# 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

## TL;DR

This study identifies a key regulatory network involving circRNA2058, miR-9226-5p, and MET that controls ovarian development and reproductive capacity in sheep.

## Contribution

The study is the first to identify the circRNA2058-miR-9226-5p-MET axis as a critical molecular switch in sheep reproduction.

## Key findings

- A core circRNA-miRNA-mRNA network was identified, with circRNA2058-miR-9226-5p-MET as a central regulatory axis.
- circRNA2058 acts as a sponge for miR-9226-5p, regulating MET expression in follicular development.
- Key signaling pathways like MAPK, JAK-STAT, and WNT were linked to reproductive differences in sheep.

## Abstract

This study investigated the genetic regulation of ovarian development in sheep with high and low fecundity. Using whole-transcriptome sequencing of ovaries from different breeds and developmental stages, the researchers constructed competing endogenous RNA (ceRNA) networks. These networks revealed key interactions between circRNAs, miRNAs, and mRNAs. A central regulatory axis, circRNA2058-miR-9226-5p-MET, was identified and experimentally validated. This axis acts as an potential critical molecular switch controlling follicular development. The findings provide important insights into the molecular mechanisms behind sheep reproduction and offer a foundation for developing new strategies to improve reproductive efficiency in sheep.

(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-fecundity sheep breed Ujumqin sheep, the high-fecundity 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: Our analysis constructed two distinct ceRNA networks: one from different fecundity groups (116 DECs, 46 DEMs, 82 DEGs) and another from different ovarian stages (186 DECs, 143 DEMs, 338 DEGs). Functional enrichment revealed key reproduction-related pathways, including Mitogen-Activated Protein Kinase(MAPK), Janus Kinase-Signal Transducer and Activator of Transcription(JAK-STAT), and WNT signaling in the fecundity comparison, and MAPK, Ras, WNT, Hippo signaling in the developmental stage comparison. Integrated analysis identified a core circRNA-miRNA-mRNA network, pinpointing circRNA2058-miR-9226-5p-MET as a central regulatory axis. The dual-luciferase assay confirmed that circRNA2058 acts as a sponge for miR-9226-5p, thereby mediating MET expression. qRT-PCR validation of randomly selected RNAs confirmed the sequencing reliability. (4) Conclusions: this study deciphers a synergistic regulatory network and identifies, for the first time, the pivotal circRNA2058-miR-9226-5p-MET ceRNA axis as an potential critical molecular switch driving follicular dominance in sheep. This discovery provides a molecular foundation for targeting core regulators of ovine reproductive efficiency and offers significant insights for innovative strategies in enhancing sheep reproduction.