Integrated Whole-Transcriptome Analysis to Elucidate the Core Regulatory Network of circRNA Involved in Ovarian Development and Reproductive Capacity Differences in Sheep: circRNA2058-miR-9226-5p-MET Axis

(1) Background: This study aims to systematically identify key candidate genes and the regulatory networks governing ovarian development in sheep breeds with divergent fecundity. Focusing on elucidating the central regulatory roles of these factors during distinct ovarian developmental stages in highly prolific breeds, the research seeks to reveal the mechanism by which multilevel regulatory networks synergistically determine ewe reproductive capacity. (2) Methods: This study utilized the ovaries from the low-fertility sheep breed Ujumqin sheep, the high-fertility sheep breed Small-tailed Han sheep, and various developmental stages of Small-tailed Han sheep as research subjects. Through whole-transcriptome sequencing analysis, differentially expressed mRNAs(DEGs) and non-coding RNAs (ncRNAs) were screened, and a ceRNA regulatory network was constructed and subjected to bioinformatic analysis. The dual-luciferase reporter gene detection system was employed to validate the targeting relationships within the obtained key circRNA-miRNA-mRNA networks. Finally, qRT-PCR was used to verify the accuracy of the sequencing results. (3) Results: The results revealed that the different fecundity groups constructed a ceRNA network comprising 116 differentially expressed circRNAs (DECs), 46 differentially expressed miRNAs (DEMs), and 82 DEGs. Similarly, the groups representing different ovarian developmental stages constructed a ceRNA network consisting of 186 DECs, 143 DEMs, and 338 DEGs. Functional enrichment analysis identified several reproduction-related signaling pathways, such as the MAPK, JAK-STAT, and WNT signaling pathways (in the different fecundity groups), and the MAPK, Ras, WNT, and Hippo signaling pathways (in the different ovarian developmental stage groups). Through comprehensive analysis of the reproduction-related pathways and screening of genes co-regulating the ceRNA networks in both comparison groups, a circRNA-miRNA-mRNA regulatory network was constructed. This analysis ultimately identified circRNA2058-miR-9226-5p-MET as playing a core regulatory role. The targeting relationship was validated using the dual-luciferase reporter assay system. The results demonstrated that miR-9226-5p mediates the expression of MET through sponging by circRNA2058. The accuracy of the sequencing results was further confirmed by qRT-PCR analysis of 8 randomly selected circRNAs and 8 miRNAs. (4) Conclusions: This study innovatively deciphered the synergistic regulatory network involving DE ncRNAs and their target genes. We identified, for the first time, a pivotal ceRNA regulatory axis: circRNA2058-miR-9226-5p-MET, which functions as a critical molecular switch driving the follicular dominance process in sheep. This discovery establishes a molecular foundation for precisely targeting core regulators of ovine reproductive efficiency, deepens our understanding of the core regulatory mechanisms governing sheep reproductive biology, and offers significant guidance for innovating strategies to enhance sheep reproductive efficiency.