GSE191213
Exploring the Pars Tuberalis (PT) and Hypothalamus (HT) transcriptome from Rasa Aragonesa ewes with different estrus and anestrus phases
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. Overall design: Comparison of the luteal, anestrous and follicular transcriptomes in ewes Hypothalamus and Pars Tuberalis tissues