The extent of the abundance of exosomal and non‐exosomal extracellular miRNAs in the bovine follicular fluid

Abstract

Follicular fluid (FF) plays an important role during follicular development and it contains several bioactive molecules including extracellular microRNAs (ECmiRNAs) that may mediate cell-cell communication during follicular development. Yet, the distribution patterns of ECmiRNAs in FF is not well characterized. This study aims to investigate the distribution of ECmiRNAs in two major fractions, namely exosomal and non-exosomal, of bovine follicular fluid (bFF). Exosomal and non-exosomal fractions from bFF were separated using Exoquick™ exosomes precipitation kit. miRNA expression was evaluated using the human miRCURY LNA™ Universal RT miRNA PCR array system. Transmission electron microscopy and immunoblotting revealed that the isolated vesicles were exosomes. The real-time PCR-based expression analysis revealed that 516 miRNAs were detected in the exosomal fraction of bFF, while 393 miRNAs were detected in the non-exosomal fraction. Among the detected miRNAs, a total of 370 miRNAs were detected in both fractions, while 145 miRNAs and 23 miRNAs were solely detected in exosomal and non-exosomal fractions, respectively. Exploratory pathway analysis showed that the genes targeted by exosomal and non-exosomal miRNAs to be involved in MAPK, Wnt, FoxO, TGF-beta, Oxytocin, ErbB, PI3K-Akt, Neurotrophin signalling pathways which are believed to be involved in follicular development, cell proliferation, and meiotic resumption. The results of our study demonstrated that besides the exosomal fraction, non-exosomal fractions can carry a significant amount of miRNAs in bFF where the exosomal fraction carries a significantly higher number of detectable miRNAs.