity and prolificacy. Taken together, our study provides insight into the transcriptional regulation in the ovaries of 2 species of goats that might serve as a key resource for understanding goat fecundity, prolificacy and genetic diversity between species.

Genome-wide analysis of microRNAs identifies the lipid metabolism pathway to be a defining factor in adipose tissue from different sheep.

MicroRNAs are short (17-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. In recent years, deep sequencing of the transcriptome is increasingly being utilized with the promise of higher sensitivity for the identification of differential expression patterns as well as the opportunity to discover new transcripts, including new alternative isoforms and miRNAs. Here, we utilized RNA-seq technology to perform a genome-wide analysis of miRNAs from the adipose tissue of the two species of sheep to look for clues that might explain the fat deposition differences between the sheep. The RNA-seq analysis detected 3132 miRNAs from the adipose tissue of the Small-tail Han and Dorset sheep, of which 2893 were defined as potential new miRNAs. In addition, 54 miRNAs were differentially expressed between the two breeds of sheep. Gene ontology and pathway analyses of the predicted target genes that negatively associated with the differentially expressed miRNAs revealed that there was less active lipid metabolism in the adipose tissue of Small Tail Han sheep. This study can help understand the underlying mechanisms responsible for the morphological differences related to fat deposition between two breeds of sheep.

Genome-wide mRNA-seq profiling reveals predominant down-regulation of lipid metabolic processes in adipose tissues of Small Tail Han than Dorset sheep.

Small Tail Han and Dorset sheep are two different sheep with distinguished morphologies in fat depositions. In order to characterize their gene expression profiles, our present study took the advantages of RNA sequencing technology with the aims to identify important genes regulating the metabolisms in adipose tissues of two different sheep. In obtained high quality sequencing reads, 85.9 (Han) and 86.1% (Dorset) were uniquely aligned to Oar v3.1 sheep reference genome, and over 76% of bases in mapped reads corresponded to mRNA. Using R package EBSeq, we identified 602 differentially expressed genes. Using the 602 genes, GO analysis showed that 30 out of 56 significantly enriched biological processes were metabolism related, of which the most significant one was triglyceride biosynthetic process. The KEGG pathway analysis indicated the down-regulation of several fat metabolic pathways. The predominant down-regulation of massive metabolic processes, particularly the lipid metabolism, in adipose tissues of Han sheep could explain, at least in part, the distinguished fat deposition between two different sheep, and our data constitute a basic picture of transcriptomes in these sheep for better understanding of underline biological mechanism in their lipid metabolisms.

Genome-wide analysis reveals the differential regulations of mRNAs and miRNAs in Dorset and Small Tail Han sheep muscles.

Sheep are highly diverse species raised for meat and other agricultural products. The aim of the present study was to investigate the genetic regulators that could control muscle growth and development in different sheep breeds. The study showed that the differentially expressed genes are involved in various cellular activities, such as metabolic cascades, catalytic function and signaling pathway. Many signaling molecules are also found to be differentially expressed, suggesting important roles of signaling pathways contributing to genetic diversity and sheep development. Analysis of miRNAs suggested important roles of miRNAs in controlling muscle differences. This study provided a genome-wide resolution of mRNA and miRNA regulations in muscles from Dorset and Han sheep.

Genome-wide transcriptome analysis between small-tail Han sheep and the Surabaya fur sheep using high-throughput RNA sequencing.

The small-tail Han sheep and the Surabaya fur sheep are two local breeds in north China, which are characterized by high-fecundity and low-prolificacy breed respectively. Significant genetic differences between these two breeds have provided increasing interests in the identification and utilization of major prolificacy genes in these sheep. High prolificacy is a complex trait, and it is difficult to comprehensively identify the candidate genes related to this trait using the single molecular biology technique. To understand the molecular mechanisms of fecundity and provide more information about high prolificacy candidate genes in high- and low-fecundity sheep, we explored the utility of next-generation sequencing technology in this work. A total of 1.8 Gb sequencing reads were obtained and resulted in more than 20 000 contigs that averaged ∼300 bp in length. Ten differentially expressed genes were further verified by quantitative real-time RT-PCR to confirm the reliability of RNA-seq results. Our work will provide a basis for the future research of the sheep reproduction.

[An update on the development of transgenic animal technology].

The animal transgenic technology has increasingly turned mature over several decades and promoted the research of transgenic technology to a new developmental phase. In this review, various kinds of transgenic technologies, including somatic cell nuclear transfer, gene transfer mediated by transposon, gene knockout mediated by RNA interference, and zinc-finger nucleases-gene targeting technology, are summarized. Recently, the success of induced pluripotent stem cells (iPS cells), which has provided an alternative way to derive pluripotent stem cells of large animals, will extend the field of transgenic animal studies. Here, we summarized the latest trends on the basis of previous studies. In addition, the characteristics of different kinds of transgenic methods in detail are discussed.