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

Abstract

•Two EV subsets (L/S) were isolated from pig seminal plasma by a SEC procedure.•A total of 1,034 proteins were identified by LC-MS/MS in seminal EVs.•Up to 737 proteins were quantified by SWATH in seminal EVs.•The two subsets of seminal EVs showed differences in proteomic profile.•Each EV subset would have different biogenesis and biological function. 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. 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. Mounting evidence shows that seminal plasma (SP), the heterogeneous fluid that surrounds sperm during/after ejaculation, plays a key role in many physiological reproductive processes, including sperm function and embryo development (1Rodriguez-Martinez H. Martinez E.A. Calvete J.J. Peña Vega F.J. Roca J. Seminal plasma: relevant for fertility?.Int. J. Mol. Sci. 2021; 22: 4368Crossref PubMed Scopus (36) Google Scholar). This fluid, mainly composed by secretions from the epididymis and accessory sex glands, contains a wide repertoire of biomolecules, as inorganic ions, hormones, lipids, nucleic acids, peptides, and proteins (1Rodriguez-Martinez H. Martinez E.A. Calvete J.J. Peña Vega F.J. Roca J. Seminal plasma: relevant for fertility?.Int. J. Mol. Sci. 2021; 22: 4368Crossref PubMed Scopus (36) Google Scholar). The last of these largely define SP function, as the modulation of sperm function, motility, capacitation, and encounter with the oocyte, as well as triggering immune responses by the female after mating or insemination, is crucial for healthy embryo development (2Rodriguez-Martinez H. Kvist U. Ernerudh J. Sanz L. Calvete J.J. Seminal plasma proteins: what role do they play?.Am. J. Reprod. Immunol. 2011; 66: 11-22Crossref PubMed Scopus (280) Google Scholar). Recent research reported that some of these SP proteins could be loaded into seminal extracellular vesicles (sEVs), where they might remain safe from the degradation by proteolytic enzymes in semen (3Lin Y. Liang A. He Y. Li Z. Li Z. Wang G. et al.Proteomic analysis of seminal extracellular vesicle proteins involved in asthenozoospermia by iTRAQ.Mol. Reprod. Dev. 2019; 86: 1094-1105Crossref PubMed Scopus (34) Google Scholar, 4Zhang X. Vos H.R. Tao W. Stoorvogel W. Proteomic profiling of two distinct populations of extracellular vesicles isolated from human seminal plasma.Int. J. Mol. Sci. 2020; 21: 7957Crossref PubMed Scopus (14) Google Scholar, 5Leahy T. Rickard J.P. Pini T. Gadella B.M. de Graaf S.P. Quantitative proteomic analysis of seminal plasma, sperm membrane proteins, and seminal extracellular vesicles suggests vesicular mechanisms aid in the removal and addition of proteins to the ram sperm membrane.Proteomics. 2020; 20e1900289Crossref PubMed Scopus (27) Google Scholar, 6Wang H. Zhu Y. Tang C. Zhou Z. Wang Z. Li Z. et al.Reassessment of the proteomic composition and function of extracellular vesicles in the seminal plasma.Endocrinology. Scopus Google Scholar). as a for in the EVs are defined as a heterogeneous of released by all and cells into the extracellular G. G. G. on the of extracellular Mol. PubMed Scopus Google Scholar). EVs as in and physiological Wang of extracellular their and for and 2019; PubMed Scopus Google Scholar, The function, and of 2020; Scopus Google including involved in vesicle during and in Reprod. 2019; PubMed Scopus Google Scholar, and the of embryo 2021; PubMed Scopus Google Scholar, H. vesicles and female Reprod. 2021; PubMed Scopus Google Scholar, A. vesicles in embryo and of the J. Reprod. Immunol. 2021; PubMed Scopus Google Scholar, et of male reproductive tract extracellular vesicles in J. Reprod. Immunol. 