Cellular Evidence and Source of Exosomes in the Biliary System of the Chinese Soft-Shelled Turtle, Pelodiscus sinensis

Abstract

Exosomes are extracellular vesicles with a size of 40–150 nm that are released from a multitude of cell types and are present in biological fluids, which are particularly rich in membrane proteins. These vesicles are present in the bile, where they function as a special regulator of the digestive system; however, their source and characteristics in normal gallbladders have never been discovered. Bile, liver, and gallbladder were obtained from healthy turtles after starvation treatment. Biliary exosomes were extracted and their morphology, particle sizes, and specific proteins were characterized by transmission electron microscope (TEM), nanoparticle tracking analysis (NTA), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) staining, and Western blotting. Furthermore, immunohistochemistry and TEM were used to investigate the distribution of exosomes in both liver and gallbladder. Biliary exosomes had a round or oval shape with apparent bilayer membranes. Biliary exosomes were uniform in size, with a range from 40 to 180 nm in diameter. Immunohistochemistry showed that the CD63 exosome marker was positive and primarily distributed in the hepatocyte junction, while the free surface of gallbladder tested negative. In addition, exosomes were present in bile but absent on the apical free surface of gallbladder. This study provides direct evidences that biliary exosomes are secreted by hepatocytes into bile canaliculus and flow with bile into the gallbladder. The source and the isolated protocol of biliary exosomes may provide technical support and a theoretical basis for the exploration of potential antiviral or anti-inflammatory properties of biliary exosomes.

Bridging the extracellular vesicle knowledge gap: insights from non-mammalian vertebrates, invertebrates, and early-diverging metazoans

“…Work in the Chinese softshell turtle showed biliary EVs originate from hepatocytes, supporting conserved EV roles in cell communication along the biliary system ( Zhu et al, 2019 ). In turtle reproductive tracts, EVs from Sertoli and telocyte sources appear to coordinate spermatogenesis and sperm maturation ( Chen and Holt, 2021 ).…”

Label-free quantitative proteomic analysis of serum exosomes from patients of renal anemia: The Good and the Bad of Roxadustat

“…All exosomes were stored at −80℃ immediately after isolation until further analysis. Transmission electron microscopy (TEM) (JEOL, 1230) and nanoparticle tracking analysis (NTA) (Particle Metrix, ZetaView PMX 110) were performed to validate the exosome isolation and purification [ 13 , 14 ].…”

Conventional and Nonconventional Sources of Exosomes–Isolation Methods and Influence on Their Downstream Biomedical Application

“…Other numerous non-mammalian eukaryotic organisms have been shown to use EXs/EXs-like vesicles; lower vertebrates, i.e. danio ( Verweij et al, 2019 ), xenopus ( Danilchik and Tumarkin, 2017 ), turtles ( Zhu et al, 2019 ), molluscs, i.e. oysters, where EXs have been found to be involved in reparation of the outer shell ( Johnstone et al, 2015 ), protozoa ( Lin et al, 2019 ) and fungi ( Rizzo et al, 2020 ).…”