Project description:Purpose: High-throughput sequencing technology were used to explore transcriptional changes in the PT and the HT tissues during follicular (F) and luteal (L) phases of estrus cycle, and anestrus phase in seasonal ewes. Real-Time Quantitative PCR Analysis (RT-qPCR) were performed to evaluate the reliability of our RNA-seq results. Methods: The PT and HT transcriptomes of 21 Rasa Aragonesa ewes (7 for each phase and tissue) were generated by Illumina Hi-Seq 2000 platform. The reads were trimmed with Trimmomatic. HISAT2were used to align lean reads to the ovine reference genome Texel Oar_v3.1 Texel Oar_v3.1 followed by samtools to be converted and sorted to bam files. StringTie was used to assemble transcripts and quantify RNA-seq. Differential gene expression were mesured using DESeq2 package in R. RT-qPCR validation of 5 housekeeping genes and 16 DEGs was performed using SYBR Premix Ex Taq II and an ABI Prism 7500 platform running the samples in triplicate. Results: RNA sequencing of HT and PT generated pproximately, 834 and 874 million paired-end reads respectively from 42 libraries (21 from each tissue). On average, the overall alignment rate to reference genome was 90%.69% of all the clean reads were uniquely mapped to the reference genome. 18895 and 18960 genes (with nonzero total read count) were expressed in PT and HT, respectively. In HT, 72 and 3 DEGs were found (p_adjusted<0.05) in the comparisons between F vs A and L vs A phases, respectively. In PT, 6, 4 and 14 DE genes were found in the comparisons F vs A, L vs A and L vs F phases, respectively. The RPL19 and RPL32 were the housekeeping genes most stable for the HT whereas the B2M and RPL27 genes were the most stable for PT. With qRT-PCR analysis, Thirteen genes from the twenty analyzed were validated confirming the reliability of our RNA-seq data. Conclusions: Reliability of RNA-seq results was validated by qRT-PCR suggesting that the results of the RNA-seq experiments were efficient and accurate. These findings showed significant possible roles of these candidate genes in regulation of seasonal reproduction and in the reproductive stages. This study affords a basic data for future research to understand, more in depth, the molecular mechanism that take place during seasonal reproduction.

Project description:Living organisms detect seasonal changes in day length (photoperiod), and alter their physiological functions accordingly, to fit seasonal environmental changes. This photoperiodic system is implicated in seasonal affective disorders and the season-associated symptoms observed in bipolar disease and schizophrenia. Thyroid-stimulating hormone beta subunit (Tshb), induced in the pars tuberalis (PT), plays a key role in the pathway that regulates animal photoperiodism. However, the upstream inducers of Tshb expression remain unknown. Here we show that late-night light stimulation acutely triggers the Eya3-Six1 pathway, which directly induces Tshb expression. Using melatonin-proficient CBA/N mice, which preserve the photoperiodic Tshb-expression response, we performed a genome-wide expression analysis of the PT under chronic short-day and long-day conditions. These data comprehensively identified long-day and short-day genes, and indicated that late-night light stimulation induces long-day genes. We verified this by advancing and extending the light period by 8 hours, which acutely induced Tshb expression, within one day. In a genome-wide expression analysis under this condition, we searched for candidate upstream genes by looking for expression that preceded Tshb’s, and identified Eya3 gene. These results elucidate the comprehensive transcriptional photoperiodic response in the PT, revealing the complex regulation of Tshb expression and unexpectedly rapid response to light changes in the mammalian photoperiodic system. Mice were separated into 2 groups. One group was maintained under the short-day conditions (light: dark = 8 h:16 h, ZT0 = lights on, ZT8 = lights off, 400 lux) and the other was housed under long-day conditions (light:dark = 16 h:8 h, ZT0 = lights on, ZT16 = lights off, 400 lux) for 2 weeks. The PTs of both groups were retrieved every 4 h for 1 day (6 time points for each group), starting at ZT0. For the experiments performed during the first day of the long-day conditions, we applied two different conditions, following 3 weeks under short-day conditions. In one, the light-onset was advanced by 8 hours (advance condition), and in the other, the dark period was delayed by 8 hours (delay condition). PTs from both groups were obtained every 4 h for 1 day, starting at the lights-on time. (Lights on for the advance condition was ZT16 as defined by the short-day condition. Lights on for the delay condition was ZT0). We sampled 25 mice at each time point. This whole procedure was repeated twice (n = 2) to obtain experimental replicates.

