Unraveling the Multi-Omic Landscape of Extracellular Vesicles in Human Seminal Plasma

Abstract

Extracellular Vesicles (EVs) from seminal plasma have achieved attention due to their potential physiopathological role in male reproductive systems. This study employed a comprehensive proteomic and transcriptomic approach to investigate the composition and molecular signatures of EVs isolated from human seminal plasma. EVs from Normozoospermic (NORMO), OligoAsthenoTeratozoospermic (OAT), and Azoospermic (AZO) subjects were isolated using a modified polymer precipitation-based protocol and characterized for size and morphology. Comprehensive proteomic analysis, using both gel-free and gel-based approaches, revealed distinct protein profiles in each group (p<0.01), highlighting potential molecules and pathways involved in sperm function and fertility. The data are available via ProteomeXchange with identifiers PXD051361 and PXD051390, respectively. Transcriptomic analysis confirmed the trend of a general downregulation of AZO and OAT compared to NORMO shedding light on regulatory mechanisms of sperm development. Bioinformatic tools were applied for functional omics analysis; the integration of proteomic and transcriptomic data provided a comprehensive understanding of the cargo content and regulatory networks present in EVs. This study contributes to elucidating the key role of EVs in the paracrine communication regulating spermatogenesis. A full understanding of these pathways not only suggests potential mechanisms regulating male fertility but also offers new insights into the development of diagnostic tools targeting male reproductive disorders.

Keywords: extracellular vesicles; intercellular communication; male reproductive tract; proteomics; reproduction; seminal plasma; transcriptomics.

Figure 1

Characterization of EVs isolated by EV-GAG-based protocol from seminal fluids. The assessment of EV content was first performed on three proband samples from three NORMO subjects (A–C); (A) NTA-estimated particle size distributions, (B) cholesterol content (expressed in fg per particle), (C) tetraspanin CD63/CD9 expression, expressed as signal-to-noise ratio (SNR). EV-pellet indicates pellet obtained by EV-GAG-mediated precipitation from seminal fluid, and SN- indicates post-precipitation supernatant. (D,E) NTA was used to compare the size and concentration of particles isolated from normozoospermic (NORMO), oligoasthenoteratozoospermic (OAT), and azoospermic (AZO) subjects. (F,G) Ultrastructural characterization of EVs from NORMO samples by TEM.

Figure 2

Representative 2D electropherograms from samples collected during the EV isolation protocol set-up. (A) EV pellet obtained without PBS washes; (B) EV pellet washed 3 times in PBS; (C) supernatant of the evGAG precipitation; (D) third PBS wash.

Figure 3

Enrichment analysis of NORMO EV protein cargo by Gene Ontology. (A) GO Cellular localization: representative scheme of the top 15 most abundant GO terms. The x-axis indicates the −log(p-Value), while the y-axis indicates different GO terms ordered by −log(p-value). (B) GO Biological Processes: visual representation of enriched GO Biological Processes (BP) terms for shotgun proteomic data of EVs isolated from human seminal fluid of NORMO subjects. Coloured bars constituting the circle graph represent the nine most enriched general BP terms. The length of the coloured bars indicates the mean of the −log(p-value) of all related specific BP terms. Salmon pink bars represent enriched specific BP terms, and the length of the bars indicate the −log(p-value) of the term. Therefore, the longer the bars, the higher the significance.

Figure 4

Proteomic differential analysis. Representative electropherograms of EV samples obtained from (a) NORMO, (b) OAT, and (c) AZO seminal fluid. Numbers indicate differential spots found by the comparative analysis.

Figure 5

Proteomic differential analysis. (A) Principal component analysis performed by %V data of the differentially abundant spots. PCA by PC1 and PC2 and the most representative spots in sample distribution (panel a). PCA graph by PC1 and PC3 (panel b). PCA graph by PC2 and PC3 (panel c). (B) Heatmap analysis performed by %V data of the differentially abundant spots, by Euclidean distance. Green bar highlights NORMO samples, blue bars the OAT samples, and the red bars the AZO samples, as reported in the legend. Spot abundance ranges from red (highly abundant) to blue (lower abundant).

Figure 6

MetaCore analysis. (A) MetaCore enrichment analysis of the three groups of proteins (highly abundant in NORMO-green; highly abundant in OAT-blue; highly abundant in AZO-red). All groups reported the comparison of GO Biological Processes and Cellular Localization. (B) Individual Process network analysis by MetaCore of the highly abundant proteins in NORMO (green), OAT (blue), and AZO (red) EV samples, respectively. Process network terms were reported on the left part of the histograms, indicating the statistical significance. At the right side of the histograms were the proteins that mostly influenced that process network.

Figure 7

EV RNA cargo: a comparative transcriptomic analysis of RNA-Seq datasets from NORMO, OAT and AZO. (A) Histograms illustrate the differentially expressed RNA counts: in green protein-coding; in blue lncRNA; and in red other RNA. (B) Venn diagram highlighting the overlap of protein-coding transcripts among the three independent datasets. (C) Visualization of transcriptome differences, by scatter plots, in EVs between NORMO and OAT; NORMO and AZO; OAT and AZO. Values depicted are the log 2 transformation of cross-sample normalized RNA-seq read counts + 1 [log₂(N + 1)]. The colour gradient, from purple to yellow, shows the density of points: yellow represents areas with the highest density of genes (many genes with similar expression levels in both conditions), while purple indicates lower density.

