中国农业科学2016，Vol.49Issue(24)：4814-4823,10.DOI:10.3864/j.issn.0578-1752.2016.24.013
蛋白质代谢通路对鸡雄性生殖细胞分化的调控
Regulatory Study of Protein Metabolism During the Differentiation Process of Chicken Male Germ Cells
摘要
Abstract
[Objective] The aim of this study was to explore the regulatory mechanism of protein metabolism during the differentiation process of chicken male germ cells and provide a basis for improving the induction system of chicken embryonic stem cells (ESCs) differentiation to male germ cells in vitro.[Method] RNA sequencing was performed using FACS-sorted cells from ESCs,PGCs(primordial germ cells) and SSCs(spermatogonial stem cells),and enrichment analysis,WEGO (Web Gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes),were carried out to find out the relevant pathways and the key genes,the expression level of which was analyzed by qRT-PCR.Moreover,NOS2 both in vitro and in vivo with NOS2 inhibitor was inhibited,and the morphologic changes of ESCs were observed and the mRNA expressions of NOS2 and other germ genes,C-kit,Cvh,Stra8,Dazl,integrin α6 and integrin f1 were detected in different groups and in different days with RT-qPCR.[Result] Final results showed that 697 differentially expressed genes were involved in biological metabolism and significantly enriched in arginine-proline metabolic pathway,tyrosine metabolic pathway and tryptophan metabolic pathway and screened some key genes,like NOS2,EAH and IDO.It was found that the expression trends of NOS2,FAH and IDO were the same as that of RNA-Seq.In inhibitory experiment in vivo,the mRNA expression of NOS2,C-kit,Cvh,Stra8,Dazl,integrin a6 and integrin β1 between blank group and control group showed no significant difference.However,in inhibited group,NOS2,C-kit,Cvh,Stra8,Dazl,integrin a6 and integrin β1 expressions were down-regulated inordinately.Moreover,in inhibitory experiment in vitro,ESCs always proliferated on the 2,4,6,8 and 10d,but disappeared the embryonic bodies in the control group.In induced group,small embryonic bodies appeared on the 2d and became bigger and increased on the 4d.Embryonic bodies started to burst in edges on the 6d,break up on the 8d and appeared spermatogonia-like cells.In inhibited group,no embryonic body appeared in the whole process and ESCs proliferated more slow than the control group.In indueed-inhibited group,no embryonic body appeared on the 2d and 4d andESCs proliferated slowly.Small embryonicbodies appeared on the 6d and the number and volume increased slightly on the 8d.On the 10d,the embryonic bodies started to break up.In vitro,NOS2,C-kit,Cvh,Stra8,Dazl,integrin a6 and integrin β1 expressions in induced group,inhibited group and induced-inhibited group were significantly down-regulated compared with the control group.[Conclusion]Inthisstudy,basedonthescreeningofarginine-prolinemetabolicpathwayandNOS2withRNA-Seq and Bioinformatics,it was found that the process of ESCs differentiation to male germ cell was inhibited after the inhibition of NOS2,which suggested that arginine-proline metabolic pathway and NOS2 has an important regulatory effect on differentiation of ESCs to male germ cells.关键词
RNA-Seq/鸡胚胎干细胞/原始生殖细胞/精原干细胞/雄性生殖细胞/一氧化氮合成酶2/抑制剂/分化Key words
RNA-Seq/ESCs/PG-Cs/SSCs/male germ cells/NOS2/inhibitor/differentiation引用本文复制引用
李东,汤贝贝,王颖洁,纪艳芹,王飞,路镇宇,王曼,张亚妮,李碧春..蛋白质代谢通路对鸡雄性生殖细胞分化的调控[J].中国农业科学,2016,49(24):4814-4823,10.基金项目
国家自然科学基金(31272429)、高等学校博士学科点专项科研基金资助课题(20103250110006)、江苏省“六大人才高峰”、江苏省优势学科 （31272429）
