111 REGULATORY microRNA ENRICHMENT AND DEGRADATION IN GRANULOSA CELLS DURING BOVINE FOLLICULAR RECRUITMENT AND DOMINANCE

Abstract

Follicular development is a result of complex hormonal and biochemical synergies that could be activated or deactivated in a spatiotemporal manner in oocytes and surrounding cells including theca, granulosa, and cumulus cells. The microRNA (miRNA), 19 to 22 nucleotides noncoding RNA, are one of the molecular cues that could play a role in posttranscriptional regulation of genes involved in follicular development. Here we aimed to understand the availability and abundance of miRNA in bovine granulosa cells (GC) derived from subordinate (SF) and leading or dominant (DF) follicles during bovine follicular recruitment and dominance at Day 3 and 7 of the oestrus cycle, respectively. For this, Simmental heifers (n = 15) were oestrus synchronized and slaughtered at 3 (n = 6) and 7 (n = 7) days after the onset of the oestrous. The SF and DF were retrieved from each animal to obtain the corresponding GC, which were subjected to miRNA-enriched total RNA isolation using the miRNeasy mini kit (Qiagen GmbH, Hilden, Germany). The integrity and quality of RNA was determined using Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA) and Nanodrop 8000 Spectrophotometer (Thermo Fisher Scientific Inc., DE, USA), respectively. The RNA was then subjected to miRNA deep sequencing using the Illumina HISEqn 2000. Raw sequence data were further processed and analysed using miRDeep2 software package. Quantification of differentially expressed (DE) miRNA was done using R software and DESEqn 2 package. MiRNA with log2 fold change difference ≥1, P-value ≤0.05, and false discovery rate ≤1 were considered to be significant. Data analysis revealed that 291 and 311 miRNA were detected in GC of SF and 312 and 314 were detected in GC of DF at Days 3 and 7, respectively. A total of 17 miRNA were DE in GC from SF compared with the DF at Day 3, of which 15 miRNA were enriched and the remaining 2 were down-regulated in SF. Similarly, at Day 7 a total of 136 miRNA was altered with 51 miRNA showed to be enriched, whereas 85 others remained low in SF compared with the DF. Nine miRNA (bta-miR-21–3p, bta-miR-221, bta-miR-708, bta-miR-214, bta-miR-335, bta-miR-155, bta-miR-199a-5p, bta-miR-21–5p and bta-miR-222) were commonly differentially expressed both at Day 3 and 7 between SF and DF. Interestingly, all of the commonly DE miRNA, except bta-miR-335, were enriched in GC of SF at both days. Gene ontological analysis indicated that majority of the DE miRNA were found to be involved in regulation of programmed cell death, cell projection morphogenesis, regulation of cell proliferation, and macromolecule biosynthesis. Therefore, the temporal abundance of mature miRNAs in GC during bovine follicular development may suggest their potential role in regulation of follicular development in general and follicular recruitment and dominance in particular.