Functional Studies on the Involvement of Fatty Acid Oxidation in the Formation of Chicken (Gallus gallus domesticus) Primordial Germ Cells
WANG Zhe, WU Rong-Feng, GENG Qing-Qing, ZHAO Zi-Duo, WU Yi-Fan, CHENG Fu-Fu, XI Yi-Fan, CHEN Xin-Yu, WANG Chun-Hui-Zi, Niu Ying-Jie, ZUO Qi-Sheng, ZHANG Ya-Ni*
Jiangsu Key Laboratory of Animal Genetics, Breeding and Molecular Design/College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
Abstract：Poultry primordial germ cells (PGCs) migrate from the blood and colonize the reproductive ridge. Therefore, PGCs can not only replace fertilized eggs for genetic modification, but also have important application prospects in germplasm resource conservation and the recovery of endangered species. In vitro culture systems for PGCs exhibit differences in supporting the growth of male and female PGCs. So it is difficult to meet the requirements of PGCs in germplasm resource conservation. In response to this problem, this study aims to explore the function of fatty acid oxidation (FAO) involved in the formation of PGCs, and provide theoretical guidance for the subsequent establishment of a perfect in vitro culture system of PGCs.The role of FAO in the differentiation of embryonic stem cells (ESCs) into PGCs was analyzed based on transcriptomics, and the optimal addition concentrations of FAO activator BMS-687453 (BMS) and inhibitor perhexiline maleate (Per) were investigated by CCK8 and EdU tests. Finally, the role of FAO in the differentiation process from ESCs to PGCs was detected by cell morphology, qRT-PCR, indirect immunofluorescence and flow cytometry.The results showed that the genes involved in FAO were differentially expressed in ESCs and PGCs by transcriptomic analysis. Moreover, it was enriched in germ cell differentiation related pathways (MAPK signaling pathway, GnRH signaling pathway, PI3K-Akt signaling pathway). qRT-PCR results showed that acetyl-Coacyltransferase 2 (ACAA2) and enoyl-CoA hydratase 1 (ECH1) were highly expressed in PGCs.CCK8 and EdU experiments determined that the optimal addition concentrations of BMS and Per were 80 and 160 nmol/L, respectively. The results of cell morphology showed that the number of blastoid bodies in BMS group was significantly higher than that in Per group at 6 d (P<0.05). In BMS group, the expression levels of chicken VASA homolog (CVH) and c-Kit proto-oncogene receptor tyrosine kinase (C-KIT) were significantly higher than those in Per group (P<0.05). Indirect immunofluorescence and flow cytometry results showed that the number of DDX4 positive cells in BMS group was significantly higher than that in Per group (P<0.05). The above results indicated that promoting FAO could promote the formation of PGCs. This study provides a theoretical basis for establishing a perfect PGCs system in vitro culture of poultry, and also helps to systematically clarify the genesis mechanism of PGCs from the perspective of cell energy metabolism.
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