206 EFFECT OF OVARIAN HYPERSTIMULATION ON CIRCULATORY microRNA PROFILE IN BOVINE FOLLICULAR FLUID AND BLOOD PLASMA

Abstract

MicroRNAs (miRNA) are noncoding small RNA that are known to play a role in posttranscriptional regulation of genes involved in various physiological processes including disease and reproduction. The circulatory forms of miRNA, which are present in body fluids, are reported to be used as biomarkers for disease and pregnancy. This study was conducted to investigate the effect of ovarian hyperstimulation on the expression pattern of circulatory miRNA in blood plasma and follicular fluid. For this, one group of Simmental heifers (n = 6) was hyper-stimulated using a superovulation (SO) protocol of 8 consecutive FSH injections over 4 days in decreasing doses, and others (n = 6) were synchronized (SY) using a standard synchronization protocol. Following this, the blood samples were collected at Day 0 (onset of oestrus) and Day 7, and follicular fluid was collected from both groups at Day 0 of the oestrous cycle. Total RNA, including small RNA, was then isolated from follicular fluid and blood plasma using a miRNeasy mini kit (Qiagen, Valencia, CA, USA) and subsequently used for circulatory miRNA expression studies using the human miRCURY LNA™ Universal RT miRNA PCR array system (Exiqon, Woburn, MA, USA). Following the miRNA PCR array run, data analysis was performed using a delta threshold cycle (ΔCT) method. Results showed that 23 miRNAs were found to be differentially expressed in blood plasma (fold change ≥2 and P ≤ 0.05) between SO and SY groups. Among these, 8 miRNAs including miR-127-3p, miR-494, miR-147, miR-134, and miR-153 were downregulated and 15 miRNAs including miR-34a, miR-103, miR-181c, miR-24-2-3p, miR-18a-3p, miR-20b-3p, and miR-708-3p were found to be upregulated in SO groups. Similarly, in follicular fluid, 71 miRNAs were found to be differentially expressed between the SO and SY groups. Of these, 33 miRNA including miR-100, miR-877, miR-659, miR-200c, miR-29b-2-3p, miR-361-5p, and miR-145 were downregulated, whereas 38 miRNAs including miR-374a, miR-720, miR-155-3p, miR-202-3p, miR-33b, miR-12-3p, and miR-224-3p were upregulated in follicular fluid aspirated from SO cows compared with the SY animals. Ingenuity pathway analysis of predicted target genes of miRNA, which are dysregulated due to ovarian hyperstimulation, showed the dominance of pathways related to neuregulin signalling, axonal guidance signalling, GNRH signalling, and Wnt β-catenin signalling pathways. In conclusion, this study revealed alternation in circulatory miRNA expression profile both in blood plasma and follicular fluid as a result of ovarian hyperstimulation.