Characterization of Extracellular Vesicles from Cilia and Epithelial Cells of Ductuli Efferentes in a Turtle (Pelodiscus sinensis)

SIMPLE SUMMARY: Ductuli efferent is a conduit passage of spermatozoa from rete testis to the epididymis. After spermiation, various extracellular vesicles are released by epithelial cells to assist and transfer micro-molecules for spermatozoa maturation. To date, there is a lack of data in ductuli efferent. In this study, we investigate the distribution, classification, and source of multivesicular bodies (MVBs) and their extracellular vesicles (EVs) in the epithelia of efferent ducts in a turtle by using light and transmission electron microscopy. Immunohistochemistry of CD63 (Cluster of differentiation 63) and ultrastructure demonstrates that ciliated and non-ciliated cells of ductuli efferent possess well developed exocytic systems. Ciliated cells and non-ciliated cells secrete extracellular vesicles via ciliary blebs and apical blebs, respectively. Results also showed numerous extracellular vesicles between these cells and indicate basolateral secretion. Moreover, these extracellular vesicles are associated with spermatozoa in the lumens of ductuli efferentes. Collectively, the present study provides cytological evidence that the ductuli efferentes (DE) epithelium secretes EVs to the lumen by (1) apical blebs, (2) ciliary blebs, and (3) from the basolateral region. Characterization and cellular distribution of these extracellular vesicles in the ductuli efferent of turtles may provide a study model to further investigate the transferring of micro-molecules via extracellular vesicles to the spermatozoa. ABSTRACT: The ductuli efferentes (DE) form a transit passage for the passage of spermatozoa from the rete testis to the epididymis. After spermiation, various epithelial secretory proteins are transferred via extracellular vesicles (EVs) to the spermatozoa for their maturation and long-term viability. The aim of the present study was to investigate the distribution, classification, and source of multivesicular bodies (MVBs) and their EVs in the epithelia of the efferentes duct in a turtle species, the soft-shelled freshwater turtle Pelodiscus sinensis by using light and transmission electron microscopy. The results showed that CD63 as a classical exosome marker was strongly immunolocalized within the apical and lateral cytoplasm of the ciliated cells (CC) and moderate to weak in the non-ciliated cells (NCC) of DE. The ultrastructure revealed that early endosome was present at the basement membrane and perinuclear cytoplasm of both CC and NCC, whereas MVBs were located over the nucleus in the cytoplasm of NCC and adjacent to the basal bodies of cilia within the CC. Many EVs, as sources of MVBs, were located within the blebs that were attached to the cilia of CC, within the apical blebs from NCC, and the lateral spaces of CC and NCC. There was ultrastructure evidence of EVs associated with spermatozoa in the lumens of DE. Collectively, the present study provides cytological evidence that the DE epithelium secreted EVs to the lumen by (1) apical blebs, (2) ciliary blebs, and (3) from the basolateral region. These EVs were associated with spermatozoa in the DE lumen of this turtle. Characterization and cellular distribution of these EVs in the DE of a turtle may provide a study model to further investigate the transferring of micromolecules via EVs to the spermatozoa.