2021; PubMed Scopus Google Scholar). The of EVs in their to their a of nucleic lipids, mainly to triggering a in them G. C. and of and extracellular Dev. PubMed Scopus Google Scholar). This is and on cells and biogenesis PubMed Scopus Google Scholar). on differences in size and the EVs released by healthy cells are into EVs of in or EVs of in plasma membrane Wang of extracellular their and for and 2019; PubMed Scopus Google Scholar). The of capable of EV subsets to a of their composition and function. Recent that EV subsets a different proteomic profile Wang of extracellular their and for and 2019; PubMed Scopus Google Scholar, L. biological and of PubMed Scopus Google Scholar, L. H. et proteomic analysis of small and large extracellular vesicles enrichment of proteins in small 2019; PubMed Scopus Google that they might have distinct biological identified G. A. and in human PubMed Scopus Google and in in J. et and of small extracellular vesicles from plasma, fluid, and seminal 2019; PubMed Scopus (27) Google remain J. H. L. X. vesicles in seminal fluid and on male in and Reprod. Sci. 2021; Scholar). on seminal EVs for of the total C. for and of extracellular of a by the and 2020; PubMed Scopus Google and research on a research that could be key of sperm including motility, capacitation, and and might G. are of from research to PubMed Scopus Google Scholar, and their in male reproductive PubMed Scopus Google Scholar, A. of male 2020; PubMed Scopus Google Scholar, L. Y. Wang Y. of in the of after the a 2020; 21: PubMed Scopus Google Scholar). In could be involved in the of sperm–oocyte L. A. vesicles of pig PubMed Scopus Google Scholar, U. H. seminal plasma on sperm function and protein by PubMed Scopus Google Scholar, J. J. Wang Y. C. W. H. et seminal plasma sperm function by into the sperm PubMed Scopus Google and in the and immune responses in the female for of and the development and of healthy Z. of gene expression by seminal in the PubMed Scopus Google Scholar). the mechanisms by could these remain to be proteins could be key In have the of sEV of their T. Rickard J.P. Pini T. Gadella B.M. de Graaf S.P. Quantitative proteomic analysis of seminal plasma, sperm membrane proteins, and seminal extracellular vesicles suggests vesicular mechanisms aid in the removal and addition of proteins to the ram sperm membrane.Proteomics. 2020; 20e1900289Crossref PubMed Scopus (27) Google Scholar, C. J. Zhou et analysis of from human seminal PubMed Scopus Google Scholar, Y. J. X. J. Y. C. et of extracellular vesicle in seminal plasma for in Reprod. 2020; PubMed Scopus Google Scholar, Z. Y. Zhou C. T. J. et of seminal plasma extracellular vesicle small in Sci. 2020; Scopus Google Scholar). In the proteomic profile of (3Lin Y. Liang A. He Y. Li Z. Li Z. Wang G. et al.Proteomic analysis of seminal extracellular vesicle proteins involved in asthenozoospermia by iTRAQ.Mol. Reprod. Dev. 2019; 86: 1094-1105Crossref PubMed Scopus (34) Google Scholar, 4Zhang X. Vos H.R. Tao W. Stoorvogel W. Proteomic profiling of two distinct populations of extracellular vesicles isolated from human seminal plasma.Int. J. Mol. Sci. 2020; 21: 7957Crossref PubMed Scopus (14) Google Scholar, 6Wang H. Zhu Y. Tang C. Zhou Z. Wang Z. Li Z. et al.Reassessment of the proteomic composition and function of extracellular vesicles in the seminal plasma.Endocrinology. Scopus Google Scholar, C. J. Zhou et analysis of from human seminal PubMed Scopus Google Scholar, L. composition and seminal plasma a of of male J. Mol. Sci. 2020; 21: PubMed Scopus Google differences in protein composition between EV subsets X. Vos H.R. Tao W. Stoorvogel W. Proteomic profiling of two distinct populations of extracellular vesicles isolated from human seminal plasma.Int. J. Mol. Sci. 2020; 21: 7957Crossref PubMed Scopus (14) Google Scholar, 6Wang H. Zhu Y. Tang C. Zhou Z. Wang Z. Li Z. et al.Reassessment of the proteomic composition and function of extracellular vesicles in the seminal plasma.Endocrinology. Scopus Google Scholar, et of distinct populations of that differentially and in Reprod. 86: Scopus Google and that sEV could be involved in a function. study the proteomic profile of sEV subsets in T. Rickard J.P. Pini T. Gadella B.M. de Graaf S.P. Quantitative proteomic analysis of seminal plasma, sperm membrane proteins, and seminal extracellular vesicles suggests vesicular mechanisms aid in the removal and addition of proteins to the ram sperm membrane.Proteomics. 