135 Differential gene expression of bovine long noncoding RNAs in single oocytes aspirated from small and large follicles

Abstract

In mammals, maternal mRNAs accumulate in the oocyte and play important roles during early embryogenesis in embryonic genome activation. Proper development is dependent upon regulation of these maternal transcripts while simultaneously activating the embryonic genome. Long noncoding RNAs (lncRNAs) regulate various cellular processes, including modulation of gene expression and epigenetic status. Recently, lncRNAs have been functionally characterised as key regulators of embryonic genome activation in humans and mice. Our laboratory identified 1535 lncRNAs in bovine oocytes using RNA-sequencing. The objective of this study was to verify our RNA-seq data and if present, characterise the expression of six highly abundant lncRNAs (2160.1, 5379.1, 18208.1, 23721, 25823.1, and 34876.2) in single oocytes aspirated from varying size follicles at different developmental stages. Single oocytes were aspirated from small (<4mm) and presumably oestrogen active (6–18mm) follicles and were denuded at the germinal vesicle (GV) or MII stage. MII stage was determined by cumulus expansion and the extrusion of the first polar body. Real-time quantitative PCR analysis, using RPL-19 as a housekeeping gene analysed using the standard curve method, revealed detectable expression for each lncRNA in single oocytes from small (SF) and presumably oestrogen active (EA) follicles at both stages. Average expression of each lncRNA from SF was used as a calibrator in normalization. Effect of follicle size and maturity stage and their interaction on the lncRNA expression were examined using a two-factorial ANOVA followed by Tukey’s HSD post hoc test. In five lncRNAs (2160.1, 5379.1, 18208.1, 25823.1, and 34876.2), maturity stage was significant (P<0.05). Moreover, we found significant interaction of stage and size in expression of lncRNA 25823.1 (P<0.05). SF oocytes at the GV stage had the highest relative expression of lncRNA 25823.1. Specific differences were detected between SF MII and SF GV oocytes, indicating higher expression in the earlier developmental stage (P<0.05). The SF oocytes at MII had a 0.63-fold decrease in relative expression with respect to the SF oocytes at GV. In addition, we found a tendency for significant interaction between maturity stage and follicle size in the expression of lncRNA 5379.1 (P<0.1). LncRNA 5379.1 exhibited the same expression pattern as lncRNA 25823.1 with SF oocytes at MII having a 0.75-fold decrease in relative expression relative to SF oocytes at GV. In summary, both lncRNA 25823.1 and 5379.1 had higher expression in oocytes aspirated from small follicles during the earlier developmental stage. The literature indicates that follicle size can be an indicator of oocyte quality and that RNAs accumulated or degraded during maturation are crucial to achieve developmental competence. Therefore, lncRNAs 25823.1 and 5379.1 shows promise to be linked with oocyte quality.