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Identification, Proteomic Profiling, and Origin of Ram Epididymal Fluid Exosome-Like Vesicles¹

Equipe Gamètes Males et Fertilité³ and Service de Spectrométrie de Masse pour la Protéomique,⁴ URM 6175 INRA-CNRS-Université de TOURS-Haras Nationaux, Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique de Nouzilly, Monnaie 37380, France

Small membranous vesicles, between 25- and 75-nm diameter, were collected by high-speed centrifugation from the ram cauda epididymal fluid and were found to be normal constituents of this fluid and of the seminal plasma. The SDS-PAGE protein pattern of these vesicles was specific and very different from that of the caudal fluid, seminal plasma, sperm extract, and cytoplasmic droplets. After two-dimensional electrophoresis separation and mass spectrometry analysis, several proteins were identified and grouped into i) membrane-linked enzymes, such as dipeptidyl peptidase IV (DPP-IV), neprilysin (NEP), phosphodiesterase-I (E-NPP3), and protein G-beta; ii) vesicle-associated proteins, such as lactadherin (MFEG8-PAS6/7) and vacuolar ATPase; iii) several cytoskeleton-associated proteins, such as actin, ezrin and annexin; and iv) metabolic enzymes. The presence of some of these proteins as well as several different hydrophobic proteins secreted by the epididymis was further confirmed by immunoblotting. These markers showed that the majority of the vesicles originated from the cauda epididymal region. The physical and biochemical characteristics of these vesicles suggest they are the equivalent of the exosomes secreted by several cell types and epithelium. The main membrane-linked proteins of the vesicles were not retrieved in the extract from cauda or ejaculated sperm, suggesting that these vesicles did not fuse with sperm in vivo.

epididymis, epididymosomes, exosomes, gamete biology, male reproductive tract, mass spectrometry, protein, proteomic, sperm, spermatozoa, sperm maturation

² Correspondence: Dr. Jean-Luc Gatti, UMR 6175 INRA-CNRS-Université de TOURS-Haras Nationaux, Physiologie de la Reproduction et des Comportements, Institut National de la Recherche Agronomique de Nouzilly, Monnaie 37380, France. FAX: 33 247 427 743; gatti{at}tours.inra.fr

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