2020; 20e1900289Crossref PubMed Scopus (27) Google Scholar). In a study in pig identified EV subsets to expression profile L. A. C. F.J. et vesicles isolated from seminal plasma different expression 2019; Scopus Google differences in their and biological function. The study aimed to define in depth the profile of pig to versus differences in protein composition between sEV size and to their proteomic the of sEV sEV were isolated using ultrafiltration and size exclusion the proteomic profiles using liquid chromatography–tandem mass spectrometry and the identified proteins quantified using sequential window acquisition of all theoretical mass The that involved were to on the of for and by the of were from were from and in an to in the semen and sperm with for of pig semen after semen the were for for SP the SP were to the of the SP samples were in the of a EVs of SP ultrafiltration and SEC for EVs is in the SP samples were for to The were for and the and were in different The were in and The were in and by for The samples from to were of the seminal EVs by SEC and of isolated EVs is in The and were to were using with of EVs were by them with and samples were loaded on SEC by with total of sequential were from and to in and to were These were to two samples SEC The non-EVs-enriched samples from and from were In samples were after EV samples from mainly in large EVs or mainly in small EVs and the non-EVs-enriched and all of were EV and proteomic The isolated sEV subsets and were using and C. for of extracellular vesicles a of the for extracellular vesicles and of the PubMed Scopus Google the for of extracellular vesicles they were in of and size distribution, by total protein and using analysis and analysis morphology, using protein markers and EV purity the last two using of this EV are The non-EVs-enriched samples were using the to they EVs or The of total protein using the to the EV samples were this of EV samples with of of of and 37 for using a a of The and size of EVs and were by using a with a and a The were with the with and to and the to The to and of were The were with of the the of in the of of the EV samples to to with The size and size of EVs were by using a the an of of EV to EV and loaded into a with a The for with The of to size using a The EV using the of the function. The to the and the were as and The of EVs and by the by et C. G. A. and of from and biological PubMed Google with samples were in for and for with the EV samples were for with the samples were with in and in the samples were using a the de a de The EV by of The were with the with and EVs protein on their membrane were to the of the for EV and The to the from the The and were on a and the on a The analysis to with size and to were using the as fluid, removal of The EVs were the of A. et a for of extracellular vesicle 2020; Scopus Google to their enrichment in proteins to the by C. for of extracellular vesicles a of the for extracellular vesicles and of the PubMed Scopus Google as protein or proteins to plasma membrane as protein proteins in and as protein of of EV with to EVs from membrane EV into were with or for samples were in to a of and in to in the EV and in the of the de of the samples to type of L-EVs, S-EVs, and samples and and samples S-EVs, and were to the proteomic profile and samples S-EVs, and were to quantify the identified proteins to differentially protein of using a S-EVs and and of total protein were with and for samples were loaded on including a The a of for the with a and for with for to protein after with for samples the were into and and respectively. samples, the into two S-EVs and and the and the of the were to the and abundant proteins, respectively. the from L-EVs into a samples were the by et A. et and by mass of proteins from two Sci. U. A. PubMed Scopus Google Scholar). this procedure from to 37 with by in 37 in a for and of the in and after in a The were for the acquisition by liquid using a to an mass in of from of loaded on a size and with for the were loaded an size in and using a from to of a of for samples and abundant proteins of samples and or abundant proteins of samples and respectively. The were on a The samples were using an to the The in in a of mass spectrometry from to and for The to for were from to for in The for were or and of with and were from the to were for after and