Abstract：The hypothalamic-pituitary-ovarian axis (HPO) is an important neuroendocrine regulatory center in mammalian reproduction. microRNAs (miRNAs) are endogenous non-coding small RNAs that regulate gene expression at the post-transcriptional level. However, the miRNA-mRNA interaction network of the reproduction-related HPO axis in Hu sheep (Ovis aries) remains understudied. In this study, the hypothalamus, pituitary and ovary tissues of Hu sheep during follicular and luteal phases in different estrous cycles were studied, and differentially expressed miRNAs were screened by small RNA-seq (sRNA-seq) sequencing technology using bioinformatics analysis. qPCR was used to detect the expression levels of miRNAs in Hu sheep tissues, to predict their target gene intersection and functional enrichment analysis, and dual luciferase reporter gene to detect target relationships. The results showed that a total of 849 known miRNAs and 632 novel miRNAs were detected in different estrous cycles (follicular and luteal phases), among which 64 differentially expressed miRNAs (30 up-regulated and 34 down-regulated) were found in the hypothalamus group during the follicular and luteal phases. There were 5 differentially expressed miRNAs (4 up-regulated and 1 down-regulated) in the follicular and luteal pituitary groups, and 43 differentially expressed miRNAs (25 up-regulated and 18 down-regulated) in the follicular and luteal ovarian groups. Functional enrichment annotation analysis of Gene Ontology (GO) and KEGG differentially expressed miRNAs by R package showed that the target genes were mainly involved in Notch signaling pathway, signal transduction, cellular communication, innate immune response and amino acid metabolism. Notch signaling pathway, pyrimidine nucleotide biosynthesis and metabolism, and amino acid metabolism play an important role in the regulation of reproduction in Huyang sheep. In addition, the relationship between differentially expressed miRNAs and target genes was analyzed by constructing miRNA-mRNA interaction networks. oar-miR-432, oar-miR-29a, oar-miR-200 and their target genes phospholipase A2 group 3 (PLA2G3), cytochrome P450 3A24 (CYP3A24), dopamine receptors 2 (DRD2), and signal transducers and activators of transcription 2 (STAT2) were enriched in signaling pathways such as brain neurotransmission, ovarian follicle growth and steroid hormone synthesis and metabolism, and may be involved in regulating the biological process of ovarian transition from follicular phase to luteal phase. The results of dual luciferase reporter gene assay showed that oar-miR-432 has a target relationship with the target gene STAT2. In this study, key miRNAs for seasonal estrus traits were screened to provide basic information for analyzing the mechanism of estrus and accelerating the selection and breeding of new strains of Hu sheep at the level of post-transcriptional regulation.
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