Abstract
BACKGROUND: Granulosa cells (GCs) play a central role in oocyte maturation and follicular development, and their function is very sensitive to oxidative stress, which can compromise female fertility. Extracellular vesicles (EVs) released by GCs serve as intercellular messengers and are known to carry bioactive molecules, including microRNAs (miRNAs), which can modulate stress responses and cellular communication within the follicular environment. Understanding the molecular mechanisms underlying oxidative stress responses in reproductive tissues is crucial for improving fertility. This study aimed to characterize and compare the miRNA profiles of both GCs and their secreted EVs following exposure to oxidative stress. Bovine GCs were treated with 5 µM H(2)O(2) for 40 min, followed by 24 h of culture. EVs were isolated from the conditioned media using size-exclusion chromatography and characterized by nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry. Small RNA sequencing was performed on both the GCs and their secreted EVs to identify stress-responsive miRNA signatures. RESULTS: Exposure to oxidative stress increased intracellular ROS levels and decreased mitochondrial activity in GCs, confirming successful stress induction. Differential expression analysis revealed no significant changes in miRNA abundance in GCs in response to oxidative stress, suggesting cellular adaptation mechanisms that preserve intracellular miRNA homeostasis. In GC derived EVs, however, 10 miRNAs were significantly more abundant, including miR-134, miR-10175-5p, miR-197, miR-2284 h-5p, miR-2284y, miR-2285av, miR-2285au, miR-369-5p, miR-2411-5p and miR-2387. These miRNAs showed log2 fold changes ranging from 3.57 to 6.89 compared to control, indicating substantial enrichment in stress-derived EVs. Functional enrichment analysis of predicted targets for the higher abundant EV-miRNAs revealed involvement in processes such as angiogenesis, cell migration as well as WNT, MAPK and Oxytocin signaling pathways, all critical for follicular function and stress adaptation. CONCLUSIONS: These findings suggest that oxidative stress alters the miRNA cargo of EVs, potentially affecting intra-follicular communication and stress adaptation The selective packaging of specific miRNAs into EVs under stress conditions indicates a sophisticated cellular mechanism for intercellular communication during adverse conditions. This dual characterization provides new insights into the regulatory role of EV-derived miRNAs in ovarian physiology under oxidative conditions and establishes the foundation for developing EVs-miRNAs as potential biomarkers for reproductive health assessment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s40659-026-00689-8.