Abstract
Seminal extracellular vesicles (EVs) include exosomes (ø 40–120 nm) and microvesicles (MVs, ø 120–1000 nm), which would be involved in multiple functional reproductive roles. The study aimed to establish which EV subtypes are present in pig semen, using a high-resolution flow cytometer to explore differences in their tetraspanin expression profile. The EVs were isolated from 12 pig ejaculates using serial ultracentrifugation and characterized by dynamic light scattering and electron microscopy for size and morphology as well as for tetraspanin expression using flow cytometry with Carboxyfluorescein succinimidyl ester (CFSE) and antibodies against CD9, CD63 and CD81. Pig semen contained a heterogeneous EV-population regarding size and morphology. Flow cytometric analysis demonstrated that the proportion of EVs expressing CD63 and CD9 was higher in MVs (P < 0.001 and P < 0.05, respectively) than in exosomes, while the opposite was true for CD81; higher (P < 0.001) in exosomes than in MVs. In conclusion, (1) the new generation of flow cytometers are able to accurately identify EVs and to gate them in two size-different populations named exosomes and MVs. (2) Tetraspanins CD9, CD63 and CD81 are present in both seminal EVs, albeit with exosomes and MVs differing in expression profiles, suggesting dissimilar cargo and binding affinity.
Highlights
Over the last years, the involvement of the cargo of mammalian extracellular vesicles (EVs), as exosomes or microvesicles (MVs), for autocrine and paracrine signaling during physiological and pathological processes has gained the interest of the scientific community[1]
The results demonstrated that the three tetraspanins CD9, CD63 and CD81, were present in both EV subtypes in all examined samples of ejaculated seminal plasma
None have proven precise enough to discriminate between EV subtypes in a single sample, which limits our understanding of the specific functions of each EV subtype[1]
Summary
The involvement of the cargo of mammalian extracellular vesicles (EVs), as exosomes or microvesicles (MVs), for autocrine and paracrine signaling during physiological and pathological processes has gained the interest of the scientific community[1]. Despite extensive research on EVs, no technique has been established to separate different EV subtypes. This hampers our capability to identify the specific cargo and function of each of the EV subtypes. Tetraspanins are highly conserved plasma membrane proteins that act as scaffolds for other proteins in particular areas of the cell plasmalemma[20]. The present study aimed quantitation of the expression of CD9, CD63 and CD81 tetraspanins in EVs present in pig seminal plasma using novel high-resolution flow cytometry. In addition to the relevance for pig reproduction, the expected results would be interesting for other mammalian species, including human, considering pig is a useful animal model for biomedical research[24]
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