脂代谢通路对鸡雄性生殖细胞分化的调控机制

The Regulatory Mechanism of the Lipid Metabolism Pathways During Male Germ Cell Differentiation in Chickens

【目的】探索家鸡雄性生殖细胞分化过程中脂代谢相关基因以及信号通路的调控机制,以期为完善体外诱导体系提供依据。【方法】采用流式细胞分选法获得纯度较高的胚胎干细胞(embryonic stem cell,ESC)、原始生殖细胞(primitive germ cells,PGC)和精原干细胞(spermatogonial stem cell,SSC),提取细胞总RNA。利用RNA-seq高通量分析方法对这三种细胞进行转录组水平测序,进行GO(Gene ontology)分析和KEGG通路富集,寻找脂代谢相关基因以及信号通路;并利用脂代谢--视黄醇代谢通路终产物视黄酸(retinoic acid,RA)以及抑制剂苯磺酰异羟肟酸(piloty’s acid)在体外诱导ESC向雄性生殖细胞分化和体外鸡胚注射后孵化,q RT-PCR(quantitative real time PCR)检测视黄醇代谢通路上差异基因的表达变化,与RNA-seq结果进行比较分析同时检测雄性生殖细胞标记基因的变化情况。【结果】GO功能显著性富集和Pathway显著性富集分析结果发现:在ESC向SSC分化过程中共存在328个基因持续参与脂代谢调控,富集到27条脂代谢通路中,包括视黄醇代谢、初级胆汁酸的合成、甾类激素的生物合成、脂肪酸代谢、甘油酯代谢以及类固醇的生物合成等通路。在视黄醇代谢通路中ADH5在PGC中特异表达,ALDH1A1在整个发育过程中持续上调,ADH5和ALDH1A1在细胞中参与视黄酸的合成;CYP26b1在整个发育过程中持续上调,参与视黄酸的降解过程;RA体外诱导ESC向SSC方向诱导试验结果表明ADH5、ALDH1A1和CYP26b1家族基因在体外RA诱导过程中变化与体内分化过程一致,RA诱导组产生的SSC样细胞显著高于控制组和Piloty’s Acid+RA组。鸡胚注射抑制剂试验结果表明视黄醇通路被抑制后孵化至18d后SSC细胞表面特异标记基因integrinα6和integrinβ1的表达水平显著的低于正常的孵化组(CON)和阴性对照组(BLANK)【结论】脂代谢--视黄醇代谢通路在家鸡雄性生殖细胞的分化过程中起重要作用,体外利用视黄醇代谢通路终产物RA进行诱导时第2天出现类胚体,第4、6天类胚体逐渐增大,第8天开始裂解,在第10天出现SSC样细胞,而在诱导过程中抑制视黄醇通路信号后则会降低SSC样细胞的产生,而在体外孵化过程中视黄醇通路抑制后也能影响SSC细胞的产生。

【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. q RT-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.