Expression profiling of microRNAs in Bovine ovarian follicular development

The study aimed to identify miRNAs expression profiles associated with growth and regression of dominant-size follicles in bovine. Follicles and corpora lutea (CL, from days 1 to 4 of the estrous cycle) were collected from abattoir ovaries and their status (healthy/atretic) was assessed by measuring steroid levels and aromatase expression. Total RNA was isolated from whole follicles at different developmental stages and from CL. An heterologous microarray (Exiqon, Denmark) approach followed by RT-qPCR validation (Qiagen, UK) was used to identify and compare miRNA profiles between large healthy follicles (diameter, 13–16 mm, n=6) and each of small (4–8 mm, n=6 pools of follicles), large atretic folllicles (13-16 mm, n=6) and CL (n=6) . RNA from the above groups was hybridized to the miRCURY LNA™ microRNA Hi-Power Labeling Kit,Hy3™/Hy5™ (Exiqon) and hybridized on the miRCURY LNA™ microRNA Array (6th gen). A total of 17 and 57 microRNAs were differentially expressed (> 2 fold, adj. P-value < 0.05) between Large Healthy and each of Small and Large Atretic follicles, respectively, a fraction of which corresponded to registered bovine miRNA sequences. A subset of 5 bovine miRNAs (miR-144, miR-202,vmiR-451, miR-652, miR-873) were confirmed by qPCR to be upregulated in Large Healthy follicles, were enriched in mural granulosa cells and their predicted targets mapped to genes involved in follicular cell proliferation and differentiation, suggesting an involvemet of this subset of microRNAs in ovarian follicle development. Moreover, a total of 11 and 22 unique miRNAs were up- and down-regulated, respectively (≥ 2.5 fold; adjusted P-value < 0.01), in corpora lutea relative Large Healthy follicles, including an upregulated cluster, miR-183-96-182, which was shown to be involved in luteal cell proliferation and steroidogenesis Six biological replicates per developmental stage (total of 24 samples) were used in a double dye microRNA microarray experiment. Samples were distributed among slides so that each experimental group was represented at least once in each slide. For each gene, mean normalized intensities (n= 6 biological replicates/group) were compared between follicle stages (SF vs LHF, LHF vs LAF and LHF vs CL).