exclusion to The were by the differences between the L-EVs versus S-EVs, S-EVs versus and L-EVs versus non-EVs-enriched samples, the as by et H. L. et of the by a for and PubMed Scopus Google for abundant and abundant of were loaded a size and with for were loaded an size in and using a from to of a of for for abundant proteins or for for abundant The analysis of in a mass The in SWATH in a from to by from to into from to The for window for the of SWATH with a of The were by The of to with the proteins in the and proteins of and proteins for and the to The analysis for of the and with the of using the evidence in the identified proteins were based on mass spectrometry by the The protein of the defined as the protein that could the with by of The were by by with SWATH and from SWATH were with a of proteins, peptides, were for In the window in and in The of and the were to total The protein were samples by total the and gene were from the enrichment function, and biological and of and pathway analysis of differentially abundant proteins using the and for and were in to the for The function, and biological were into were using and were in to were for SP The SP samples were to procedure for EV subsets samples were in L-EVs, in small and non-EVs-enriched and The isolated EVs were and proteomic were in the samples the procedure samples and and samples S-EVs, and were for and samples S-EVs, and were to quantify the identified proteins to differentially samples were using the The to for and to differentially abundant proteins samples and analysis and of protein profiles were by T. J. for of using analysis and PubMed Scopus Google Scholar). or with to the differentially abundant proteins between The to to the of EV The of the by the The were by and differences between samples and were by were protein in in samples from pig SP revealed that the in samples in samples the showed that EVs were in samples in L-EVs samples analysis that EVs were in samples of in samples of by showed the samples vesicles in size from to with the size for The samples a morphologically heterogeneous of vesicles in size from to with the size that The showed both and samples subsets of EVs with or protein were in subset size differences were between and samples, revealed the of small EVs in the samples and some large EVs in the samples analysis revealed that both EV subsets EV markers such as and for and L-EVs, or and EV enrichment in both EV subsets, as the of and for and and that to from the samples, and proteomic analysis in and EV The LC-MS/MS from type of and the samples of L-EVs and S-EVs total of proteins were in the and proteins of and proteins for total of 1,034 proteins with an were identified as into and of them showed a of be enrichment analysis The biological analysis a of biological proteins involved in processes, and or In to reproductive processes, the analysis revealed that proteins were involved in and The analysis revealed an enrichment of proteins with and the proteins to and Quantitative using the SWATH the identified proteins, 737 were the quantified proteins were in the samples, S-EVs, L-EVs, and analysis of the total and between and non-EVs-enriched samples and samples from and non-EVs-enriched samples, and between and non-EVs-enriched with the of the the of quantified proteins two with the samples and the the and non-EVs-enriched samples This in into two with the samples and the with the non-EVs-enriched samples would be with differential expression analysis to the proteins that showed the differences between and were as and the of the differentially abundant proteins is in the differentially abundant proteins in the of the samples EVs and were with non-EVs-enriched samples, proteins were abundant and two abundant in proteins were abundant and abundant in samples the differentially abundant proteins in the EV samples, were two abundant proteins, and abundant proteins and total of proteins were abundant and abundant in with shows the proteins the abundant differences between proteins with the differentially abundant between the two subsets of isolated extracellular vesicles large and small from pig seminal plasma The proteins