174 MicroRNA EXPRESSION PROFILE IN BOVINE CUMULUS–OOCYTE COMPLEXES DURING LATE OOGENESIS

Abstract

During late oogenesis, the mammalian oocyte synthesizes and stores mRNA necessary to guide the early stages of embryo development before the activation of embryonic transcription. The oocyte also contains many post-transcriptional regulatory mechanisms that coordinate mRNA stability and translation before specific activation. MicroRNAs (miRNAs) are short noncoding RNAs (17–25 nucleotides) that repress translation of target genes through sequence complementation and have recently been identified in murine oocytes. The objective of the current study was to identify and characterize the expression of miRNAs in bovine cumulus–oocyte complexes (COC) during late oogenesis as a potential mechanism for post-transcriptional regulation of mRNA in developing bovine oocytes. Ovaries from beef cattle (mixed populations) were obtained at a local abattoir. The COC were aspirated from 2- to 10-mm follicles and were pooled from each of 5 replicate collections for RNA extraction (n = 2241 total COC). Small RNA in the 16- to 27-bp range was isolated and used to construct cDNA libraries for sequencing, producing 2529 successful sequences that were clustered based on matching 14 consecutive bases to the most common member of the cluster. The consensus sequences of the clusters were screened for mitochondrial RNA, rRNA, tRNA, and snoRNA contaminants, leading to removal of 774 (31%) sequences from consideration. The remaining 1755 putative miRNA sequences were compared with known miRNA in miRBase, revealing 62 bovine COC miRNA clusters matching previously known sequences and 4 with no match. The cluster with the largest number of sequences identified in bovine COC matched the sequence of the let-7 miRNA family (657 sequences or 37% of putative miRNA). Within the let-7 family, let-7b (459 sequences or 26%) was the most abundant followed by let-7i (135 sequences or 8%). The four clusters that did not match sequences in miRBase represent putative novel miRNA. One of these four clusters had relatively high expression in bovine COCs (308 sequences or 18%), whereas the other 3 clusters had relatively low expression (total of 55 combined sequences or 3%). Expression of several putative miRNAs (let-7b, let-7i, miR-106a, and the abundant novel miRNA) in bovine COC were confirmed using TaqMan miRNA assays. These results demonstrate the presence of miRNA within bovine COC during late oogenesis, which suggests that these post-transcriptional regulatory elements may play a role in coordinating mRNA stability and translation in bovine oocytes.