Xu Yixin, Zhou Tuanping, Shao Li, Zhang Bei, Liu Kailu, Gao Chao, Gao Li, Liu Jiayin, Cui Yugui, Chian Ri-Cheng
State Key Laboratory of Reproductive Medicine, Center for Clinical Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China; Department of Obstetrics and Gynecology, McGill University, Montreal, Canada.
State Key Laboratory of Reproductive Medicine, Center for Clinical Reproductive Medicine, The First Affiliated Hospital of Nanjing Medical University, Nanjing, PR China.
Gene Expr Patterns. 2017 Nov;25-26:46-58. doi: 10.1016/j.gep.2017.05.002. Epub 2017 Jun 2.
Cumulus cells (CCs) are considered as an important source to predict oocyte quality. Despite numerous candidate genes in CCs have been identified for embryonic developmental competence, the results are inconsistent. The next generation RNA-sequencing was used to investigate the transcriptomic differences in CCs from in vitro matured oocytes did or did not develop to blastocyst stage following in vitro fertilization (IVF). In our study, the corresponding mouse oocytes were traced using a single-cell tracking system, and CCs were pooled into groups based on the embryonic developmental outcomes. In vivo matured oocytes with blastocyst development were set as a reference group. The transcriptomic differences in mouse CCs from in vitro maturated oocytes with or without blastocyst formation were tested by RNA-sequencing. Real-time PCR was used to verify the expression levels of those candidate genes. A total of 103 transcripts were significantly up-regulated, and 97 down-regulated, in the CCs with the oocytes developed to blastocyst stage. The bioinformatics study showed that those genes were involved in tube morphogenesis, cell-cell signaling and cell projection formation. Nine genes were selected from the most significantly changed transcripts after comparison with the reference group: Arrb1, Atp2c1, Cdh5, Cntnap1, Mkln1, Lgr4, Rhobtb1, Smc2 and Six2, as the candidate target genes. They were associated with the regulation of G-protein coupled receptors, Wnt and MAPK signaling, actin filaments and cell adhesion. Real-time PCR verified the up-regulation of all 9 genes, and significantly increased of Rhobtb1, Mkln1, Smc2, Arrb1, Atp2c1, Cdh5 and Lgr4. Based on RNA-sequencing, we found the changes in gene transcription of mouse CCs that were critical for the communication between CCs and oocytes. The results could provide novel insights on non-invasively predicting the oocyte quality and improving developmental competence.
卵丘细胞(CCs)被认为是预测卵母细胞质量的重要来源。尽管已在CCs中鉴定出众多与胚胎发育能力相关的候选基因,但其结果并不一致。采用新一代RNA测序技术研究体外成熟卵母细胞在体外受精(IVF)后能否发育至囊胚阶段的CCs中的转录组差异。在我们的研究中,使用单细胞追踪系统追踪相应的小鼠卵母细胞,并根据胚胎发育结果将CCs分组。将发育为囊胚的体内成熟卵母细胞设为参照组。通过RNA测序检测体外成熟的有或无囊胚形成的小鼠CCs中的转录组差异。采用实时PCR验证这些候选基因的表达水平。在卵母细胞发育至囊胚阶段的CCs中,共有103个转录本显著上调,97个转录本下调。生物信息学研究表明,这些基因参与了管形态发生、细胞间信号传导和细胞突起形成。与参照组比较后,从变化最显著的转录本中选取9个基因:Arrb1、Atp2c1、Cdh5、Cntnap1、Mkln1、Lgr4、Rhobtb1、Smc2和Six2作为候选靶基因。它们与G蛋白偶联受体、Wnt和MAPK信号传导、肌动蛋白丝及细胞黏附的调节相关。实时PCR验证了所有9个基因的上调,且Rhobtb1、Mkln1、Smc2、Arrb1、Atp2c1、Cdh5和Lgr4显著增加。基于RNA测序,我们发现了小鼠CCs基因转录的变化,这些变化对于CCs与卵母细胞之间的通讯至关重要。这些结果可为无创预测卵母细胞质量和提高发育能力提供新的见解。