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Photoperiodic time-series mRNASeq assays of the sheep pars tuberalis

ABSTRACT: Accurate timing and anticipation of seasonal changes are required to initiate physiological adaptations over the course of the year such as hibernation, changes in metabolism, fattening and reproductive activity. To achieve this, organisms have evolved complex seasonal timekeeping systems that rely on day length sensing (photoperiodism). The aim of this project is to investigate the role of the circadian clock in photoperiodism within the crucial photoperiod sensing tissue the pars tuberalis.

ORGANISM(S): Ovis aries

PROVIDER: GSE144677 | GEO | 2020/02/04

REPOSITORIES: GEO

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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: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.

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Photoperiodic time-series mRNASeq assays of the sheep pars tuberalis

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