|Cushman, Robert - Bob
|Smith, Timothy - Tim
Submitted to: Animal Reproduction Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/3/2012
Publication Date: 1/1/2012
Citation: Miles, J.R., McDaneld, T.G., Wiedmann, R.T., Cushman, R.A., Echternkamp, S.E., Vallet, J.L., Smith, T.P.L. 2012. MicroRNA expression profile in bovine cumulus–oocyte complexes: Possible role of let-7 and miR-106a in the development of bovine oocytes. Animal Reproduction Sciences. 130(1-2):16-26.
Interpretive Summary: Abnormal embryonic development is a key mechanism that decreases fertility in cattle. The quality and competence of an oocyte affect early embryonic development; therefore, proper maturation of the oocyte is necessary for good fertility in cattle. During oocyte maturation, many maternal mRNAs accumulate within the oocyte, which guide the early stages of embryonic development. Many of these maternal mRNAs are stored for an extended period of time and thus require post-transcriptional regulatory mechanisms to coordinate mRNA stability prior to activation as well as selective degradation. MicroRNAs (miRs) are highly conserved non-coding RNAs that bind to complementary sequence of target mRNAs and regulate gene expression through translational repression or mRNA degradation. The objective of the current study was to examine miR expression in bovine cumulus oocyte complexes (COCs) in a comprehensive fashion based on sequencing. A cDNA library was constructed from pooled COCs, which identified 67 miR sequence clusters that matched previously reported miRs in the miRBase and 5 miR sequence clusters that had no match in the miRBase. Validation of selected miRs as well as some biogenic transcripts involved in miR processing was performed in denuded oocytes and COCs using real-time PCR. The results confirm that miRs are present in bovine COCs during late oogenesis. These identified miRs may play a role in regulating maternal mRNAs in developing cattle oocytes, which may affect oocyte quality and competence and, ultimately, fertility in cattle.
Technical Abstract: The expression of microRNAs (miRs) in bovine cumulus-oocyte complexes (COCs) during late oogenesis was profiled to determine the potential for regulation of maternal mRNAs by this class of small RNAs. A cDNA cloning and sequencing strategy resulted in 1812 putative miR sequences, representing 72 distinct sequence clusters. Comparison of the cluster sequences to miRBase determined that 67 (93%) of the bovine COC miR corresponded to previously identified miR sequences and validated that the cloning successfully identified the miR population in these cells. The remaining 5 clusters (7% of total miRs) had no match in the miRBase and potentially represented novel miR. Two of these putative novel clusters match sequences we have also observed in swine muscle but which are not in the current build of the miRBase, further supporting that they are potential miR. The miR cluster with the most members from the cDNA sequences (685 sequences, 38% of total miRs) matched the let-7 family and indicates this family of miR is the most highly expressed in bovine COC. Using real-time PCR assays and independent sources of oocytes and COCs from various sized follicles, we were able to confirm expression of let-7b and let-7i, as well as miR-106a. These results establish the presence of miR during late oogenesis, supporting the hypothesis that maternally-derived mRNAs in developing cow oocytes may be influenced or regulated by microRNAs.