Effect of vitrification on the mRNA transcriptome of bovine oocytes.
Effect of vitrification on the mRNA transcriptome of bovine oocytes.
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DOI：
10.1111/rda.12942
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发表时间：
2017-03
期刊：
影响因子：
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通讯作者：
N. Wang;C. Li;H-B Zhu;H. Hao;H.Y. Wang;C-L Yan;S. Zhao;W. Du;D. Wang;Y. Liu;
中科院分区：
文献类型：
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作者：
N. Wang;C. Li;H-B Zhu;H. Hao;H.Y. Wang;C-L Yan;S. Zhao;W. Du;D. Wang;Y. Liu;
玻璃化冷冻会降低哺乳动物卵母细胞的发育能力，这与玻璃化冷冻后卵母细胞的异常表达密切相关。然而，玻璃化冷冻对RNA测序(RNA-seq)法检测的卵母细胞转录机制的影响尚未明确。在本研究中，用Smart-seq2分析了新鲜的牛卵母细胞和玻璃化冷冻的牛卵母细胞的mRNA转录本，用DEseq2确定了差异表达的基因(调整后的p值为0.05，最小倍数变化为2)。然后根据基因本体论(GO)和基因组(KEGG)数据库搜索差异表达的mRNAs。最后，用实时定量聚合酶链式反应(qRT-PCR)验证10个候选基因的mRNA表达。在每个新鲜或玻璃化的卵母细胞样本中检测到大约12,000个基因。其中，102个基因在玻璃化组中的表达水平差异显著：12个主要与细胞周期、受精和葡萄糖代谢有关的基因上调，90个主要涉及线粒体、核糖体蛋白、细胞骨架、跨膜蛋白、细胞周期和钙离子的基因下调。GO分析表明，这些基因主要集中在膜结合细胞器、大分子复合体和胞内部分。随机选择的10个候选基因的mRNA表达水平与RNA-SEQ结果一致。综上所述，我们的结果表明，玻璃化冷冻通过下调基因来影响牛卵母细胞的mRNA转录组，这是玻璃化冷冻卵母细胞发育能力下降的原因之一。我们的发现将有助于确定提高玻璃化卵母细胞效率的方法。
Vitrification has been shown to decrease the developmental capacity of mammalian oocytes, and this is closely associated with the abnormal mRNA expressions of vitrified oocytes. However, the effect of vitrification on transcriptional machinery of oocytes examined by RNA sequencing (RNA-seq) has yet to be defined. In the present study, the mRNA transcriptomes of fresh and vitrified bovine oocytes were analysed by Smart-seq2 with the differently expressed genes determined by DEseq2 (an adjusted p-value of .05 and a minimum fold change of 2). The differentially expressed mRNAs were then searched against the Gene Ontology (GO) and Genomes (KEGG) database. Finally, the mRNA expressions of 10 candidate genes were validated using quantitative real-time PCR (qRT-PCR). Approximately 12,000 genes were detected in each sample of fresh or vitrified oocytes. Of these, the expression levels of 102 genes differed significantly in vitrified groups: 12 genes mainly involved in cell cycle, fertilization and glucose metabolism were upregulated, and 90 genes mainly involved in mitochondria, ribosomal protein, cytoskeleton, transmembrane protein, cell cycle and calcium ions were downregulated. GO analysis showed that these genes were mainly enriched in terms of membrane-bounded organelles, macromolecular complex, and intracellular part. The mRNA expression levels of 10 candidate genes selected randomly were in agreement with the results of the RNA-seq. In conclusion, our results showed that vitrification affected the mRNA transcriptome of bovine oocytes by downregulating genes, which contributed to the decreased developmental capacity of vitrified oocytes. Our findings will be useful in determining approaches to improve the efficiency of vitrified oocytes.