{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T07:26:57Z","timestamp":1788852417169,"version":"build-2803163510"},"reference-count":43,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2019,11,1]],"date-time":"2019-11-01T00:00:00Z","timestamp":1572566400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["31872433, 31272521"],"award-info":[{"award-number":["31872433, 31272521"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>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.<\/jats:p>","DOI":"10.3390\/ani9110888","type":"journal-article","created":{"date-parts":[[2019,11,1]],"date-time":"2019-11-01T12:30:50Z","timestamp":1572611450000},"page":"888","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Characterization of Extracellular Vesicles from Cilia and Epithelial Cells of Ductuli Efferentes in a Turtle (Pelodiscus sinensis)"],"prefix":"10.3390","volume":"9","author":[{"given":"Imran","family":"Tarique","sequence":"first","affiliation":[{"name":"MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yifei","family":"Liu","sequence":"additional","affiliation":[{"name":"MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuebing","family":"Bai","sequence":"additional","affiliation":[{"name":"MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9658-3690","authenticated-orcid":false,"given":"Abdul","family":"Haseeb","sequence":"additional","affiliation":[{"name":"MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ping","family":"Yang","sequence":"additional","affiliation":[{"name":"MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yufei","family":"Huang","sequence":"additional","affiliation":[{"name":"MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenjia","family":"Qu","sequence":"additional","affiliation":[{"name":"MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruizhi","family":"Wu","sequence":"additional","affiliation":[{"name":"MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Waseem Ali","family":"Vistro","sequence":"additional","affiliation":[{"name":"MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6087-7029","authenticated-orcid":false,"given":"Quisheng","family":"Chen","sequence":"additional","affiliation":[{"name":"MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2019,11,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1016\/j.micron.2012.06.010","article-title":"The ultrastructural characteristics of the spermatozoa stored in the cauda epididymidis in Chinese soft-shelled turtle Pelodiscus sinensis during the breeding season","volume":"44","author":"Bian","year":"2013","journal-title":"Micron"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1148","DOI":"10.1016\/j.theriogenology.2008.01.028","article-title":"Seasonal effects on apoptosis and proliferation of germ cells in the testes of the Chinese soft-shelled turtle, Pelodiscus sinensis","volume":"69","author":"Zhang","year":"2008","journal-title":"Theriogenology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"358","DOI":"10.1002\/j.1939-4640.2002.tb02243.x","article-title":"Expression and regulation of aquaporins 1, 8, and 9 in the testis, efferent ducts, and epididymis of adult rats and during postnatal development","volume":"23","author":"Badran","year":"2002","journal-title":"J. Androl."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"122","DOI":"10.1002\/jez.2002","article-title":"Morphological and ultrastructural study of the efferent ductules in the Chinese soft-shelled turtle Pelodiscus sinensis","volume":"325","author":"Waqas","year":"2016","journal-title":"J. Exp. Zool A Ecol. Genet. Physiol."},{"key":"ref_5","first-page":"1","article-title":"Fluid and electrolyte reabsorption in the ductuli efferentes testis","volume":"53","author":"Clulow","year":"1998","journal-title":"J. Reprod Fertil Suppl."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"3584","DOI":"10.1073\/pnas.1817018116","article-title":"Motile cilia of the male reproductive system require miR-34\/miR-449 for development and function to generate luminal turbulence","volume":"116","author":"Yuan","year":"2019","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_7","unstructured":"Skinner, M.K. (2018). Efferent Ductules: Structure and Function. Encyclopedia of Reproduction (Second Edition), Academic Press."