Comprehensive proteomics analysis of exosomes derived from human seminal plasma

Abstract

SummaryExosomes are membranous nanovesicles of endocytic origin that carry and transfer regulatory bioactive molecules and mediate intercellular communication between cells and tissues. Although seminal exosomes have been identified in human seminal plasma, their exact composition and possible physiologic function remain unknown. The objective of this study was to perform a comprehensive proteomics analysis of exosomes derived from human seminal plasma. Seminal exosomes were isolated and purified from 12 healthy donors using a 30% sucrose cushion‐based exosome‐isolation protocol, followed by characterization by western blot, transmission electron microscopy, and nanoparticle tracking analysis before performing extensive liquid chromatography tandem mass spectrometry proteomics analysis. The identified proteins were analyzed by bioinformatics analysis, and seminal exosomes‐associated proteins were selectively validated by western blot. A total of 1474 proteins were identified in all seminal exosomes samples, with Gene Ontology analysis demonstrating that these identified seminal exosomes‐associated proteins were mostly linked to ‘exosomes,’ ‘cytoplasm,’ and ‘cytosol.’ Bioinformatics analysis indicated that these proteins were mainly involved in biologic processes, including metabolism, energy pathways, protein metabolism, cell growth and maintenance, and transport. Of these identified proteins, PHGDH, LGALS3BP, SEMG1, ACTB, GAPDH, and the exosomal‐marker protein ALIX were validated by western blot. This study provided a more comprehensive description of the seminal exosomes proteome and could also be a resource for further screening of biomarkers and comparative proteomics studies, including those associated with male infertility and prostate cancer.

Proteomic analysis of seminal extracellular vesicle proteins involved in asthenozoospermia by iTRAQ

“…We identified a total of 3,699 proteins in seminal extracellular vesicles, which is a substantially greater number than the 139 human prostasome proteins previously presented by Utleg et al (), 440 prostasome proteins reported by Poliakov et al (Poliakov et al, ), and 1,474 proteins in seminal extracellular vesicles recently identified by Cundong Liu (Yang et al, ). There are 794 proteins accounting 21.47% of 3,699 total proteins had been previously identified in at least one study, remarkably, 2,905 no‐overlapping proteins identified in this study had not been identified in previous studies (Figure S1), The no‐overlapping proteins including ACE, PPAP2A, and SMYD2 were validated by western blot in our study (Figure ), and GO analysis shown that most percentage of no‐overlapping proteins enriched localized in “Exosomes”, “lysosome” and “cytoplasm” (Figure S2), these results indicated that the nonoverlapping proteins were most probably derived from seminal extracellular vesicles, and also suggested that the iTRAQ method is more highly sensitive than the previous proteomic technology.…”

“…We identified a total of 3,699 proteins in seminal extracellular vesicles, which is a substantially greater number than the 139 human prostasome proteins previously presented by Utleg et al (2003), 440 prostasome proteins reported by Poliakov et al (Poliakov et al, 2009), and 1,474 proteins in seminal extracellular vesicles recently identified by Cundong Liu (Yang et al, 2017).…”

“…It has been reported that human semen contains a large number of exosome-like vesicles, which were originally called prostasomes (Ronquist, 2012), because they were first described as a secretory product of the prostate, the diameter of prostasomes ranges from 30 to 500 nm (Poliakov, Spilman, Dokland, Amling, & Mobley, 2009;Sullivan, Frenette, & Girouard, 2007). However, these seminal exosome-like vesicles likely originate from the male genital tract, such as epididymis, prostate, seminal vesicles, and contain exosomes and microvesicles, so we used the term "seminal extracellular vesicles" (Poliakov et al, 2009;Vojtech et al, 2014;Yang et al, 2017). Seminal extracellular vesicles have already been implicated as promotion of spermatozoa motility (Arienti et al, 1999;Carlsson, Ronquist, Stridsberg, & Johansson, 1997;Fabiani, Johansson, Lundkvist, & Ronquist, 1994;Fabiani, Johansson, Lundkvist, & Ronquist, 1995;;Schwarz et al, 2013;Stegmayr & Ronquist, 1982), immunodulation/immunomodulation (Kelly et al, 1991), antibacterial activity (Carlsson, Pahlson, Bergquist, Ronquist, & Stridsberg, 2000), regulation of capacitation process (Cross, 1996), and antioxidant protection (Saez, Motta, Boucher, & Grizard, 1998;Saez, Motta, Boucher, & Grizard, 2000).…”

Expressions of miR‐525‐3p and its target gene SEMG1 in the spermatozoa of patients with asthenozoospermia

“…The prostatsomes can transfer the substance to the spermatozoa, such as DNA and CD38(Park et al ., ; Ronquist et al ., ). Our previous study found that SEMG1 protein is contained in exosomes extracted from seminal plasma (Yang, et al ., ). So we speculated that SEMG1 mRNA and protein could be incorporated in ejaculated spermatozoa by exosomes from prostate, though, further research is required to investigate it.…”

Sperm and Seminal Plasma Proteomics: Molecular Changes Associated with Varicocele-Mediated Male Infertility

“…Apart from sperm and seminal plasma protein studies, the focus has currently shifted to understand the physiological function of the exosomes. In 2017, Yang et al [73] profiled the seminal exosomal proteins in fertile donors. Exosomal proteins were associated with protein metabolism, energy pathway and transport.…”