Exploring differentially expressed genes in hypothalamic, pars tuberalis and pineal gland transcriptomes in different sexual behavior phenotypes in rams using RNA-Seq

Reproductive seasonality is a limiting factor in sheep production. Sexual behavior is a key element in reproductive efficiency, and this function is regulated by the hypothalamus-pituitary-gonadal (HPG) axis. To understand the mechanisms of sexual behavior, transcriptomic sequencing technology was used to identify differentially expressed genes (DEGs) in the hypothalamus (HT), pars tuberalis (PT) and pineal gland (PG) in Rasa Aragonesa rams with different sexual behavior. Bioinformatics analysis of the 16,401 identified genes by RNA-Seq revealed 103 and 12 DEGs in the HT and the PG, respectively, at a false discovery rate (FDR) of 5% with an absolute value of expression ≥ 1 (log2FC). However, no DEGs were found in the PT. Functional annotation and pathway enrichment analysis showed that DEGs of HT were enriched mainly in neuroactive ligand-receptor interactions and signaling pathways, including notable candidate genes such as MTNR1A, CHRNA2, FSHB, LHB, GNRHR, AVP, PRL, PDYN, CGA, GABRD, and TSHB, which play a crucial role in sexual behavior. The GnRH and cAMP signaling pathways were also highlighted. In addition, gene set enrichment analysis (GSEA) identified potential pathways, dominated mainly by biological process category, that could be responsible for the differences in sexual behavior observed in rams. The intracellular protein transport and pattern specification process were enriched within the PT and the transcription factor binding and protein ubiquitination pathways for the PG. Thus, these pathways together may play an important role in the regulation of the sexual behavior in Rasa Aragonesa rams through the hypothalamic-pituitary-gonadal axis. The validation of 5 DEGs using reverse transcription quantitative polymerase chain reaction (RT-qPCR) showed expression patterns like the found with RNA-Seq. Overall, these results contribute to understanding the genomic basis of sexual behavior in rams. Our study demonstrates that multiple networks and pathways orchestrate sexual behavior in sheep.Male sexual behavior is a key factor in reproduction, especially in seasonal breeders such as sheep. The identification of differentially expressed genes (DEGs) in brain regions involved in male reproduction and sexual behavior between rams with different sexual activity by RNA high-throughput sequencing can provide useful information to the sheep meat industry. This work aimed to determine the possible molecular mechanisms underlying the sexual behavior of Rasa Aragonesa rams by investigating transcriptional changes in the hypothalamus (HT), pars tuberalis (PT) and pineal gland (PG) between active (A) and nonactive (NA) rams. Comparative analysis revealed 103 and 12 DEGs between the A vs. NA comparison in the HT and the PG, respectively, but no DEGs were found in the PT. Gene ontology (GO) enrichment analysis of DEGs in HT samples revealed significant pathways, associated mainly with neuroactive ligand-receptor interactions, and the GnRH and cAMP signaling pathways. Furthermore, gene set enrichment analysis (GSEA) detected many overrepresented pathways related to sexual behavior via an interaction network within the hypothalamic-pituitary-gonadal axis. These data will be helpful for further investigations to look for mutations or functional single nucleotide polymorphisms (SNPs) that may be used for genetic assisted selection to improve sexual behavior in sheep.