Uptake Assay of Ram Seminal Plasma Extracellular Vesicles to Sperm.

A novel immunoassay for ApoB-100, the main protein component of lipoproteins, enables the development of methods to enrich extracellular vesicles from human plasma while depleting both lipoproteins and free proteins.

In mammals, seminal plasma is a complex fluid surrounding spermatozoa, composed of secretions from the male reproductive tract. It plays a crucial role in modulating sperm function, but it remains unclear whether the components that regulate sperm physiology, travel freely or within extracellular vesicles secreted by the reproductive tract and accessory glands. This study evaluated three methodologies-ultracentrifugation (UC), size-exclusion chromatography (SEC), and polyethylene glycol precipitation (PEG)-for isolation of ram seminal plasma extracellular vesicles enriched fractions (SP-EVs), assessing their efficiency in terms yield, morphology, protein profile, and functionality. Western blot confirmed the presence of EV-specific markers (CD9, CD63, and HSP70), minimal cytoplasmic and lipoprotein contamination. SEC, particularly the second fraction (P2), yielded SP-EVs with conserved morphology, apparently reduced aggregation, and a unique protein profile enriched in low molecular weight proteins, compatible with most capacitation-modulating proteins. In contrast, UC and PEG resulted in higher particle concentration and aggregation. By CFSE labeling of SP-EVs, all preparations exhibited a targeted binding pattern to spermatozoa, with distinct patterns localized to midpiece, head and post-acrosomal regions. Additionally, western blot analysis showed that SP-EVs transport and transfer binder of sperm proteins (RSVP20 and RSVP14) to spermatozoa, with RSVP20 showing the highest incorporation, particularly from the P2 fraction. SPINK3, despite being detected in SP-EVs, was not incorporated, indicating selective protein delivery. These findings may be important to understand the role of seminal plasma extracellular vesicles on sperm, and significant for improving the efficiency of reproductive biotechnologies, as these ram SP-EVs enrichment fractions can deliver functional proteins to spermatozoa.

<p dir="ltr">Extracellular vesicles (EVs) are membrane-enclosed nanoparticles secreted by all cell types. They carry a variety of bioactive molecules, including lipids, proteins, and nucleic acids, which can act as signaling messengers to other cells. The two main types of EVs under investigation are microvesicles and exosomes. Microvesicles are formed by outward budding of the plasma membrane, while exosomes originate from inward budding within multivesicular bodies and are released upon fusion with the plasma membrane. EVs are found in various biological fluids such as blood, saliva, and urine, as well as in cell culture media. Their roles in intercellular communication have made them a focus of research in fundamental biology, biomarker discovery, and therapeutic development, including engineering EVs for clinical applications.</p><p dir="ltr">Studying EVs often requires their isolation, which presents several challenges. The physical and molecular similarities between EV subtypes make them difficult to separate, and common isolation protocols often co-isolate contaminants such as protein aggregates and lipoproteins. In Study I, we compared five fundamentally different EV isolation techniques, each based on distinct EV properties such as density, size, affinity, and hydrophobicity. These methods were applied to two biofluids, cell culture supernatant and plasma, at varying volumes to assess whether the optimal isolation method depends on the biofluid type. Plasma, for instance, contains high levels of lipoproteins that overlap with EVs in size and density, complicating isolation. EV isolates were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Bioanalyzer, flow cytometry, and proteomic profiling. All methods successfully enriched for EVs, but the composition and quality of the isolates varied depending on the biofluid, sample volume, and isolation technique. This study emphasized the importance of method selection, as each technique isolates different EV subpopulations with different contaminants, which can affect study outcomes and comparability. It also highlighted that some components initially considered as contaminants may be biologically relevant.</p><p dir="ltr">In Studies II and III, we explored the therapeutic potential of dendritic cell (DC)- derived EVs, particularly their use in cancer immunotherapy. Using EVs loaded with the model antigen ovalbumin (OVA), we investigated strategies to enhance their immunogenicity. In Study II, we focused on the immune checkpoint molecule PD-L1, which was present on our EVs. PD-L1 present on Tumor-derived EVs is known to suppress T cell activation, but its role on therapeutic DC-EVs was unclear. We generated PD-L1-deficient (PD-L1-/-) EVs and compared their immunogenicity to wild-type EVs in multiple mouse tumor models. While most models showed a trend toward stronger immune activation with PD-L1-/- EVs, a significant improvement was observed only in a prophylactic tumor model. These findings suggest that removing PD-L1 may enhance the efficacy of EV-based immunotherapies.</p><p dir="ltr">In Study III, we tested whether targeting EVs to B cells could further improve their immunogenicity. A soluble fusion protein (C1C2-D123) was developed to bind phosphatidylserine on EVs and CD21 on B cells. This protein successfully directed EVs to B cells in vitro and in vivo without altering their biodistribution. In an in vivo immunization model, EVs decorated with the fusion protein induced a higher frequency of antigen-specific CD8+ T cells compared to non-targeted EVs, supporting B cell targeting as a strategy to enhance immune responses.</p><p dir="ltr">In conclusion, this thesis highlights the importance of EV isolation strategy and cargo engineering in optimizing EV-based therapies. It demonstrates that both the method of isolation and the molecular composition of EVs significantly influence their biological and therapeutic potential.</p><h3>List of scientific papers</h3><p dir="ltr">I. Rosanne E. Veerman, <b>Loes Teeuwen</b>, Paulo Czarnewski, Gözde Güclüler Akpinar, AnnSofi Sandberg, Xiaofang Cao, Maria Pernemalm, Lukas M. Orre, Susanne Gabrielsson, Maria Eldh. Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin. J Extracellular Vesicles. Volume 10, Epub e12128, (2021). <a href="https://doi.org/10.1002/jev2.12128" rel="noreferrer" target="_blank">https://doi.org/10.1002/jev2.12128</a></p><p dir="ltr">II. <b>Teeuwen*, L.</b>, Steiner*, L., Reinhardt, C., Offens, A., Kuipers, J.E., Martínez- Martínez, D., Mazouin, J., Chambers, B.J., Güçluler Akpinar, G., Gabrielsson, S. Removal of PD-L1 on extracellular vesicles for cancer vaccination modulates anti-tumor responses in a murine immunotherapy model. [Manuscript]</p><p dir="ltr">III. Annemarijn Offens, <b>Loes Teeuwen</b>, Gozde Gucluler Akpinar, Loïc Steiner, Sander Kooijmans, Doste Mamand, Hannah Weissinger, Alexander Kall, Maria Eldh, Oscar P.B. Wiklander, Samir El-Andaloussi, Mikael C.I. Karlsson, Pieter Vader, Susanne Gabrielsson. A fusion protein that targets antigen-loaded extracellular vesicles to B cells enhances antigen-specific T cell expansion. J Controlled Release, Volume 382, 113665 (2025). <a href="https://doi.org/10.1016/j.jconrel.2025.113665" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.jconrel.2025.113665</a></p><p dir="ltr">* Shared first authorship.</p>

