Reproduction Abstracts

Little is known about the involvement of miRNAs during terminal follicle differentiation in the monovular ovary. This study aimed to characterise miRNAs involved in the follicle-luteal transition in bovine. Microarray analyses were performed on RNA from ovulatory-size follicles (n=6) and early corpora lutea (n=6) obtained at an abattoir. Exiqon’s miRCURY LNA microRNA Array, sixth generation was used and results were validated by qPCR. A total of ten and 24 miRNAs were upregulated and downregulated (greater than twofold; Benjamini and Hochberg adjustment, P<0.05), respectively, in luteal relative to follicular tissues. Among top upregulated miRNAs in the CL (greater than fivefold) were the cluster miR-182-96-183 and miR-132. To investigate the functional involvement of these miRNAs we used an in vitro model of forskolin-induced bovine granulosa cell luteinisation. Levels of miR-132 and miR-96 increased more than twofold within the first 4 days of luteinisation. Transfecting these cells with specific LNA inhibitors or mimics miR-132 and miR-96 led, respectively, to abolished expression and a significant increase in the levels of these miRNAs (P<0.001) within 2 days. However, the induced changes in miRNA levels during luteinisation did not have any significant effect on granulosa cell growth curves or on transcript levels of predicted mRNA targets including FOXO1, ADCY6, and CDKN1A. In summary, we have identified the miR-182-96-183 cluster and miR-132 as candidate miRNAs for an involvement in the follicular–luteal transition in the monovular bovine ovary. We are currently testing further the effects of these miRNAs during luteinisation in vitro, and we are investigating the actual distribution of these miRNAs within luteal tissues.