Sequential miRNA-mRNA profiling of regulatory pathways during in vitro maturation of canine oocytes

The acquisition of meiotic and developmental competence in oocytes is a highly regulated process that includes post-transcriptional mechanisms mediated by microRNAs (miRNAs). To characterize these changes, we compared mRNA and miRNA expression profiles between non-matured, and oocytes submitted to IVM in canines using RNA sequencing. The oocytes were obtained from antral follicles dissected from canine ovaries. Total RNA was isolated from pooled oocytes from each condition and subjected to parallel mRNA and miRNA sequencing, followed by differential expression and pathway enrichment analyses. Our findings reveal transcriptional change after in vitro culture, showing significant differential expression (FDR < 0.05, |log₂FC| > 1). Non-matured oocytes displayed an expression pattern associated with transcriptional preparation and structural organization, while IVM oocytes showed expression patterns consistent with cytoplasmic remodeling and metabolic activation. The miRNA profile changed with maturation, accompanied by a selective increase in miRNAs associated with post-transcriptional control of the cell cycle and structural pathways. Enrichment analysis revealed significant changes in signaling networks associated with cell adhesion, cytoskeletal organization, and communication. Components of the Hippo signaling pathway displayed altered transcript abundance following IVM, suggesting that structural and regulatory cues present within the oviduct may not be fully reproduced in culture. Our findings provide new insights into the molecular constraints of canine IVM that may serve as biomarkers for improving oocyte quality and reproductive technology outcomes in dogs.