Zuo Qisheng, Li Dong, Zhang Lei, Elsayed Ahmed Kamel, Lian Chao, Shi Qingqing, Zhang Zhentao, Zhu Rui, Wang Yinjie, Jin Kai, Zhang Yani, Li Bichun
Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, China.
Key Laboratory of Animal Breeding Reproduction and Molecular Design for Jiangsu Province, College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, China; College of Veterinary medicine, Suez Canal University, Ismailia, 41522, Egypt.
PLoS One. 2015 Feb 6;10(2):e0109469. doi: 10.1371/journal.pone.0109469. eCollection 2015.
Here, we explore the regulatory mechanism of lipid metabolic signaling pathways and related genes during differentiation of male germ cells in chickens, with the hope that better understanding of these pathways may improve in vitro induction. Fluorescence-activated cell sorting was used to obtain highly purified cultures of embryonic stem cells (ESCs), primitive germ cells (PGCs), and spermatogonial stem cells (SSCs). The total RNA was then extracted from each type of cell. High-throughput analysis methods (RNA-seq) were used to sequence the transcriptome of these cells. Gene Ontology (GO) analysis and the KEGG database were used to identify lipid metabolism pathways and related genes. Retinoic acid (RA), the end-product of the retinol metabolism pathway, induced in vitro differentiation of ESC into male germ cells. Quantitative real-time PCR (qRT-PCR) was used to detect changes in the expression of the genes involved in the retinol metabolic pathways. From the results of RNA-seq and the database analyses, we concluded that there are 328 genes in 27 lipid metabolic pathways continuously involved in lipid metabolism during the differentiation of ESC into SSC in vivo, including retinol metabolism. Alcohol dehydrogenase 5 (ADH5) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) are involved in RA synthesis in the cell. ADH5 was specifically expressed in PGC in our experiments and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) persistently increased throughout development. CYP26b1, a member of the cytochrome P450 superfamily, is involved in the degradation of RA. Expression of CYP26b1, in contrast, decreased throughout development. Exogenous RA in the culture medium induced differentiation of ESC to SSC-like cells. The expression patterns of ADH5, ALDH1A1, and CYP26b1 were consistent with RNA-seq results. We conclude that the retinol metabolism pathway plays an important role in the process of chicken male germ cell differentiation.
在此，我们探究鸡雄性生殖细胞分化过程中脂质代谢信号通路及相关基因的调控机制，希望对这些通路的深入了解能够改善体外诱导效果。利用荧光激活细胞分选技术获得了高度纯化的胚胎干细胞（ESC）、原始生殖细胞（PGC）和精原干细胞（SSC）培养物。然后从每种细胞类型中提取总RNA。采用高通量分析方法（RNA测序）对这些细胞的转录组进行测序。利用基因本体论（GO）分析和KEGG数据库来鉴定脂质代谢途径及相关基因。视黄醇代谢途径的终产物视黄酸（RA）可诱导ESC体外分化为雄性生殖细胞。采用定量实时PCR（qRT-PCR）检测视黄醇代谢途径中相关基因表达的变化。根据RNA测序和数据库分析结果，我们得出结论，在ESC体内分化为SSC的过程中，27条脂质代谢途径中的328个基因持续参与脂质代谢，包括视黄醇代谢。乙醇脱氢酶5（ADH5）和醛脱氢酶1家族成员A1（ALDH1A1）参与细胞内RA的合成。在我们的实验中，ADH5在PGC中特异性表达，醛脱氢酶1家族成员A1（ALDH1A1）在整个发育过程中持续增加。细胞色素P450超家族成员CYP26b1参与RA的降解。相反，CYP26b1的表达在整个发育过程中下降。培养基中的外源性RA诱导ESC分化为类SSC细胞。ADH5、ALDH1A1和CYP26b1的表达模式与RNA测序结果一致。我们得出结论，视黄醇代谢途径在鸡雄性生殖细胞分化过程中起重要作用。
