BackgroundNumerous long non-coding RNAs (lncRNAs) have been identified and their roles in gene regulation in humans, mice, and other model organisms studied; however, far less research has been focused on lncRNAs in farm animal species. While previous studies in chickens, cattle, and pigs identified lncRNAs in specific developmental stages or differentially expressed under specific conditions in a limited number of tissues, more comprehensive identification of lncRNAs in these species is needed. The goal of the FAANG Consortium (Functional Annotation of Animal Genomes) is to functionally annotate animal genomes, including the annotation of lncRNAs. As one of the FAANG pilot projects, lncRNAs were identified across eight tissues in two adult male biological replicates from chickens, cattle, and pigs.ResultsComprehensive lncRNA annotations for the chicken, cattle, and pig genomes were generated by utilizing RNA-seq from eight tissue types from two biological replicates per species at the adult developmental stage. A total of 9393 lncRNAs in chickens, 7235 lncRNAs in cattle, and 14,429 lncRNAs in pigs were identified. Including novel isoforms and lncRNAs from novel loci, 5288 novel lncRNAs were identified in chickens, 3732 in cattle, and 4870 in pigs. These transcripts match previously known patterns of lncRNAs, such as generally lower expression levels than mRNAs and higher tissue specificity. An analysis of lncRNA conservation across species identified a set of conserved lncRNAs with potential functions associated with chromatin structure and gene regulation. Tissue-specific lncRNAs were identified. Genes proximal to tissue-specific lncRNAs were enriched for GO terms associated with the tissue of origin, such as leukocyte activation in spleen.ConclusionsLncRNAs were identified in three important farm animal species using eight tissues from adult individuals. About half of the identified lncRNAs were not previously reported in the NCBI annotations for these species. While lncRNAs are less conserved than protein-coding genes, a set of positionally conserved lncRNAs were identified among chickens, cattle, and pigs with potential functions related to chromatin structure and gene regulation. Tissue-specific lncRNAs have potential regulatory functions on genes enriched for tissue-specific GO terms. Future work will include epigenetic data from ChIP-seq experiments to further refine these annotations.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5037-7) contains supplementary material, which is available to authorized users.

Formation of the inner cell mass (ICM) and trophectoderm (TE) marks the first differentiation event in mammalian development. These two cell types have completely divergent fates for the remainder of the developmental process. The molecular mechanisms that regulate ICM and TE formation are poorly characterized in horses. The objective of this study was to establish the transcriptome profiles of ICM and TE cells from horse blastocysts using RNA sequencing (RNA-seq). A total of 12 270 genes were found to be expressed in either lineage. Global analysis of the transcriptome profiles by unsupervised clustering indicated that ICM and TE samples presented different gene expression patterns. Statistical analysis indicated that 1662 genes were differentially expressed (adjusted P < 0.05 and fold change > 2) between ICM and TE. Genes known to be specific to the ICM and TE were expressed primarily in their respective tissue. Transcript abundance for genes related to biological processes important for horse blastocyst formation and function is presented and discussed. Collectively, our data and analysis serve as a valuable resource for gene discovery and unraveling the fundamental mechanisms of early horse development.

J.M.R. was supported by an Austin Eugene Lyons Fellowship awarded by the University of California, Davis. The Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (awarded to P.J.R.; R01HD070044) and the Fertility Laboratories of Colorado partly supported the research presented in this manuscript.

Molecular changes occurring during mammalian oocyte maturation are partly regulated by cytoplasmic polyadenylation (CP) and affect oocyte quality, yet the extent of CP activity during oocyte maturation remains unknown. Single bovine oocyte RNA sequencing (RNA-Seq) was performed to examine changes in transcript abundance during in vitro oocyte maturation in cattle. Polyadenylated RNA from individual germinal-vesicle and metaphase-II oocytes was amplified and processed for Illumina sequencing, producing approximately 30 million reads per replicate for each sample type. A total of 10,494 genes were found to be expressed, of which 2,455 were differentially expressed (adjusted P<0.05 and fold change >2) between stages, with 503 and 1,952 genes respectively increasing and decreasing in abundance. Differentially expressed genes with complete 3’-untranslated-region sequence (279 increasing and 918 decreasing in polyadenylated transcript abundance) were examined for the presence, position, and distribution of motifs mediating CP, revealing enrichment (85%) and lack there of (18%) in up- and down-regulated genes, respectively. Examination of total and polyadenylated RNA abundance by quantitative PCR validated these RNA-Seq findings. The observed increases in polyadenylated transcript abundance within the RNA-Seq data are likely due to CP, providing novel insight into targeted transcripts and resultant differential gene expression profiles that contribute to oocyte maturation.