<p dir="ltr">Extracellular vesicles (EVs) are membrane-enclosed nanoparticles secreted by all cell types. They carry a variety of bioactive molecules, including lipids, proteins, and nucleic acids, which can act as signaling messengers to other cells. The two main types of EVs under investigation are microvesicles and exosomes. Microvesicles are formed by outward budding of the plasma membrane, while exosomes originate from inward budding within multivesicular bodies and are released upon fusion with the plasma membrane. EVs are found in various biological fluids such as blood, saliva, and urine, as well as in cell culture media. Their roles in intercellular communication have made them a focus of research in fundamental biology, biomarker discovery, and therapeutic development, including engineering EVs for clinical applications.</p><p dir="ltr">Studying EVs often requires their isolation, which presents several challenges. The physical and molecular similarities between EV subtypes make them difficult to separate, and common isolation protocols often co-isolate contaminants such as protein aggregates and lipoproteins. In Study I, we compared five fundamentally different EV isolation techniques, each based on distinct EV properties such as density, size, affinity, and hydrophobicity. These methods were applied to two biofluids, cell culture supernatant and plasma, at varying volumes to assess whether the optimal isolation method depends on the biofluid type. Plasma, for instance, contains high levels of lipoproteins that overlap with EVs in size and density, complicating isolation. EV isolates were characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), Bioanalyzer, flow cytometry, and proteomic profiling. All methods successfully enriched for EVs, but the composition and quality of the isolates varied depending on the biofluid, sample volume, and isolation technique. This study emphasized the importance of method selection, as each technique isolates different EV subpopulations with different contaminants, which can affect study outcomes and comparability. It also highlighted that some components initially considered as contaminants may be biologically relevant.</p><p dir="ltr">In Studies II and III, we explored the therapeutic potential of dendritic cell (DC)- derived EVs, particularly their use in cancer immunotherapy. Using EVs loaded with the model antigen ovalbumin (OVA), we investigated strategies to enhance their immunogenicity. In Study II, we focused on the immune checkpoint molecule PD-L1, which was present on our EVs. PD-L1 present on Tumor-derived EVs is known to suppress T cell activation, but its role on therapeutic DC-EVs was unclear. We generated PD-L1-deficient (PD-L1-/-) EVs and compared their immunogenicity to wild-type EVs in multiple mouse tumor models. While most models showed a trend toward stronger immune activation with PD-L1-/- EVs, a significant improvement was observed only in a prophylactic tumor model. These findings suggest that removing PD-L1 may enhance the efficacy of EV-based immunotherapies.</p><p dir="ltr">In Study III, we tested whether targeting EVs to B cells could further improve their immunogenicity. A soluble fusion protein (C1C2-D123) was developed to bind phosphatidylserine on EVs and CD21 on B cells. This protein successfully directed EVs to B cells in vitro and in vivo without altering their biodistribution. In an in vivo immunization model, EVs decorated with the fusion protein induced a higher frequency of antigen-specific CD8+ T cells compared to non-targeted EVs, supporting B cell targeting as a strategy to enhance immune responses.</p><p dir="ltr">In conclusion, this thesis highlights the importance of EV isolation strategy and cargo engineering in optimizing EV-based therapies. It demonstrates that both the method of isolation and the molecular composition of EVs significantly influence their biological and therapeutic potential.</p><h3>List of scientific papers</h3><p dir="ltr">I. Rosanne E. Veerman, <b>Loes Teeuwen</b>, Paulo Czarnewski, Gözde Güclüler Akpinar, AnnSofi Sandberg, Xiaofang Cao, Maria Pernemalm, Lukas M. Orre, Susanne Gabrielsson, Maria Eldh. Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin. J Extracellular Vesicles. Volume 10, Epub e12128, (2021). <a href="https://doi.org/10.1002/jev2.12128" rel="noreferrer" target="_blank">https://doi.org/10.1002/jev2.12128</a></p><p dir="ltr">II. <b>Teeuwen*, L.</b>, Steiner*, L., Reinhardt, C., Offens, A., Kuipers, J.E., Martínez- Martínez, D., Mazouin, J., Chambers, B.J., Güçluler Akpinar, G., Gabrielsson, S. Removal of PD-L1 on extracellular vesicles for cancer vaccination modulates anti-tumor responses in a murine immunotherapy model. [Manuscript]</p><p dir="ltr">III. Annemarijn Offens, <b>Loes Teeuwen</b>, Gozde Gucluler Akpinar, Loïc Steiner, Sander Kooijmans, Doste Mamand, Hannah Weissinger, Alexander Kall, Maria Eldh, Oscar P.B. Wiklander, Samir El-Andaloussi, Mikael C.I. Karlsson, Pieter Vader, Susanne Gabrielsson. A fusion protein that targets antigen-loaded extracellular vesicles to B cells enhances antigen-specific T cell expansion. J Controlled Release, Volume 382, 113665 (2025). <a href="https://doi.org/10.1016/j.jconrel.2025.113665" rel="noreferrer" target="_blank">https://doi.org/10.1016/j.jconrel.2025.113665</a></p><p dir="ltr">* Shared first authorship.</p>

