Cross-species validation of conserved miR-504/PDHA1 and miR-500/EGLN2 interactions in sheep oocyte development

The quality of in vitro maturation (IVM) is a major factor limiting the efficiency of in vitro fertilization (IVF) and early embryonic development in livestock. This study aimed to validate, in sheep oocytes, the functional relevance of two conserved core miRNA-mRNA interactions, miRNA-504 (miR-504)/pyruvate dehydrogenase alpha 1 (PDHA1) and miRNA-500 (miR-500)/Egl nine homolog 2 (EGLN2), previously identified in porcine oocytes. Using sheep cumulus-oocyte complexes (COCs) as the experimental model, we combined bioinformatic analysis, dual-luciferase reporter assays, transfection of miRNA mimics and inhibitors, quantitative reverse transcription PCR (qRT-PCR), Western blotting (WB), fluorescence staining, IVM, IVF, and in vitro culture (IVC). Through these approaches, we systematically evaluated the effects of the two regulatory axes on sheep oocyte maturation quality, redox status, mitochondrial function, and subsequent early embryonic development, and further assessed the effects of combined intervention. The results showed that miR-504 and miR-500 were highly conserved among multiple mammalian species, with highly consistent seed sequences. Dual-luciferase assays confirmed that miR-504 and miR-500 directly targeted PDHA1 and EGLN2, respectively. Functional analyses showed that overexpression of either miR-504 or miR-500 suppressed cumulus expansion and increased apoptosis, with apoptosis rates increasing from 14.26% to 15.53% in the corresponding control groups to 18.08% and 19.46%, respectively. At the same time, intracellular reactive oxygen species (ROS) levels increased significantly, whereas glutathione (GSH) content and mitochondrial membrane potential decreased significantly. The expression of key developmental factors, including growth differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), and cyclin-dependent kinase 1 (CDK1), also declined. These findings indicate that both pathways impair developmental competence by disrupting cytoplasmic maturation. Developmental phenotype analysis further showed that inhibition of miR-504 or miR-500 increased cleavage rate, morula rate, and blastocyst rate. Blastocyst cell numbers were significantly higher in the miR-504 inhibitor and miR-500 inhibitor groups than in the control group (p < 0.05). Molecular analyses showed that miR-504 negatively regulated PDHA1 and its associated energy metabolic pathway, whereas miR-500 negatively regulated the EGLN2/hypoxia-inducible factor-1α (HIF-1α) pathway. In addition, the combined treatment group receiving both miR-504 inhibitor and miR-500 inhibitor outperformed either single-treatment group in morula formation, blastocyst formation, and blastocyst cell number (p < 0.05), indicating an enhanced effect of combined inhibition in sheep oocytes. In summary, this study supports cross-species conservation at the level of the core miRNA-mRNA interactions of miR-504/PDHA1 and miR-500/EGLN2 and supports their functional relevance in sheep oocytes. These findings provide new evidence for understanding the molecular mechanisms that regulate mammalian oocyte quality and offer a theoretical basis for improving in vitro embryo production (IVP) in sheep.