Abstract

MicroRNAs (miRNAs) are members of noncoding small RNAs (~21–25 nucleotides long) that inhibit translation and/or induce degradation of protein-coding mRNAs that contain complementary sequences to miRNAs by interacting with the 32 untranslated region. They are known to influence the expression of hundreds of genes in numerous biological processes including development. In order to get insight into the regulation of miRNAs during bovine early development, we applied a heterologous approach to identify and predict bovine miRNAs differentially regulated during oocyte maturation. For this purpose, the miRCURYTM locked nucleic acids (LNA) array (Exiqon, Vedbaek, Denmark), consisting of 454 capture probes for human, mouse, and rat miRNAs, as registered and annotated in the miRBase release 8.0 at TheWellcomeTrust Sanger Institute, was used to compare the miRNA expression profile between bovine immature and matured oocytes. Total RNAs with miRNAs were isolated from three independent pools (each containing 200 oocytes) of immature and in vitro-matured oocytes using an miRNeasy mini kit (Qiagen, Hilden, Germany). The isolated miRNAs were amplified using a Global MicroRNA amplification kit (System Bioscience LLC, Mountain View, CA, USA) to obtain enough targets for hybridization. The RNA samples from immature and matured oocytes were labelled with either Hye3^™ or Hye5^™ dyes and hybridized on three independent slides. After 16 h of incubation at 60°C, slides were sequentially washed and scanned using an Axon GenePix 4000B scanner (Axon Instruments, Foster City, CA, USA). Species-specific GenePix^® Array Lists (GAL; Molecular Devices Corp., Sunnyvale, CA, USA) files of human, mouse, and rat were used independently. Following normalization using GProcessor 2.0a, probes with an average value of ≥1.5-fold change were considered to be differentially regulated. From the total miRNA probes (454) spotted on the array, about 400 were detected in bovine oocytes. Analysis of the array data using a human, mouse, and rat files indicated that a total of 33, 46, and 76 clones were differentially regulated during oocyte maturation, respectively. Of these, 11 miRNAs were found to come out as differentially regulated in all three array analyses. Accordingly, immature oocytes were found to be enriched with miR-208, miR-125a, miR-130b, miR-25, miR-375, and miR-382, while mature oocytes were enriched with miR-122a, miR-101, miR-206, miR-515-5P, and miR-101. The candidate miRNAs were predicted to target mRNA products of genes related to growth regulating proteins, transcription, and tumor necrosis factors. Investigation of the corresponding target mRNAs and protein products during oocyte maturation will enable insight into miRNA-controlled regulation of maternal gene expression.