BackgroundInfection and graft-versus-host disease (GvHD) are the major causes for mortality and morbidity of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Plasma-derived extracellular vesicles (EVs) contain disease-related proteins, DNAs and RNAs, and have recently been suggested as potential biomarker candidates for transplantation complications. However, EV isolation from small plasma volumes in clinical biomarker studies using conventional methods is challenging. We therefore investigated if EVs isolated by novel automated acoustic trapping could be developed as potential biomarkers for allo-HSCT complications by performing a clinical proof-of-principle study.ResultsPlasma samples were collected from twenty consecutive patients with high-risk/relapsed hematologic malignancies undergoing allo-HSCT before transplantation and post-transplant up to 12 weeks. EVs were isolated from small plasma sample volumes (150 μl) by an automated, acoustofluidic-based particle trapping device, which utilizes a local λ/2 ultrasonic standing wave in a borosilicate glass capillary to capture plasma EVs among pre-seeded polystyrene microbeads through sound scatter interactions. We found that EVs could be reliably isolated from all plasma samples (n = 173) and that EV numbers increased more than 2-fold in the majority of patients after transplantation. Also, sufficient quantities of RNA for downstream microRNA (miRNA) analysis were obtained from all samples and EV miRNA profiles were found to differ from whole plasma profiles. As a proof of principle, expression of platelet-specific miR-142-3p in EVs was shown to correlate with platelet count kinetics after transplantation as expected. Importantly, we identified plasma EV miRNAs that were consistently positively correlated with infection and GvHD, respectively, as well as miRNAs that were consistently negatively correlated with these complications.ConclusionsThis study demonstrates that acoustic enrichment of EVs in a clinical biomarker study setting is feasible and that downstream analysis of acoustically-enriched EVs presents a promising tool for biomarker development in allo-HSCT. