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 β1 were detectedin 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, FAH 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 α6 and integrin β1 between blank group and control group showed no significant difference. However, in inhibited group, NOS2, C-kit, Cvh, Stra8, Dazl, integrin α6 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 induced-inhibited group, no embryonic body appeared on the 2d and 4d and ESCs proliferated slowly. Small embryonic bodies 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 α6 and integrin β1 expressions in induced group, inhibited group and induced-inhibited group were significantly down-regulated compared with the control group. 【Conclusion】In this study, based on the screening of arginine-proline metabolic pathway and NOS2 with RNA-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.

Key words: RNA-Seq, ESCs, PGCs, SSCs, male germ cells, NOS2, inhibitor, differentiation