abundant in L-EVs and S-EVs samples are in and respectively. in a enrichment of differentially abundant proteins were to differences between the two subsets of EV and with SP proteins In the the abundant proteins in both EV samples were with the abundant proteins in the non-EVs-enriched The revealed that the abundant proteins in the EV samples to and development the abundant proteins in the non-EVs-enriched samples were to and reproductive processes, the revealed that the abundant proteins in EV samples were involved in embryo and reproductive in The abundant proteins in non-EVs-enriched samples were involved in reproductive in in and in In this and were abundant in and and in in analysis revealed that of the differentially abundant proteins between EV samples and non-EVs-enriched samples were involved in and analysis revealed that abundant proteins in EV samples were to and abundant in non-EVs-enriched samples were to and degradation enrichment analysis of differentially abundant proteins between and samples to differences between the two EV The biological analysis showed abundant proteins in samples were involved in processes, and abundant proteins in samples were involved in immune and function analysis showed abundant proteins in samples and abundant proteins in S-EVs and differentially abundant proteins involved in processes, abundant in samples were and in The pathway analysis abundant proteins in samples to and in samples were to the and the proteins quantified in pig with in the and to the proteins identified as EVs markers in both the of pig were and in the and respectively. In sEV proteins identified in the study were in EV for EV research is the of a procedure to different EV subsets in The study aimed to two subsets of using that as an and a of is to small from large where the small remain in the and the large are in the G. with an for extracellular PubMed Scopus Google Scholar, C. A. et profiles in of extracellular and PubMed Scopus Google Scholar, L. Wang A. et of to cells extracellular Sci. U. A. PubMed Scopus Google Scholar). The two isolated EV subsets were in using a of of C. for of extracellular vesicles a of the for extracellular vesicles and of the PubMed Scopus Google including morphology, and concentration, showed differences between EVs of the two subsets, some size between the two to be using G. A. G. and in by extracellular Mol. PubMed Scopus Google Scholar). analysis revealed that EVs from both subsets showed a of to EV-specific protein this showed that of the two sEV subsets in EVs distinct with proteins is a in aimed the protein composition of that the a on the of such S.P. J. Y. A. et of for the and of extracellular vesicles from protein and in human 2020; PubMed Scopus Google Scholar). In the the sEV subsets were isolated using a procedure and This procedure for EV from a wide of for extracellular in and J. PubMed Scopus Google including semen J. et from are for sperm 2011; PubMed Scopus Google for of large of EVs with a high degree of purity size exclusion for extracellular vesicle enrichment and proteomic analysis from relevant 2019; Scopus Google Scholar). The of a high abundant protein in pig SP C. Martinez E.A. H. et of the seminal plasma PubMed Scopus Google in the sEV samples would that the EV samples in the study have by proteins could be for the of EVs by proteomic Proteomic this of the differentially abundant proteins between and non-EVs-enriched samples, in the in non-EVs-enriched vesicles a of mainly proteins, are to cells to a et al.Reassessment of 2019; PubMed Scopus Google Scholar). In the of to the of EVs in to a of their in both physiological and C. Proteomic profiling in extracellular vesicles for and 2021; Scopus Google Scholar, of extracellular and PubMed Scopus Google Scholar). the proteomic profile of well in proteins (3Lin Y. Liang A. He Y. Li Z. Li Z. Wang G. et al.Proteomic analysis of seminal extracellular vesicle proteins involved in asthenozoospermia by iTRAQ.Mol. Reprod. Dev. 2019; 86: 1094-1105Crossref PubMed Scopus (34) Google Scholar, 4Zhang X. Vos H.R. Tao W. Stoorvogel W. Proteomic profiling of two distinct populations of extracellular vesicles