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Effect of vitrification on the microRNA transcriptome in mouse blastocysts

ABSTRACT: Vitrification is commonly used in the cryopreservation of mammalian blastocysts to overcome the temporal and spatial limitations of embryo transfer. Previous studies have shown that the implantation ability of vitrified blastocysts is impaired and that microRNAs (miRNAs) regulate the critical gene for embryo implantation. However, little information is available about the effect of vitrification on the miRNA transcriptome in blastocysts, which partially explains the impaired implantation ability of vitrified blastocysts. In the present study, the miRNA transcriptomes in fresh and vitrified mouse blastocysts were analyzed by microRNA Taqman assay based method, and the results were validated using quantitative real-time PCR (qRT-PCR). Then, the differentially expressed miRNAs were assessed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Overall, 760 known mouse miRNAs were detected in the vitrified and fresh mouse blastocysts. Of these, the expression levels of five miRNAs differed significantly: in the vitrified blastocysts, four miRNAs (mmu-miR-199a-5p, mmu-miR-329-3p, mmu-miR-136-5p and mmu-miR-16-1-3p) were upregulated, and one (mmu-miR-212-3p) was downregulated. The expression levels of all miRNAs measured by the microRNA Taqman assay based method and qRT-PCR were consistent. The four upregulated miRNAs were predicted to regulate 877 candidate target genes, and the downregulated miRNA was predicted to regulate 231 genes. The biological analysis further showed that the differentially expressed miRNAs mainly regulated the implantation of embryos. In conclusion, the results of our study showed that vitrification significantly altered the miRNA transcriptome in mouse blastocysts, which may decrease the implantation potential of vitrified blastocysts.

ORGANISM(S): Mus musculus

PROVIDER: GSE62581 | GEO | 2016/12/31

SECONDARY ACCESSION(S): PRJNA264470

REPOSITORIES: GEO

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Project description:We investigated functions of miRNAs at the level of the whole transcriptome of primary neurons. We transfected mouse E17.5 forebrain primary neuronal cultures (at four to six days of in vitro development) with miRNA mimics and inhibitors. After approximately 48 h post transfection we profiled the effect of these transfections on gene expression with Illumina mRNA microarrays. Cultures were transfected with mimics and inhibitors of five mouse miRNAs (mmu-miR-124, mmu-miR-434-3p, mmu-miR-143, mmu-miR-145 and mmu-miR-25) and with a mimic of a non-mouse miRNA (cel-miR-67). Direct widespread inhibition of gene expression by the perturbed miRNAs was evident when gene expression in cultures transfected with miRNA mimics was compared to those transfected with the inhibitors (or to matched mock transfected cultures): 3-prime UTRs of downregulated transcripts were significantly enriched in seed matching sites for the perturbed miRNAs. Interestingly, analysis of differential gene expression in mock transfected cells (identified through comparison of mock transfected cultures with matched untreated cultures) revealed that genes inhibited by miRNAs were enriched in genes upregulated in mock transfected cultures. This inhibition was the most efficient by the two neuronal miRNAs under investigation (mmu-miR-124 and mmu-miR-434-3p). To investigate if miRNA mediated inhibition of stress induced genes (i.e. stress associated with transfections) was also the case in other stresses, we profiled gene expression changes triggered by chronic neuronal depolarisation. For this, we treated the cultures with KCl (15 mM, 48 h) and compared them to matched untreated cultures. We found that genes upregulated by KCl had a significant intersection with those upregulated by the mock transfection. Moreover, we also found that genes upregulated by KCl had a significant intersection with genes inhibited by mmu-miR-124 and mmu-miR-434-3p. Therefore we concluded that neuronal miRNAs stabilise the neuronal transcriptome by inhibiting stress inducible genes.

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Effect of vitrification on the microRNA transcriptome in mouse blastocysts

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