207 CIRCULATORY microRNA SIGNATURES IN FOLLICULAR FLUID IN RELATION TO THE GROWTH STATUS OF BOVINE OOCYTES

Abstract

Cell-to-cell communication within the follicle involves many signalling molecules, and this process is believed to be mediated by secretion and uptake of exosomes that contained several bioactive molecules including circulatory microRNAs (miRNA). The present study was conducted to investigate the circulating miRNA expression pattern in exosome and nonexosomal portion of follicular fluid (FF) in follicles with fully grown or growing oocytes. For this, the FF and cumulus–oocyte complexes (COC) were retrieved from 5- to 8-mm individual follicles from ovaries obtained from local abattoir. Then, the oocytes were subjected to brilliant cresyl blue (BCB) staining and classified as BCB+ (fully grown oocytes) and BCB– (growing oocytes). Accordingly, the corresponding FF was classified as BCB+ and BCB– based on their oocyte source. Following this, the exosomes were trapped from each FF categories using ExoQuick™ (SBI System Bioscience). Thus, total RNA was isolated from exosomal and nonexosomal portion of the FF using miRNeasy mini kit (Qiagen) and subjected to miRNA expression studies. The human miRCURY LNA™ Universal RT miRNA PCR array system (Exiqon) was used for miRNA expression profiling. Data analysis was performed using a comparative threshold cycle (ΔCT) method. The results revealed that 26 miRNAs were found to be differentially expressed (fold change ≥2 and P < 0.05) between the exosomal portion of FF from fully grown and growing oocyte groups. Among these, 17 miRNA including miR-608, miR-654-5p, miR-640, miR-582-5p, miR-449b, miR-155 were upregulated and 9 miRNA including miR-373, miR-526b*, miR-33a*, miR-30b, miR-29a* were downregulated in exosomal portion of FF of growing oocyte groups. The ingenuity pathway analysis of genes predicted to be targeted by those miRNAs were found to be involved in WNT/β catenine, purine metabolism, protein ubiquitination, and cAMP-mediated signalling pathways. Similarly, 36 miRNA were differentially expressed between the non-exosomal portion of FF of fully grown and growing oocytes. From those, 27 miRNA including let-7i*, miR-328, miR-223, miR-19b-1*, miR-423-5p, miR-29c, miR-659 were upregulated, whereas the expression level of 9 miRNA including miR-381, miR-18a*, miR-30e*, miR-934, and miR-302c was downregulated in the nonexosomal portion of FF of growing oocyte groups. In addition, the ingenuity pathway analysis indicated that the genes predicted to be targeted by these miRNA were found to be involved in NRF2-mediated oxidative stress response, tight junction signalling, and protein ubiquitination pathways. In conclusion, this study detected the presence of exosome or non-exosome-mediated circulation of miRNA in the bovine follicular fluid and oocyte growth-dependent variation of circulatory miRNA in the follicular environment.