isolated from human seminal plasma.Int. J. Mol. Sci. 2020; 21: 7957Crossref PubMed Scopus (14) Google Scholar, 6Wang H. Zhu Y. Tang C. Zhou Z. Wang Z. Li Z. et al.Reassessment of the proteomic composition and function of extracellular vesicles in the seminal plasma.Endocrinology. Scopus Google Scholar, C. J. Zhou et analysis of from human seminal PubMed Scopus Google Scholar, A. de H. J. from and a differential protein expression PubMed Scopus Google proteomic analysis of in is in et T. Rickard J.P. Pini T. Gadella B.M. de Graaf S.P. Quantitative proteomic analysis of seminal plasma, sperm membrane proteins, and seminal extracellular vesicles suggests vesicular mechanisms aid in the removal and addition of proteins to the ram sperm membrane.Proteomics. 2020; 20e1900289Crossref PubMed Scopus (27) Google and et T. proteomic profiling of extracellular vesicles in the proteins to sequential sperm with differences between and 2020; PubMed Scopus Google an proteomic analysis of a total of and proteins, in and respectively. the of this is the proteomic analysis of pig sEVs. a study in sEV proteins using mass spectrometry U. H. seminal plasma on sperm function and protein by PubMed Scopus Google Scholar). In the a total of proteins were identified and 737 of them were quantified by of LC-MS/MS and respectively. analysis of the proteins identified in both is the of would for a and of the EV of the of pig sEV with the for EVs from of and in and to proteins in pig that have reported in of these EV proteins, such as the and C. Martinez E.A. H. et of the seminal plasma PubMed Scopus Google Scholar, X. Rickard J.P. G. de Graaf S.P. Seminal plasma proteins as markers of sperm 2019; PubMed Scopus Google or the protein A. A. et of with proteomic and quantified in seminal PubMed Scopus Google Scholar, H. Y. of the and in J. Mol. Sci. 2020; 21: PubMed Scopus Google are involved in sperm function. evidence the that SP proteins are to both sperm function and the female immune (2Rodriguez-Martinez H. Kvist U. Ernerudh J. Sanz L. Calvete J.J. Seminal plasma proteins: what role do they play?.Am. J. Reprod. Immunol. 2011; 66: 11-22Crossref PubMed Scopus (280) Google Scholar). is as to these modulating SP proteins in the SP or are in where they might remain safe from SP on this have the of two subsets of as well as the from SP the of study the using a or depth of Wang et H. Zhu Y. Tang C. Zhou Z. Wang Z. Li Z. et al.Reassessment of the proteomic composition and function of extracellular vesicles in the seminal plasma.Endocrinology. Scopus Google the of human SP from with EVs and The as extracellular The of of the two subsets of with that of non-EVs-enriched quantified proteins were in all This could be by (1Rodriguez-Martinez H. Martinez E.A. Calvete J.J. Peña Vega F.J. Roca J. Seminal plasma: relevant for fertility?.Int. J. Mol. Sci. 2021; 22: 4368Crossref PubMed Scopus (36) Google proteins and proteins that were by the cells (2Rodriguez-Martinez H. Kvist U. Ernerudh J. Sanz L. Calvete J.J. Seminal plasma proteins: what role do they play?.Am. J. Reprod. Immunol. 2011; 66: 11-22Crossref PubMed Scopus (280) Google the between this from the of a to differentially abundant proteins differences were for proteins between The of L-EVs in proteins mainly is these to small including and in their membrane L. into the function of in the of PubMed Scopus Google Scholar). These proteins are involved in EV biogenesis and to PubMed Scopus Google Scholar, The biogenesis and PubMed Scopus (36) Google Scholar, 2019; PubMed Scopus Google Scholar). proteins in L-EVs were an for of protein involved in the multivesicular pathway of during and 2011; PubMed Scopus Google and and two proteins involved in vesicular and membrane fusion C. fusion by and plasma membrane PubMed Scopus (27) Google Scholar, A. T. of to from the plasma membrane and to PubMed Google Scholar). The in L-EVs of small and proteins, could that L-EVs were mainly by the mechanisms of by fusion of multivesicular bodies to the plasma membrane or by from the plasma membrane A. on biogenesis and Mol. Sci. PubMed Scopus Google by the apocrine the for seminal EV for S-EVs J.

Topics & Concepts