Extracellular vesicles isolated from porcine seminal plasma exhibit different tetraspanin expression profiles

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.

Chicken seminal fluid lacks CD9‐ and CD44‐bearing extracellular vesicles

“…The electron microscopy of the samples depicted scattered presence of round particles (Figure a–c) whose ultrastructural aspect was mostly compatible with lipid droplets rather than with EVs (Figure a–c), compared to findings in mammals (Alvarez‐Rodriguez, Ljunggren, et al, ; Barranco et al, ). A further striking finding was their scarcity and low size (Figure d–f), irrespective of the samples assessed, either from original SF or following isolation/enrichment by use of the Total Exosome Precipitation Reagent.…”

“…-c) whose ultrastructural aspect was mostly compatible with lipid droplets rather than with EVs (Figure 1a-c), compared to findings in mammals (Alvarez-Rodriguez,Ljunggren, et al, 2019;Barranco et al, 2019). A further striking finding was their scarcity and low size(Figure 1d-f), irrespective of the samples assessed, either from original SF or following isolation/enrichment by use of the Total Exosome Precipitation Reagent.The flow cytometric sorting procedure was able to clearly separate the distinct particular surface markers for the EVs in the positive control (porcine exosomes).…”

MiRNAs in the Peri-Implantation Period: Contribution to Embryo–Maternal Communication in Pigs

“…This observation was the first hint that EVs can be active mediators of embryo-maternal communication during early pregnancy in pigs. To date, EVs have been isolated from different body fluids in different species, including porcine seminal plasma [55] and uterine lumen flushings [22]. Recently, intensive secretion of EVs by endometrial and trophoblast cells was detected during the peri-implantation period in pigs.…”

Comparative Evaluation of Methods for Isolating Small Extracellular Vesicles Derived from Pancreatic Cells

“…was not exclusive to the sEVs. Recent studies reported that microvesicles also expressed CD63 36,37 . A combined strategy of several sEVs marker proteins such as CD63, TSG101 and CD9 may re ect the sEVscontaining fractions more accurately.…”