ABSTRACT Background: Prostasomes, extracellular vesicles (EVs) abundantly present in seminal plasma, express distinct tetraspanins (TS) and galectin-3 (gal-3), which are supposed to shape their surface by an assembly of different molecular complexes. In this study, detergent-sensitivity patterns of membrane-associated prostasomal proteins were determined aiming at the solubilization signature as an intrinsic multimolecular marker and a new parameter suitable as a reference for the comparison of EVs populations in health and disease. Methods: Prostasomes were disrupted by Triton X-100 and analyzed by gel filtration under conditions that maintained complete solubilization. Redistribution of TS (CD63, CD9, and CD81), gal-3, gamma-glutamyltransferase (GGT), and distinct N-glycans was monitored using solid-phase lectin-binding assays, transmission electron microscopy, electrophoresis, and lectin blot. Results: Comparative data on prostasomes under normal physiology and conditions of low sperm count revealed similarity regarding the redistribution of distinct N-glycans and GGT, all presumed to be mainly part of the vesicle coat. In contrast to this, a greater difference was found in the redistribution of integral membrane proteins, exemplified by TS and gal-3. Accordingly, they were grouped into two molecular patterns mainly consisting of overlapped CD9/gal-3/wheat germ agglutinin-reactive glycoproteins and CD63/ GGT/concanavalin A-reactive glycoproteins. Conclusions: Solubilization signature can be considered as an all-inclusive distinction factor regarding the surface properties of a particular vesicle since it reflects the status of the parent cell and the extracellular environment, both of which contribute to the composition of spatial membrane arrangements. https://phaidrabg.bg.ac.rs/o:25139 doi:10.48101/ujms.v126.7673 info:eu-repo/semantics/article Janković, Tamara Danilović Luković, Jelena Miler, Irena Mitić, Ninoslav Hajduković, Ljiljana Janković, Miroslava eng extracellular vesicles, detergent sensitivity, CD63, CD9, gamma-glutamyl transferase, molecular patterns, normozoospermia, oligozoospermia 2021 Upsala journal of medical sciences 126 application/pdf 2696173 bytes Assembly of tetraspanins, galectin-3, and distinct N-glycans defines the solubilization signature of seminal prostasomes from normozoospermic and oligozoospermic men http://creativecommons.org/licenses/by/4.0/legalcode