The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU) forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt), disrupts the homolog of the Caenorhabditis elegans (C. elegans) unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans.

BackgroundUse of RNA-Seq presents unique benefits in terms of gene expression analysis because of its wide dynamic range and ability to identify functional sequence variants. This technology provides the opportunity to assay the developing embryo, but the paucity of biological material available from individual embryos has made this a challenging prospect.ResultsWe report here the first application of RNA-Seq for the analysis of individual blastocyst gene expression, SNP detection, and characterization of allele specific expression (ASE). RNA was extracted from single bovine blastocysts (n = 5), amplified, and analyzed using high-throughput sequencing. Approximately 38 million sequencing reads were generated per embryo and 9,489 known bovine genes were found to be expressed, with a high correlation of expression levels between samples (r > 0.97). Transcriptomic data was analyzed to identify SNP in expressed genes, and individual SNP were examined to characterize allele specific expression. Expressed biallelic SNP variants with allelic imbalances were observed in 473 SNP, where one allele represented between 65-95% of a variant’s transcripts.ConclusionsThis study represents the first application of RNA-seq technology in single bovine embryos allowing a representation of the embryonic transcriptome and the analysis of transcript sequence variation to describe specific allele expression.

Embryo morphokinetics suggests that the timing of the first embryonic cell divisions may predict the developmental potential of an embryo; however, correlations between embryonic morphokinetics and physiology are not clear. Here, we used RNA sequencing to determine the gene expression profile of in vitro-produced early- and late-dividing bovine embryos and their respective blastocysts, and compared these profiles to in vivo-produced blastocysts to identify differentially expressed genes (DEGs). Principal component analysis revealed that fast- and slow-dividing embryos possess similar transcript abundance over the first cleavages. By the blastocyst stage, however, more DEGs were observed between the fast- and slow-dividing embryo groups, whereas blastocysts from the slow-dividing group were more similar to in vivo-produced blastocysts. Gene ontology enrichment analysis showed that the slow-dividing and in vivo-produced blastocysts shared biological processes related to groups of up- or down-regulated genes when compared to the fast-dividing blastocysts. Based on these DEG results, we characterized the relationship between developmental kinetics and energy metabolism of in vitro-produced bovine embryos. Embryos from fast- and slow-dividing groups exhibited different pyruvate and lactate metabolism at 22 hr post-in vitro culture (hpc), glucose consumption at 96 hpc, and glutamate metabolism at 168 hpc. Glycogen storage was similar between cleavage-stage and morulae groups, but was higher in the blastocysts of the slow-dividing group. On the other hand, blastocysts of the fast-dividing group had a higher concentration of lipids. Taken together, these data identify transcriptomic and metabolic differences between embryos with different morphokinetics, suggesting that sorting embryos based on cleavage speed may select for different metabolic patterns. Mol. Reprod. Dev. 83: 324-336, 2016. © 2016 Wiley Periodicals, Inc.

Our objective was to determine whether oxidative damage of rhesus macaque sperm induced by reactive oxygen species (ROS) in vitro would affect embryo development following intracytoplasmic sperm injection (ICSI) of metaphase II (MII) oocytes. Fresh rhesus macaque spermatozoa were treated with ROS as follows: 1 mM xanthine and 0.1 U/ml xanthine oxidase (XXO) at 37°C and 5% CO₂ in air for 2.25 h. Sperm were then assessed for motility, viability, and lipid peroxidation. Motile ROS-treated and control sperm were used for ICSI of MII oocytes. Embryo culture was evaluated for 3 days for development to the eight-cell stage. Embryos were fixed and stained for signs of cytoplasmic and nuclear abnormalities. Gene expression was analyzed by RNA-Seq in two-cell embryos from control and treated groups. Exposure of sperm to XXO resulted in increased lipid peroxidation and decreased sperm motility. ICSI of MII oocytes with motile sperm induced similar rates of fertilization and cleavage between treatments. Development to four- and eight-cell stage was significantly lower for embryos generated with ROS-treated sperm than for controls. All embryos produced from ROS-treated sperm demonstrated permanent embryonic arrest and varying degrees of degeneration and nuclear fragmentation, changes that are suggestive of prolonged senescence or apoptotic cell death. RNA-Seq analysis of two-cell embryos showed changes in transcript abundance resulting from sperm treatment with ROS. Differentially expressed genes were enriched for processes associated with cytoskeletal organization, cell adhesion, and protein phosphorylation. ROS-induced damage to sperm adversely affects embryo development by contributing to mitotic arrest after ICSI of MII rhesus oocytes. Changes in transcript abundance in embryos destined for mitotic arrest is evident at the two-cell stage of development.