Project description:The preservation of locally endangered breeds is essential for ecosystem services that affect both society and the environment. Reproductive fitness becomes a crucial consideration in this regard. MicroRNAs (miRNAs) are small noncoding RNA molecules with a key role in post-transcriptional regulation. They usually act in tissues, where they are produced, but when released in extracellular fluid, they are termed circulating miRNAs (c-miRNAs). C-miRNAs might be potential biomarkers, whose profile changes under different physiological states. The purpose of this work is to establish a connection between distinctive variations in c-miRNAs’ expression and specific estrus cycle phases in Frabosana-Roaschina sheep, an endangered Piedmontese breed. Two trials of twenty ewes with different reproductive efficiency (nulliparous in the first trial, pluriparous in the second trial) were sampled on alternate days, after synchronization, for blood, saliva and feces. Ultrasound scans were performed during the induced estrus cycle. Animals were also video recorded to assess behaviors. Play behaviors and no sexual behaviors were detected in the first trial; sexual behaviors and no play behaviors in the second one. Based on plasma trends of 17β-estradiol and progesterone and ultrasound images, two moments were identified for miRNAs analyses: the beginning of the follicular phase (day 2) and the beginning of luteal phase (day 11). C-miRNAs of 6 representative animals from second trial were sent to be sequenced. Analyses on the sequencing data have highlighted twelve c-miRNAs differentially expressed (DE), comparing day 11 with day 2: five miRNAs resulted upregulated, whereas seven miRNAs are downregulated. An enrichment analysis, based on predicted targets, using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) databases was performed. Many of these genes regulate reproductive pathways with the possible involvement of miRNAs. In the end, qRT-PCR validation of DE miRNAs in all ewes was performed and differences in gene expression between the two sampling points and the two trials emerged, in line with literature. The investigation of these miRNAs’ involvement in the regulation of the estrus could improve reproductive performance and welfare of Frabosana-Roaschina.

Project description:Transcriptomics and functional bioinformatics were used to investigate the potential interactions of undernutrition and the presence of the conceptus at the time of maternal recognition of pregnancy on uterine indicators of metabolism and reproduction. Adult Rasa Aragonesa ewes were allocated to one of two planes of nutrition during 28 days: maintenance energy intake (control; 5 cyclic, 6 pregnant ewes) providing 7.8 MJ of metabolisable energy per ewe and 0.5 maintenance intake (undernourished; 6 cyclic, 7 pregnant ewes) providing 3.9 MJ of metabolisable energy per ewe. RNA from uterine tissue was harvested at slaughter on day 14 of estrus or pregnancy, and hybridized to the Agilent 15K Sheep Microarray chip. Functional bioinformatics analyses were performed using the Dynamic Impact Approach (DIA) and Ingenuity Pathway Analysis. Among metabolic pathways, citrate cycle, oxidative phosphorylation, pentose and glucuronate interconversions and biosynthesis of unsaturated fatty acids were upregulated in control pregnant compared with control cyclic ewes. However, these pathways were not altered in undernourished ewes. The presence of an embryo in undernourished ewes upregulated fatty acid and glycogenic amino acid metabolism. RIG-I and Toll like receptors and chemokine signaling pathways were upregulated by the presence of the embryo in both control and undernourished ewes, but in the latter group the impact was lower. Undernutrition alone upregulated carbohydrate metabolism, but different pathways were altered in cyclic versus pregnant ewes. Citrate cycle, glycolysis/gluconeogenesis, and pentose and glucuronate interconversions were upregulated in undernourished cyclic compared with control cyclic ewes. Glycolysis/gluconeogenesis, pyruvate and propanoate metabolism, beta-alanine and phenylalanine metabolism were upregulated in undernourished pregnant compared with control pregnant ewes, whereas biosynthesis of unsaturated fatty acids and replication and repair were downregulated. Undernutrition alone led to an overall weak activation of immune system pathways both in cyclic and pregnant ewes. However, undernourished cyclic compared with control cyclic ewes had a high activation of NOD-like and RIG-1 like receptor signaling pathways, whereas undernourished pregnant compared with control pregnant ewes only had a weak upregulation of T cell receptor signaling pathways. Overall, data revealed that metabolic and immune adaptations of the uterus to nutrient restriction are dependent on the presence of the conceptus.

Dataset Information

Exploring the Pars Tuberalis (PT) and Hypothalamus (HT) transcriptome from Rasa Aragonesa ewes with different estrus and anestrus phases

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