EV-Derived Small Non-Coding RNAs from Porcine Follicular Fluid Regulate Follicle Development using Pandora sequence

Junhe Hu^1*Shuting Peng¹Wei Deng¹Zhenzhen Guo¹Juan Wu¹Xiansheng Tan¹Zhi Zeng¹Anqun Wang²

• ¹Hunan University of Humanities, Science and Technology, Loudi, China
• ²Shuangfeng County Yiqun Cultivation and Breeding Farmers' Professional Cooperative, Loudi city, Hunan province, China

Background: 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. Methods: 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. Results: Firstly, SncRNA composition analysis demonstrated distinct proportional distributions: PiRNA constituted 43% in group L versus 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 versus 0.3% in group L, and 5′ tsRNA representing 13.8% in S versus 9.5% in group L. Secondly, differential expression analysis identified 8 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 4 highly downregulated piR-1374439 , piR-949425, SnoRNA-6 and SnoRNA-7. Finally, GO enrichment and KEGG pathway of these ten 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. Conclusion: 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.