# EV-derived small non-coding RNAs from porcine follicular fluid regulate follicle development using Pandora sequence

## TL;DR

This study shows that small RNA molecules in pig follicular fluid help control oocyte development, with differences between small and large follicles that could improve reproductive technologies.

## Contribution

The study identifies size-dependent SncRNA profiles and their regulatory roles in follicle development, offering new biomarkers for reproductive technologies.

## Key findings

- Porcine follicular fluid EVs contain distinct SncRNA profiles in small and large follicles, with piRNA and tsRNA showing significant differences.
- Eight upregulated and four downregulated SncRNAs were identified in large follicles, linked to oocyte maturation and developmental pathways.
- GO and KEGG analyses revealed key roles in oocyte meiosis, embryonic development, insulin, and Notch signaling pathways.

## Abstract

Follicular fluid extracellular vesicles (EVs) have emerged as critical mediators of intercellular communication during oocyte development. This study investigates the role of small non-coding RNAs (SncRNAs) within porcine follicular fluid extracellular vesicles in regulating oocyte maturation, with a focus on functional disparities between small (group S: < 3 mm) and large (group L:>6 mm) follicles.

Extracellular vesicles were isolated from porcine follicular fluid via ultracentrifugation and characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and nanoflow cytometry for CD63 and CD81 positive expression. Pandora sequencing of SncRNAs identified differentially expressed SncRNAs (piRNAs, rRNAs, tsRNAs, miRNAs, and snoRNAs) across follicle sizes using Pandora sequence.

Firstly, SncRNA composition analysis demonstrated distinct proportional distributions: PiRNA constituted 43% in group L vs. 51.8% in S follicles, while tsRNA showed an inverse trend (25.3% in group L vs. 14.2% in group S). Subtype-specific differences were prominent in tsRNAs, with 3′ tsRNA accounting for 7.8% in group S follicles vs. 0.3% in group L, and 5′ tsRNA representing 13.8% in S vs. 9.5% in group L. Secondly, differential expression analysis identified eight highly upregulated SncRNAs in group L follicles, including upregulated piR-1060463, piR-1205752, miR-10b, miR-29b, miR-100, miR-221-3p and SnoRNA-4, SnoRNA-5, alongside four highly downregulated piR-1374439, piR-949425, SnoRNA-6 and SnoRNA-7. Finally, GO enrichment and KEGG pathway of these 10 significantly different expressed piRNAs, miRNAs and SnoRNAs targeting genes highlighted critical roles in oocyte meiosis, in utero embryonic development, insulin signaling pathway and Notch signaling pathway between group L and group S, which is closely related to oocyte and follicle development. Strikingly, GO of these targets gene is rich in utero embryonic development (p-value 0.003, count ~20) and mitochondrial transport (p-value 0.001, count ~30), and these target genes with insulin signaling pathway (p-value ~0.01, count ~18) and oocyte meiosis pathway (p-value 0.02, count ~8), which converged on follicular development and oocyte maturation pathways.

Our findings demonstrate that follicular fluid EVs SncRNAs orchestrate oocyte developmental competence through size-dependent profiles and pathway activation. These results provide novel insights into improving assisted reproductive technologies in swine and offer potential biomarkers for ovarian follicular selection.