Pregnancy success results from the interaction of multiple factors, among them are folliculogenesis and early embryonic development. Failure during these different processes can lead to difficulties in conception. Alternatives to overcome these problems are based on assisted reproductive techniques. Extracellular vesicles are cell-secreted vesicles present in different body fluids and contain bioactive materials, such as messenger RNA, microRNAs (miRNAs), and proteins. Thus, our hypothesis is that extracellular vesicles from follicular fluid from 3–6 mm ovarian follicles can modulate bovine embryo development in vitro. To test our hypothesis follicular fluid from bovine ovaries was aspirated and small-extracellular vesicles (<200 nm) were isolated for further analysis. Additionally, small-extracellular vesicles (EVs) were utilized for functional experiments investigating their role in modulating messenger RNA, microRNA as well as global DNA methylation and hydroxymethylation levels of bovine blastocysts. EVs from 3–6 mm follicles were used for RNA-seq and miRNA analysis. Functional annotation analysis of the EVs transcripts revealed messages related to chromatin remodeling and transcriptional regulation. EVs treatment during oocyte maturation and embryo development causes changes in blastocyst rates, as well as changes in the transcription levels of genes related to embryonic metabolism and development. Supplementation with EVs from 3–6 mm follicles during oocyte maturation and early embryo development (until the 4-cell stage) increased the levels of bta-miR-631 (enriched in EVs from 3–6 mm follicles) in embryos. Interestingly, the addition of EVs from 3–6 mm follicles induced changes in global DNA methylation and hydroxymethylation levels compared to embryos produced by the standard in vitro production system. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of 3–6 mm follicles during oocyte maturation and early embryo development can partially modify metabolic and developmental related genes as well as miRNA and global DNA methylation and hydroxymethylation, suggesting that EVs play an important role during oocyte maturation and early embryo development in vitro.
Intramuscular fat (IMF) content is related to insulin resistance, which is an important prediction factor for disorders, such as cardiovascular disease, obesity and type 2 diabetes in human. At the same time, it is an economically important trait, which influences the sensorial and nutritional value of meat. The deposition of IMF is influenced by many factors such as sex, age, nutrition, and genetics. In this study Nellore steers (Bos taurus indicus subspecies) were used to better understand the molecular mechanisms involved in IMF content. This was accomplished by identifying differentially expressed genes (DEG), biological pathways and putative regulatory factors. Animals included in this study had extreme genomic estimated breeding value (GEBV) for IMF. RNA-seq analysis, gene set enrichment analysis (GSEA) and co-expression network methods, such as partial correlation coefficient with information theory (PCIT), regulatory impact factor (RIF) and phenotypic impact factor (PIF) were utilized to better understand intramuscular adipogenesis. A total of 16,101 genes were analyzed in both groups (high (H) and low (L) GEBV) and 77 DEG (FDR 10%) were identified between the two groups. Pathway Studio software identified 13 significantly over-represented pathways, functional classes and small molecule signaling pathways within the DEG list. PCIT analyses identified genes with a difference in the number of gene-gene correlations between H and L group and detected putative regulatory factors involved in IMF content. Candidate genes identified by PCIT include: ANKRD26, HOXC5 and PPAPDC2. RIF and PIF analyses identified several candidate genes: GLI2 and IGF2 (RIF1), MPC1 and UBL5 (RIF2) and a host of small RNAs, including miR-1281 (PIF). These findings contribute to a better understanding of the molecular mechanisms that underlie fat content and energy balance in muscle and provide important information for the production of healthier beef for human consumption.
BackgroundPositively correlated with carcass weight and animal growth, the ribeye area (REA) and the backfat thickness (BFT) are economic important carcass traits, which impact directly on producer’s payment. The selection of these traits has not been satisfactory since they are expressed later in the animal’s life and multigene regulated. So, next-generation technologies have been applied in this area to improve animal’s selection and better understand the molecular mechanisms involved in the development of these traits. Correlation network analysis, performed by tools like WGCNA (Weighted Correlation Network Analysis), has been used to explore gene-gene interactions and gene-phenotype correlations. Thus, this study aimed to identify putative candidate genes and metabolic pathways that regulate REA and BFT by constructing a gene co-expression network using WGCNA and RNA sequencing data, to better understand genetic and molecular variations behind these complex traits in Nelore cattle.ResultsThe gene co-expression network analysis, using WGCNA, were built using RNA-sequencing data normalized by transcript per million (TPM) from 43 Nelore steers. Forty-six gene clusters were constructed, between them, three were positively correlated (p-value< 0.1) to the BFT (Green Yellow, Ivory, and Light Yellow modules) and, one cluster was negatively correlated (p-value< 0.1) with REA (Salmon module). The enrichment analysis performed by DAVID and WebGestalt (FDR 5%) identified eight Gene Ontology (GO) terms and three KEGG pathways in the Green Yellow module, mostly associated with immune response and inflammatory mechanisms. The enrichment of the Salmon module demonstrated 19 GO terms and 21 KEGG pathways, related to muscle energy metabolism, lipid metabolism, muscle degradation, and oxidative stress diseases. The Ivory and Light yellow modules have not shown significant results in the enrichment analysis.ConclusionWith this study, we verified that inflammation and immune response pathways modulate the BFT trait. Energy and lipid metabolism pathways, highlighting fatty acid metabolism, were the central pathways associated with REA. Some genes, as RSAD2, EIF2AK2, ACAT1, and ACSL1 were considered as putative candidate related to these traits. Altogether these results allow us to a better comprehension of the molecular mechanisms that lead to muscle and fat deposition in bovine.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5345-y) contains supplementary material, which is available to authorized users.
Mineral contents in bovine muscle can affect meat quality, growth, health, and reproductive traits. to better understand the genetic basis of this phenotype in nelore (Bos indicus) cattle, we analysed genome-wide mRNA and miRNA expression data from 114 muscle samples. The analysis implemented a new application for two complementary algorithms: the partial correlation and information theory (pcit) and the regulatory impact factor (Rif), in which we included the estimated genomic breeding values (GeBVs) for the phenotypes additionally to the expression levels, originally proposed for these methods. We used PCIT to determine putative regulatory relationships based on significant associations between gene expression and GeBVs for each mineral amount. then, Rif was adopted to determine the regulatory impact of genes and miRnAs expression over the GeBVs for the mineral amounts. We also investigated over-represented pathways, as well as pieces of evidences from previous studies carried in the same population and in the literature, to determine regulatory genes for the mineral amounts. for example, NOX1 expression level was positively correlated to Zinc and has been described as Zinc-regulated in humans. Based on our approach, we were able to identify genes, miRnAs and pathways not yet described as underlying mineral amount. the results support the hypothesis that extracellular matrix interactions are the core regulator of mineral amount in muscle cells. putative regulators described here add information to this hypothesis, expanding the knowledge on molecular relationships between gene expression and minerals. Besides nutritional quality, mineral amount affects meat quality in many ways. For example, the tenderization process of the skeletal muscle is driven by the action of the calcium-dependent protease calpain 1-4. Minerals also affect reproduction, as copper, zinc, selenium and manganese supplementation improves pregnancy rate 5 ; as well as health and growth performance 6,7 in beef cattle. Mineral homeostasis regulation partially depends on genetic factors, among others 8. Thus, understanding the genetic aspects linked to mineral amount in bovine muscle can
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