Figure 8

Differential gene expression between NORMO, OAT, and AZO. (A) Principal component analysis (PCA) showing distinct separation of NORMO with respect to the other two groups. (B) Heatmap with hierarchical clustering represents the expression levels from three groups: AZO (red), OAT (blue), and NORMO (green). The colour scale indicates gene expression intensity, with red representing higher expression and blue indicating lower expression. (C) The Volcano plot contrasting gene expression between OAT and NORMO (upper panel) and AZO vs. NORMO (lower panel) groups. The log2 fold changes on the x-axis show the magnitude of expression differences, while the −log10 p-values on the y-axis indicate statistical significance. Genes that are significantly differentially expressed, surpassing set thresholds for fold change and p-value, are labelled and coloured to stand out from non-significant ones (green) The fold change threshold has been set to zero. (D) The image presents a bar chart displaying the number of differentially expressed (DE) genes between three study groups: AZO, NORMO, and OAT. The chart shows comparisons between groups, indicating the direction of expression change. The tallest bar represents many genes downregulated in OAT compared to NORMO, while the smallest bar indicates a single gene downregulated in AZO compared to OAT.

Figure 9

Gene enrichment analysis for the biological process ontology (GO) terms. The y-axis lists the GO terms. The top 100 differentially expressed genes (DEGs) were included. The x-axis quantifies the gene count associated with each term. The bars are coloured based on a combined score, indicated by the heatmap legend on the right, with red denoting higher scores and blue representing lower scores. KEGG pathway. Chart from a KEGG pathway enrichment analysis. The enriched terms along the y-axis represent various biological pathways. The x-axis shows the gene count that correlates with each pathway.

Figure 10

Network of interactions of proteins involved in the fertilization pathway generated by STRING. (A) A general overview. Specifically Alpha-1B-Glycoprotein (A1BG), Disintegrin and metalloproteinase domain-containing protein 2 (ADAM2), A-Kinase Anchor Protein 3, 4 (AKAP3, AKAP4), Aldehyde dehydrogenase 2 (ALDH2), Aldolase A (ALDOA), BAG cochaperone 6 (BAG6), Voltage-dependent L-type calcium channel subunit beta-3 (CACNB3), Cation channel sperm-associated protein 1, beta, gamma (CATSPER1, CATSPERB, CATSPERG), CD9 antigen (CD9), Calmegin (CLGN), Clusterin (CLU), Cysteine-rich secretory protein 1, 2, 3 (CRISP1, CRISP2, CRISP3), Glyceraldehyde-3-phosphate dehydrogenase, testis-specific (GAPDHS), Heat shock protein family A member 4 (HSPA4), Endoplasmin (HSP90B1), Stress-70 protein, mitochondrial (HSPA9), Insulin-like growth factor 1 (IGF1), Lactate dehydrogenase A, C (LDHA, LDHC), Lactadherin (MFGE8), Macrophage migration inhibitory factor (MIF), Outer dense fibre protein 4 (ODF4), Glycodelin (PAEP), Phosphoglycerate kinase 2 (PGK2), Sperm acrosome membrane-associated protein 1, 4 (SPACA1, SPACA4), Semenogelin 1 (SEMG1), Sorbitol dehydrogenase (SORD), Coiled-coil domain containing 54 (CCDC54), Sperm Adhesion Molecule 1 (SPAM1), Osteopontin (SPP1), and complex protein 1 subunit alpha (TCP1) are shown. (B) Network of selected and investigated interactions of ADAM2, CLGN, CRISP1, CRISP2, CRISP3, MIF, GAPDHS, PGK2, SPAM1, SPP1, and PAEP proteins generated by STRING. Each node represents a protein, while edges delineate the type of interconnection (network caption).

Figure 11

Gene expression analysis by ddPCR. Relative expression of Cysteine Rich Secretory Protein 1-2-3 (CRISP1-2-3), Sperm Adhesion Molecule 1 (SPAM1), ADAM Metallopeptidase Domain 2 (ADAM2), Macrophage Migration Inhibitory Factor (MIF), Parkinsonism-associated deglycase (PARK7), Ocitrate dehydrogenase (NADP(+)) 1, cytosolic (IDH1), Chloride intracellular channel 4 (CLIC4), Prostate stem cell antigen (PSCA), and Kallikrein-related peptidase 3 (KLK3) in seminal EVs of normozoospermic subjects. Normalization of target genes was performed using GAPDH and RNY4 as reference genes. Gene expression was performed by ddPCR. Graphical diagrams are plotted as box-whisker plots, where boxes show the interquartile range with median and mean values, and whiskers represent min and max confidence intervals. Outliers, plotted as individual dots, represent out-of-range values. * p < 0.05; ** p < 0.01; *** p < 0.001.