Omics & gene networks

Long-read sequencing reveals extracellular vesicles-induced transcriptional changes in bovine cumulus-oocyte complexes

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Abstract

Extracellular vesicles (EVs) within the ovarian follicle are increasingly recognized as functional conveyors of molecular signals that coordinate communication between cumulus cells and the oocyte. These vesicle-borne cues contribute to the acquisition of oocyte competence in vivo, yet how EVs influence RNA-level responses during in vitro culture remains insufficiently defined. Understanding these interactions is particularly relevant for pre-in vitro maturation (Pre-IVM) systems, where reconstituting elements of the follicular microenvironment may enhance developmental potential. Conventional short-read RNA sequencing has advanced knowledge of cumulus-oocyte complex (COC) biology, but its reliance on RNA fragmentation restricts the recovery of full-length transcripts and constrains the evaluation of transcript integrity. Long-read sequencing technologies-particularly Oxford Nanopore-overcome these limitations by enabling direct profiling of unfragmented polyadenylated RNA molecules, offering a more complete and biologically faithful representation of transcript diversity and gene expression. Here, we applied long-read nanopore RNA sequencing to investigate differences in transcript abundance in bovine COCs exposed to EVs isolated from small ovarian follicles (S-EVs) during a standard Pre-IVM system. COCs were aspirated from 3-6 mm follicles and assigned to two groups: (1) Control, cultured under Pre-IVM conditions without EV supplementation, and (2) S-EV Treatment, cultured under identical conditions with the addition of follicle-derived EVs. Each group consisted of three biological replicates. Total RNA was extracted, libraries were prepared following standard protocols, and sequencing was performed on the Oxford Nanopore MinION platform. Downstream analyses followed the nf-core/nanopore-rnaseq workflow, providing a standardized pipeline for basecalling, quality control, alignment to the Bos taurus reference genome, transcript quantification, and differential expression analysis. Transcript profiling detected expression of 12,531 genes across all libraries. Differential expression analysis (false discovery rate ≤ 0.05) identified 4,758 genes exhibiting significant expression differences between Control and S-EV-treated COCs, demonstrating that exposure to follicle-derived EVs was associated with widespread differences in transcript abundance during the Pre-IVM interval. Functional enrichment analyses revealed distinct biological patterns between conditions: genes with higher expression in Control COCs were predominantly associated with metabolic transport and cytoskeletal organization, whereas genes more highly expressed following EV supplementation were enriched for processes related to mRNA metabolism, calcium signaling, and cell-cycle activity. These findings suggest that EVs are associated with coordinated changes in gene expression. The presence of RNA molecules within vesicle cargo provides a biologically plausible mechanism underlying the observed RNA-level differences, as transferred transcripts may directly influence gene expression in recipient cells. Collectively, these results support the role of follicular EVs as active components of the in vitro culture environment and provide a foundation for further investigation of EV-mediated communication in oocyte-supporting cells. Together, these findings contribute to the understanding of follicle-derived signals reflected at the transcriptomic level during early in vitro culture and support efforts to approximate physiological conditions in Pre-IVM systems.

Keywords: 2026

How to Cite:

da Silveira, J., Fernandes, A. & Souza, L., (2026) “Long-read sequencing reveals extracellular vesicles-induced transcriptional changes in bovine cumulus-oocyte complexes”, World Congress on Genetics Applied to Livestock Production Digital Archive 2026(1): 2295818. doi: https://doi.org/10.31274/wcgalp.24374

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