Abstract: 【Objective】This research explores the regulatory mechanism of the lipid metabolic signaling pathways and its related genes during the process of chickens’ male germ cell differentiation to provide a basis for improving the efficiency of the in vitro induction system. 【Method】Fluorescence activated cell sorting (FACS) was used to obtain highly purified ESC (embryonic stem cell), PGC (Primitive germ cells) and SSC (spermatogonial stem cells), to extract total RNA from each type of cell. High throughput analysis methods-RNA-seq and Microarray were used to sequence the transcriptome level of obtained cells. Gene ontology analysis (GO) and the KEGG database were used to look for lipid metabolism signaling pathways and related genes. RA (Retinoic acid), the end-product of retinol metabolism pathway, was used to induce ESC in vitro differentiation into male germ cell combination with Piloty’s Acid. qRT-PCR was used to detect the expression changes of the genes involved in the retinol metabolic pathways. 【Result】From the results of RNA-seq, we found that there were 328 genes in 27 lipid metabolic pathways that were continuously involved in lipid metabolism regulation in the process of ESC differentiation into SSC in vivo. Some of these pathways, included retinol metabolism. primary bile acid synthesis, steroid hormone biosynthesis, metabolism of fatty acid metabolism, glyceride metabolism and the steroid biosynthesis pathway. In the retinol metabolism pathways; ADH5 was expressed in PGC specifically; ALDH1A1 increased persistently throughout the entire development process; both two genes were involved in retinoic acid synthesis in the cell. CYP26b1 was involved in the degradation of retinoic acid and down expressed throughout the entire development process. The experiment that induced ESC differentiation to SSC with RA shows that the changing process of ADH5, ALDH1A1 and CYP26b1 family genes were consistent with RNA-seq results. SSC-like cells generated in RA induced group was significantly higher than the control group and Piloty's Acid + RA group. The results of injecting the inhibitor into embryos showed that retinol pathway was inhibited and the expression levels of SSC-specific cell surface marker gene :integrinβ1 and integrinα6 was significantly lower than CON group and BLANK group after 18 days of incubation. 【Conclusion】 According to the above results the lipid metabolism-retinol metabolism pathway plays an important role in the process of chicken male germ cell differentiation. The process of inducing ESC to germ line cells differentiation by RA showed that there were some small embryos in D2 which became larger in D4 and D6 before these embryos dissociated in D8. The spermatogonial stem cell like cells appeared in D10 reducing the number of SSC-like cells when retinoid path signal was suppressed in the process of induction, so as in the process of incubation when the pathway be suppressed.

Key words: chicken, RNA-seq, male germ cells, retinoic acid, lipid metabolism