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1038\/37352","article-title":"A role for oestrogens in the male reproductive system","volume":"390","author":"Hess","year":"1997","journal-title":"Nature"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2667","DOI":"10.1007\/s00018-011-0689-3","article-title":"Membrane vesicles, current state-of-the-art: Emerging role of extracellular vesicles","volume":"68","author":"Gyorgy","year":"2011","journal-title":"Cell Mol. Life Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"13","DOI":"10.1093\/jb\/mvj128","article-title":"Exosomes: A Common Pathway for a Specialized Function","volume":"140","author":"Simoes","year":"2006","journal-title":"J. Biochem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1007\/s00018-014-1710-4","article-title":"Tumor-derived exosomes in oncogenic reprogramming and cancer progression","volume":"72","author":"Saleem","year":"2015","journal-title":"Cell Mol. Life Sci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"2869","DOI":"10.1002\/ijc.29664","article-title":"Immuno-based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer","volume":"137","author":"Duijvesz","year":"2015","journal-title":"Int. J. Cancer"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"20121","DOI":"10.1074\/jbc.273.32.20121","article-title":"Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes","volume":"273","author":"Escola","year":"1998","journal-title":"J. Biol. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"26913","DOI":"10.3402\/jev.v3.26913","article-title":"Minimal experimental requirements for definition of extracellular vesicles and their functions: A position statement from the International Society for Extracellular Vesicles","volume":"3","author":"Hill","year":"2014","journal-title":"J. Extracell Vesicles"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"317","DOI":"10.1002\/aja.1001720406","article-title":"Maturation of Spermatozoa in the Epididymis of the Chinese-Hamster","volume":"172","author":"Yanagimachi","year":"1985","journal-title":"Am. J. Anat."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"34","DOI":"10.1038\/cdd.2014.130","article-title":"Exosomes as divine messengers: Are they the Hermes of modern molecular oncology?","volume":"22","author":"Braicu","year":"2015","journal-title":"Cell Death Differ."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"415","DOI":"10.1016\/j.ceb.2004.06.003","article-title":"Exosomes: Endosomal-derived vesicles shipping extracellular messages","volume":"16","author":"Fevrier","year":"2004","journal-title":"Curr. Opin. Cell Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"871","DOI":"10.1111\/j.1600-0854.2008.00734.x","article-title":"Exosome function: From tumor immunology to pathogen biology","volume":"9","author":"Schorey","year":"2008","journal-title":"Traffic"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1016\/j.acthis.2013.11.004","article-title":"Immunolocalization of estrogen and androgen receptors in the caput epididymidis of the fat sand rat (Psammomys obesus): Effects of seasonal variations, castration and efferent duct ligation","volume":"116","author":"Menad","year":"2014","journal-title":"Acta Histochem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"151","DOI":"10.1016\/0022-4731(74)90121-6","article-title":"Demonstraion of an adrogen binding protein (ABP) in rabbit testis: Secretion in efferent duct fluid and passage into epididymis","volume":"5","author":"French","year":"1974","journal-title":"J. Steroid Biochem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1002\/aja.1001630404","article-title":"Microvasculature of the mouse testis and excurrent duct system","volume":"163","author":"Suzuki","year":"1982","journal-title":"Am. J. Anat."},{"key":"ref_22","first-page":"541","article-title":"Lectin histochemical evaluation of glycoconjugates in dog efferent ductules","volume":"188","author":"Wakui","year":"1996","journal-title":"J. Anat."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1002\/ar.1092110309","article-title":"Fluid-phase and adsorptive endocytosis in ciliated epithelial cells of the rat ductuli efferentes","volume":"211","author":"Hermo","year":"1985","journal-title":"Anat. Rec."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2176","DOI":"10.1002\/ar.21282","article-title":"Proximal Testicular Ducts of the Mediterranean Gecko (Hemidactylus turcicus)","volume":"293","author":"Rheubert","year":"2010","journal-title":"Anat. Rec."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/S0940-9602(04)80009-8","article-title":"Morphology of the male reproductive duct system of Caiman crocodilus (Crocodylia, Alligatoridae)","volume":"186","author":"Guerrero","year":"2004","journal-title":"Ann. Anat. Anat. Anz. Off. Organ Anat. Ges."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"312","DOI":"10.1002\/jmor.10251","article-title":"Excurrent duct system of the male turtle Chrysemys picta","volume":"261","author":"Holmes","year":"2004","journal-title":"J. Morphol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1111\/j.1463-6395.2007.00278.x","article-title":"Efferent ductules of the fan-throated lizard Sitana ponticeriana Cuvier: Light and transmission electron microscopy study","volume":"88","author":"Akbarsha","year":"2007","journal-title":"Acta Zool."