Dynamin serves a crucial role in lipid absorption in endodermal epithelial cells of Japanese quail yolk sac membrane

• Main Authors: Cheng-Ting Tung, 童政庭
• Other Authors: 丁詩同
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/6favqg

碩士 === 國立臺灣大學 === 動物科學技術學研究所 === 106 === During avian embryonic development mid and late stages are critical, of which the yolk sac membrane (YSM) extends from the avian abdomen to embrace the whole yolk, and endodermal epithelial cells (EECs) within the YSM are liable for the absorption and utilization of the lipids. However, the mechanism of lipids absorption and utilization by YSM are still unclear. In this study, we investigated lipid absorption mechanism in EECs, in attempt to make contributions to poultry industry. The lipids in the yolk are mostly composed of the very low density lipoprotein (VLDL), whose uptake mainly depends on the clathrin-mediated endocytosis (CME) on the cell membranes, CME relies on the coordination of lot of proteins to help membrane invagination, and the vesicle formation through the regulation of dynamin (DNM). In addition, we focused on the role of DNM in the mechanism of lipids utilization in EECs. We utilized Japanese quails as an animal model, to examine mRNA expression patterns of sterol O-acyltransferase 1 (SOAT1), apolipoprotein B (ApoB), families of cell death-inducing DFFA-like effector (CIDE) and DNM in YSM along the development and to establish the pathway of lipids absorption, modification and lipoprotein assembly for the regulation of DNM in YSM. The mRNA expression of DNM1 reached a peak at day 10 of incubation, SOAT1, CIDEA and ApoB expression were expressed aboundently during incubation day 10-14,16 and 18 respectively, and all the results were significantly different with those at day 2 (P < 0.05), suggesting DNM1 helps the lipids absorption in YSM. Next, a DNM inhibitor: dynasore was injected into fertilized eggs of Japanese quail at incubation day 3, and the phenotypes were observed at day 10. Results demonstrated the complete development percentage of embryos were significantly lower in the dynasore group than the controls, sham and vehicle-groups (P<0.05). The activities of DNMs were also verified by the absorptions of fluorescence labeled-VLDL (DiI-yVLDL) in EECs. Fluorescent signals in EECs were decreased significantly after pre-treatments of dynasore for 20 and 30 minutes. The absorptions of VLDL at various levels (800, 1050, and 1300 µg/mL of DiI-yVLDL) were blocked in EECs with the pre-treated dynasore. Third, we examine if the mRNA levels of DNM1, SOAT1, ApoB and CIDEA were affected in EECs, due to the lack of lipid resources. EECs were pre-treated with dynasore for 3 hours to inhibit the VLDL absorption for 24 hours before the mRNA extraction. Nonetheless, all these gene expression levels were not altered. Finally, we tested treating EECs with S-Nitroso-L-glutathione (GSNO, a DNM activator) for 30 minutes, and results showed that the intake of DiI-yVLDL was increased significantly by GSNO. In conclusion, DNM indeed serves a critical role of the lipids absorption in YSM. Furthermore, we successfully manipulated the DNM activity in EECsm despite that the mRNA expression of DNM, SOAT1, ApoB and CIDEA were not changed in EEC, suggesting the amount and activities of these target gene should be analyzed by other methods. These results delineated the mechanism of lipids utilization in EECs, and way improve the avian embryonic development in the future.