Integrative analysis of circRNA, miRNA, and mRNA profiles to reveal ceRNA regulation in geese ovarian development from the birth /laying to ceased periods

Laying performance is a key metric for assessing avian reproductive efficiency. Ovarian tissues from Wanxi White Geese (WWGs) at different laying stages (birth period, laying period, and ceased period) were used in this study as research subjects. mRNA and circRNA expression profiles of ovarian tissues were constructed across various laying phases using transcriptome sequencing technology and bioinformatics approaches. The results revealed a total of 504 differentially expressed circRNAs (DEcircRNAs) (BO vs LO), 369 DEcircRNAs (LO vs CO), and 306 DEcircRNAs (BO vs CO) across different laying stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses demonstrated that DEcircRNA target genes were significantly enriched in processes including ovarian granulosa-cell proliferation, apoptosis, differentiation, and folliculogenesis. Concurrently, these targets were prominently implicated in phosphoinositide metabolism and played key roles in the GnRH, MAPK, and p53 signaling pathways. The expression of DEGs and proteins in ovarian tissues at different laying periods was detected by qRT-PCR technology and Western blot technology respectively. The results showed that the sequencing results were true and reliable. A ceRNA (circRNA-miRNA-mRNA) network was successfully constructed, and four key ceRNA regulatory axes implicated in ovarian development and steroid hormone synthesis/secretion were identified, namely novel_circ_107999-miR-1 44-y-NR2F2/TGM2, novel_circ_072697-miR-143-x-FMN2, novel_circ_031722-miR-101-x-CASP2/RPN 2, and novel_circ_100886-miR-101-y-PTX3/SEMA3 E/HSD3B1. The dual-luciferase reporter gene assay confirmed a targeting relationship between the novel_circ_072697-miR-143-x-FMN2 axis. In conclusion, this study system-atically explored the coordinated regulatory mechanisms of DEcircRNAs and their target genes, providing a theoretical foundation for identifying the key genes and molecular mechanisms underlying ovarian development in WWGs across different laying stages. These findings advance our understanding of the biological features of seasonal reproduction in this species and offer valuable insights for enhancing avian reproductive performance.