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/j.bcmd.2005.12.001","article-title":"Exosomes biological significance: A concise review","volume":"36","author":"Johnstone","year":"2006","journal-title":"Blood Cells Mol. Dis."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/j.tcb.2008.11.003","article-title":"Shedding microvesicles: Artefacts no more","volume":"19","author":"Cocucci","year":"2009","journal-title":"Trends Cell Biol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"3481","DOI":"10.1038\/emboj.2011.286","article-title":"Endosome maturation","volume":"30","author":"Huotari","year":"2011","journal-title":"EMBO J."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"35562","DOI":"10.1038\/srep35562","article-title":"Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells","volume":"6","author":"Chen","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1053\/gast.2001.26263","article-title":"Intestinal epithelial cells secrete exosome-like vesicles","volume":"121","author":"Raposo","year":"2001","journal-title":"Gastroenterology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1074\/mcp.M112.021303","article-title":"Two distinct populations of exosomes are released from LIM1863 colon carcinoma cell-derived organoids","volume":"12","author":"Tauro","year":"2013","journal-title":"Mol. Cell Proteom."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"193","DOI":"10.1007\/s00018-017-2595-9","article-title":"Current knowledge on exosome biogenesis and release","volume":"75","author":"Hessvik","year":"2018","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_35","first-page":"36","article-title":"Analysis of Apical and Basolateral Secreted Exosomes from Human Proximal Tubule Epithelial Cells (Ptec) under Normal and Diseased Conditions","volume":"22","author":"Wang","year":"2017","journal-title":"Nephrology"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Ivell, R., and Holstein, A.-F. (1997). The Role of Apocrine Released Proteins in the Post-Testicular Regulation of Human Sperm Function. The Fate of the Male Germ Cell, Springer.","DOI":"10.1007\/978-1-4615-5913-9"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"394","DOI":"10.1002\/mrd.90023","article-title":"Nature\u2019s ingenuity: Bypassing the classical secretory route via apocrine secretion","volume":"63","author":"Hermo","year":"2002","journal-title":"Mol. Reprod. Dev."},{"key":"ref_38","first-page":"88","article-title":"The sperm journey in the excurrent duct: Functions of microvesicles on sperm maturation and gene expression along the epididymis","volume":"12","author":"Sullivan","year":"2015","journal-title":"Anim. Reprod."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"R21","DOI":"10.1530\/REP-13-0058","article-title":"Epididymosomes, prostasomes, and liposomes: Their roles in mammalian male reproductive physiology","volume":"146","author":"Sullivan","year":"2013","journal-title":"Reproduction"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"19242","DOI":"10.18632\/oncotarget.8092","article-title":"Cellular evidence for nano-scale exosome secretion and interactions with spermatozoa in the epididymis of the Chinese soft-shelled turtle, Pelodiscus sinensis","volume":"7","author":"Chen","year":"2016","journal-title":"Oncotarget"},{"key":"ref_41","first-page":"521","article-title":"The role of Na+-H+ exchange in fluid and solute transport in the rat efferent ducts","volume":"84","author":"Hansen","year":"1999","journal-title":"Exp. Physiol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"654","DOI":"10.1038\/ncb1596","article-title":"Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells","volume":"9","author":"Valadi","year":"2007","journal-title":"Nat. Cell Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"4797","DOI":"10.1210\/en.2002-220531","article-title":"Induction of Reproductive Tract Developmental Abnormalities in the Male Rat by Lowering Androgen Production or Action in Combination with a Low Dose of Diethylstilbestrol: Evidence for Importance of the Androgen-Estrogen Balance","volume":"143","author":"Rivas","year":"2002","journal-title":"Endocrinology"}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/9\/11\/888\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T13:31:02Z","timestamp":1760189462000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/9\/11\/888"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,11,1]]},"references-count":43,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2019,11]]}},"alternative-id":["ani9110888"],"URL":"https:\/\/doi.org\/10.3390\/ani9110888","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,11,1]]}}}