| Objective To screen the functional microRNAs (miRNAs) during the initiation of primordial follicle development and explore the potential mechanisms.Methods Given the potential role of miRNAs in the ovary yet no one has ever studied them specifically in the process of initiation of primordial follicle development, their expression was profiled in day3and day5mice ovary by microarray. The differentially expressed miRNAs were then selected, validated by Real-Time PCR and then subjected to a global analysis of miRNA-regulated signaling pathways and related genes on the basis of miRNA expression profile and bioinformatic interpretation using KEGG and GO database. MiR-145was detected by In situ hybridization in3day and5day old mice ovary. MiR-145antagomir was used to silence the expression of miR-145, Confocal Microscopy, Real-Time PCR were applied to detect the interference efficiency. The HE staining, immunohistochemistry, Western Blot, in vitro ovarian cultures, immunohistochemistry and follicle counting were used to evaluate the effect of miR-145antagomir on the primordial follicle initiation. The target genes of miR-145were validated by Real-Time PCR, immunohistochemistry, Western Blot and Luciferase activity assay. The influence of down-regulated miR-145on the protein levels of Smad pathway, non-Smad signaling pathway and apoptosis were detected by Western Blot.Results15miRNAs (miR-122, miR-145, miR-380-3p, miR-680, miR-484, miR-496, miR-17, miR-1906, miR-136*, miR-125-3p, etc) were upregulated in day5samples and9miRNAs (miR-302*, miR-466g, miR-466f-3p, miR-500, miR-382*, miR-574-3p, miR-1196, etc) were downregulated. Eight miRNAs among these filtered ones were validated to be significantly different between the3-d-old and5-d-old mice ovaries (P<0.05). We searched for potential mRNA targets of these24differentially expressed miRNAs using computational prediction algorithms (TargetScan5.1). These putative target genes were submitted to GO analysis, the results revealed that the down-regulated miRNAs target genes significant GOs included cell adhesion, apoptosis, cell cycle, protein modification process, DNA repair, angiogenesis, potassium ion transport, etc. The up-regulated miRNAs target genes significant GOs contained cell adhesion, cell cycle, apoptosis, small GTPase mediated signal transduction, protein transport, intracellular signaling cascade, RNA splicing, etc. The pathway enrichment analysis revealed that48significant signal transduction pathways were regulated by up-regulated miRNA and29were significantly regulated by downregulated miRNAs with a p-value and FDR less than0.001. The miRNA-mRNA regulatory networks based on TGF-β signaling pathway related genes, being established. MiR-145was expressed predominantly in granulosa cells (GC) of primary follicles, no staining was observed in oocytes and pregranulosa cells of primordial follicles. Histological examination and immunohistochemistry of the ovaries after4days of incubation indicated that antagomir group with a concentration of4μM induced a significant reduction of primordial, primary and secondary follicle compared to the scrambled antagomir group (AN group)(P<0.05), miR-145antagomir significantly downregulated zp1, zp2and zp3mRNA expression as well as the follicle number (P<0.05). Western Blot, real-time PCR and Luciferase activity assay analysis revealed that Tgfbr2was the target gene of miR-145. Further study demonstrated that the miR-145antagomir up-regulated the expression of Tgfbr2, then activated the Smad signaling pathway but not P38MAPK or JNK pathway. The apoptosis associated protein including BCL-2was decreased while BAX and cleaved CASPASE3were increased.Conclusion MiRNAs and their regulated signaling pathways are involved in the process of primordial follicle initiation. MiR-145is one of the important miRNA which targets to Tgfbr2to regulate the initiation of primordial follicle development and maintain the primordial follicle pool.
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