Aims: MicroRNAs(miRNAs) are present extracellular vesicles (EV) in the follicular fluid (FF) and play a role in oocyte and early embryonic development. We identified several miRNAs in the FFs (miR-148a, miR-151-5p, miR-199a-3p, miR-29b, and miR-425-5p) that play essential roles in proper embryonic development and DNA demethylation. However, the molecular mechanism underlying miRNA synthesis in granulosa cells (GCs) and miRNA secretion from GCs into FFs remain unclear.
Methods: FFs and GCs were collected from small antral follicles (3-5 mm in diameter) of ovaries derived from 10 individual cows. The frequency of miRNAs in FFs and GCs was determined using small RNAseq. In addition, the mRNAs in the corresponding GCs were subjected to RNAseq. Frequency of miRNAs compared between FFs and the corresponding GCs. Correlations between pairs of miRNAs within 10 FFs or 10 GCs samples were calculated. Ten FFs and GCs samples were rated based on the frequency of each miRNA and divided into the top 3 rich and bottom 3 poor samples to obtain differentially expressed genes (DEGs) using RNAseq data. The top upstream regulators and Kyoto Encyclopedia Genes and Gnome pathways of the DEGs for each miRNA were predicted using Ingenuity Pathway Analysis and DAVID functional annotation, respectively.
Results and conclusion: A total of 352 miRNAs were detected in FF samples and 304 miRNAs in GCs samples. Only nine miRNAs were correlated between GCs and FFs, indicating a selective secretion mechanism. In addition, we found that groups of miRNAs were significantly correlated in both FFs and GCs. The FF data is useful for predicting the miRNA secretion mechanism, and those of GCs are useful for exploring miRNA synthesis mechanisms. In addition, upstream regulators and KEGG pathways associated with high expression levels of each miRNA were predicted, which could help determine key factors regulating follicular conditions.