ORCID Profile
0000-0002-9977-2347
Current Organisation
Colorado State University
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Publisher: CSIRO Publishing
Date: 2014
DOI: 10.1071/RDV26N1AB113
Abstract: In cattle, follicles grow in a wave-like pattern, with typically 2 or 3 waves per oestrous cycle. During each wave, one follicle of a cohort becomes dominant (DF), whereas the remaining subordinate follicles (SF) in the cohort undergo atresia. If the endocrine conditions are appropriate (low progesterone), the dominant follicle goes on to ovulate. In order to unravel the molecular mechanisms associated with ovulation and follicular atresia, here we aimed to investigate the expression of short regulatory microRNA (miRNA) in granulosa cells of DF and SF using deep sequencing. For this, Simmental heifers (n = 7) were synchronized according to standard protocols and slaughtered at Day 19 of the oestrous cycle. Follicles were categorized as DF (≥12 mm n = 5) and SF (≤10 mm n = 78). Granulosa cells from both follicle groups were used for total RNA (enriched with miRNA) isolation using miRNeasy mini kit (Qiagen GmbH, Hilden, Germany). The RNA concentration and integrity were measured using Nano Drop 8000 spectrophotometer (Nano Drop, Wilmington, DE, USA) and Agilent, 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively. Libraries were constructed by GATC BioTech AG (Konstanz, Germany) and sequenced on Illumina HISEqn 2000. Prediction of both known and novel miRNA was done using miRDeep2 software packages. Quantification of differentially expressed miRNA was done using R software and DESEqn 2 packages. The MiRNA with log2 fold change difference ≥1, P-value ≤0.05, and false discovery rate of ≤0.1 were considered to be significant. Results showed that 318 and 322 known miRNA were detected in DF and SF, respectively. It was shown that 28 miRNA including bta-miR-122, bta-miR-139, and bta-miR-375, and 35 others including bta-miR-138, bta-miR-20b, and bta-miR-33a were uniquely detected in DF and SF, respectively. In addition to the known annotated miRNA, 20 and 24 novel miRNA were detected in DF and SF, respectively. Expression analysis revealed that 65 miRNA were differentially expressed in granulosa cells of SF compared with DF. Thirty miRNA including bta-miR-409a (involved in cell death by targeting genes BCL2l11, BIRC5, and PTEN) and bta-miR-335 (involved in cell proliferation, migration, and differentiation) are up-regulated in SF, whereas 35 miRNA including the miR-183 cluster (bta-mir-183, bta-miR-182, and bta-mir-96) involved in apoptosis inhibition are down-regulated in SF. The pathway analysis of potential target genes of differentially expressed miRNA is found to be involved in pathways, namely Wnt signalling, MAPK signalling, TGF-β signaling, and other pathways related to cell proliferation and apoptosis. In conclusion, the presence of stage-specific miRNA in granulosa cells support the potential role of miRNA in posttranscriptional regulation of genes during follicular development, mainly ovulation and follicular atresia.
Publisher: CSIRO Publishing
Date: 2014
DOI: 10.1071/RDV26N1AB111
Abstract: Follicular development is a result of complex hormonal and biochemical synergies that could be activated or deactivated in a spatiotemporal manner in oocytes and surrounding cells including theca, granulosa, and cumulus cells. The microRNA (miRNA), 19 to 22 nucleotides noncoding RNA, are one of the molecular cues that could play a role in posttranscriptional regulation of genes involved in follicular development. Here we aimed to understand the availability and abundance of miRNA in bovine granulosa cells (GC) derived from subordinate (SF) and leading or dominant (DF) follicles during bovine follicular recruitment and dominance at Day 3 and 7 of the oestrus cycle, respectively. For this, Simmental heifers (n = 15) were oestrus synchronized and slaughtered at 3 (n = 6) and 7 (n = 7) days after the onset of the oestrous. The SF and DF were retrieved from each animal to obtain the corresponding GC, which were subjected to miRNA-enriched total RNA isolation using the miRNeasy mini kit (Qiagen GmbH, Hilden, Germany). The integrity and quality of RNA was determined using Agilent 2100 Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA, USA) and Nanodrop 8000 Spectrophotometer (Thermo Fisher Scientific Inc., DE, USA), respectively. The RNA was then subjected to miRNA deep sequencing using the Illumina HISEqn 2000. Raw sequence data were further processed and analysed using miRDeep2 software package. Quantification of differentially expressed (DE) miRNA was done using R software and DESEqn 2 package. MiRNA with log2 fold change difference ≥1, P-value ≤0.05, and false discovery rate ≤1 were considered to be significant. Data analysis revealed that 291 and 311 miRNA were detected in GC of SF and 312 and 314 were detected in GC of DF at Days 3 and 7, respectively. A total of 17 miRNA were DE in GC from SF compared with the DF at Day 3, of which 15 miRNA were enriched and the remaining 2 were down-regulated in SF. Similarly, at Day 7 a total of 136 miRNA was altered with 51 miRNA showed to be enriched, whereas 85 others remained low in SF compared with the DF. Nine miRNA (bta-miR-21–3p, bta-miR-221, bta-miR-708, bta-miR-214, bta-miR-335, bta-miR-155, bta-miR-199a-5p, bta-miR-21–5p and bta-miR-222) were commonly differentially expressed both at Day 3 and 7 between SF and DF. Interestingly, all of the commonly DE miRNA, except bta-miR-335, were enriched in GC of SF at both days. Gene ontological analysis indicated that majority of the DE miRNA were found to be involved in regulation of programmed cell death, cell projection morphogenesis, regulation of cell proliferation, and macromolecule biosynthesis. Therefore, the temporal abundance of mature miRNAs in GC during bovine follicular development may suggest their potential role in regulation of follicular development in general and follicular recruitment and dominance in particular.
No related grants have been discovered for Samuel Gebremedhn.