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The proteome of large or small extracellular vesicles in pig seminal plasma differs and defines sources and biological functions. Spectral Library

ABSTRACT: Seminal plasma contains many and morphologically heterogeneous extracellular vesicles (sEVs) which, released by cells of the testis, epididymis and accessory sex glands, are involved in male and female reproductive processes. The goal of this study was to describe the proteome of porcine sEVs isolated by a size exclusion chromatography (SEC)-based procedure and size-classed as small (S-EVs) or large (L-EVs). The sEV-subsets were characterized for their total protein concentration, morphology, size distribution, EV-specific protein markers and purity. A total of 988 proteins were identified by LC-MS/MS and 737 of them were quantified by SWATH in S-EVs, L-EVs and EVs-free samples. The differential expression analysis revealed 197 differentially abundant proteins between both EV-subsets, and 37 and 199 between S-EVs and L-EVs vs EVs-free samples, respectively. The gene ontology (GO) enrichment analysis of differentially abundant proteins suggested, by the type of protein detected, that S-EVs would be released mainly 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, the protein types detected might imply L-EVs are released by fusion of multivesicular bodies with the plasma membrane and would be involved in sperm physiological processes, such as capacitation and avoidance of oxidative stress. In conclusion, this study provides a procedure capable of isolating of subsets of EVs from pig seminal plasma with a high degree of purity and shows differences in the proteomic profile between EV-subsets, suggesting different sources and biological functions.

INSTRUMENT(S): TripleTOF 6600

ORGANISM(S): Sus Scrofa Domesticus (domestic Pig)

TISSUE(S): Seminal Vesicle

SUBMITTER: Luz Valero

LAB HEAD: Jordi Roca

PROVIDER: PXD039833 | Pride | 2023-02-15

REPOSITORIES: Pride

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Project description:Background: Acute coronary syndromes (ACS) are associated with aberrant gene expression and epigenetic mechanisms. In particular, de novo DNA methylation is typically linked to gene silencing, but its role in heart disease remains not fully understood. Extracellular vesicles (EVs) are active components in cellular communication for their ability to carry a plethora of signalling biomolecules, thus representing a promising new diagnostic/therapeutic approach in cardiovascular diseases (CVDs). Indeed, there is the need of novel biomarkers for ACS prediction and timely detection. Purpose: We hypothesized that specific epigenetic signals can be carried by EVs. In this regard, we isolated and characterized circulating EVs from ACS patients and evaluated their potential role to influence DNA methylation in target cells. Methods: Circulating EVs were recovered, by ultracentrifugation, from plasma samples of 19 ACS patients and 50 healthy subjects (HS). Nanoparticle tracking analysis (NTA) and western blot (WB) were used to confirm the EVs integrity and purity. Peripheral blood mononuclear cells (PBMCs) of volunteer donors were treated with both ACS and HS derived EVs and genomic DNA was extracted to perform epigenome wide analysis through Reduced Representation Bisulfite Sequencing. ShinyGO, PANTHER, and STRING tools were interrogated to perform GO and PPI network analyses. Flow Cytometry, qRT-PCR, and WB analysis were also performed to evaluate and validate both intra-vesicular and intra-cellular signals. Results: Plasma ACS-derived EVs showed a significant up-regulation of DNA methyltransferases (DNMTs) gene expression levels as compared to HS (P<0.001). Specifically, de novo methylation transcripts, as DNMT3A and DNMT3B were significantly increased in plasma ACS-EVs. DNA methylation analysis of PBMCs from volunteer donors treated with HS- and ACS-derived EVs showed that RNF166 and CCND3 genes resulted the most hyper- and hypo-methylated, respectively, after by ACS-EV treatment. In addition, PPI network analysis specifically evidenced the subnetwork with SRC, as a hub gene, connecting it to NOTCH1, FOXO3, CDC42, IKBKG, RXRA, DGKG, known as important genes already involved in the onset of CVDs. Surprising, other novel genes, BAIAP2, SYP, CHL1, and SHB, which were hypomethylated, were found significantly overexpressed in PBMCs (P<0.005), underlying the fundamental modulating properties of EV cargo in atherosclerosis. Conclusions: These findings support the significant role of ACS plasma-derived EVs, inducing de novo DNA methylation signals, and modulating specific signaling pathways in target cells.

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The proteome of large or small extracellular vesicles in pig seminal plasma differs and defines sources and biological functions. Spectral Library

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