The Proteome of Large or Small Extracellular Vesicles in Pig Seminal Plasma Differs, Defining Sources and Biological Functions

Abstract

Seminal plasma contains many morphologically heterogeneous extracellular vesicles (sEVs). These are sequentially released by cells of the testis, epididymis, and accessory sex glands and involved in male and female reproductive processes. This study aimed to define in depth sEV subsets isolated by ultrafiltration and size exclusion chromatography, decode their proteomic profiles using liquid chromatography-tandem mass spectrometry, and quantify identified proteins using sequential window acquisition of all theoretical mass spectra. The sEV subsets were defined as large (L-EVs) or small (S-EVs) by their protein concentration, morphology, size distribution, and EV-specific protein markers and purity. Liquid chromatography-tandem mass spectrometry identified a total of 1034 proteins, 737 of them quantified by SWATH in S-EVs, L-EVs, and non-EVs-enriched samples (18-20 size exclusion chromatography-eluted fractions). The differential expression analysis revealed 197 differentially abundant proteins between both EV subsets, S-EVs and L-EVs, and 37 and 199 between S-EVs and L-EVs versus non-EVs-enriched samples, respectively. The gene ontology enrichment analysis of differentially abundant proteins suggested, based on the type of protein detected, that S-EVs could be mainly released through an apocrine blebbing pathway and be involved in modulating the immune environment of the female reproductive tract as well as during sperm-oocyte interaction. In contrast, L-EVs could be released by fusion of multivesicular bodies with the plasma membrane becoming involved in sperm physiological processes, such as capacitation and avoidance of oxidative stress. In conclusion, this study provides a procedure capable of isolating subsets of EVs from pig seminal plasma with a high degree of purity and shows differences in the proteomic profile between EV subsets, indicating different sources and biological functions for the sEVs.

Keywords: extracellular vesicles; pig; proteomics; seminal plasma; subsets.

Graphical abstract

Fig. 1

Flow chart showing the handling of boar seminal plasma prior to isolation of extracellular vesicle subsets. RT, room temperature. The drawing was created using the software of BioRender.com.

Fig. 2

Schematic summary of the size exclusion chromatography procedure to isolate the two subsets of extracellular vesicles (EVs) from boar seminal plasma and the experimental workflow used for the analysis of EVs. The resulting EVs were characterized for total protein concentration, morphology, size, presence of specific protein markers, and purity. The drawing was created using the software of BioRender.com

Fig. 3

Characterization of extracellular vesicle (EVs) subsets, namely, small (S-EVs) or large (L-EVs), isolated from pig seminal plasma (SP) samples (n = 3; three ejaculates per sample; one ejaculate per male pig).A, violin plot displaying the total protein concentration in both SP-EV subsets. The dashed line shows the median and dotted lines the 25 to 75% quartiles. B, representative histogram of particle size distribution of S-EVs and L-EVs assessed by nanoparticle tracking analysis. C, particle size distribution of S-EVs and L-EVs analyzed by dynamic light scattering (red, S-EVs; blue, L-EVs) in terms of intensity and volume. The black and gray lines represent the average of intensity size distribution of S-EVs and L-EVs, respectively. D, representative images of the morphology of S-EVs and L-EVs assessed by transmission electron microscopy. E, representative histogram of CFSE/CD63/HSP90β/ALB expression in S-EVs and L-EVs assessed by flow cytometry. ALB, albumin; CFSE, carboxyfluorescein succinimidyl ester; CNT, control; HSP90β, heat shock protein 90β.

Fig. 4

Proteomic profiling of the two subsets of pig seminal extracellular vesicles (EVs) (small [S-EVs]andlarge [L-EVs]) and ofnon-EV-enrichedsamples (n = 3; three ejaculates per each sample; one ejaculate per individual).A, two-dimensional principal component analysis evaluating differences on the quantified proteins in S-EVs, L-EVs, and non-EVs-enriched samples. B, heatmap depicting the abundance patterns of 737 proteins quantified in the S-EVs, L-EVs, and non-EVs-enriched samples. Each column and row represents an individual sample and protein, respectively. Relative protein levels are depicted in color scale: red indicates more abundance and blue indicates less abundance. C, volcano plot showing the differentially abundant proteins between the pairwise compared S-EVs, L-EVs, and non-EVs-enriched samples. The x-axis shows the log₂ fold change of the comparation, while the y-axis shows the −log₁₀ of the calculated probability (p value). The dots indicate the proteins that are most abundant in each sample. The horizontal red line indicates p-value = 0.05, and the vertical green line indicates log₂ fold change = ±2. D, Venn diagram showing the number of differentially abundant proteins (with log₂ fold change > 2; p-value ˂ 0.05) between EVs samples (S-EVs or L-EVs) and non-EVs-enriched samples of pig seminal plasma. (1) More abundant in EVs samples and (2) more abundant in non-EVs-enriched samples.

Fig. 5

Gene Ontology enrichment analysis of differential abundant proteins between extracellular vesicles (EVs) samples (small EVs [S-EVs] or large EVs [L-EVs]) andnon-EVs-enrichedsamples of pig seminal plasma.A, the terms are ranked by their enrichment scores that were overrepresented in the highly abundant proteins of EVs samples (140 proteins; green) or in the non-EVs-enriched samples (73 proteins; blue). B, reproductive processes in which the enriched are involved.

Fig. 6

Gene Ontology enrichment analysis of differentially abundant proteins between the two subsets of extracellular vesicles (EVs) isolated from pig seminal plasma (small [S-EVs] or large [L-EVs]). Terms are ranked by their enrichment scores, which were overrepresented in the highly abundant proteins of S-EVs (29 proteins; pink) or L-EVs (168 proteins; blue) samples.

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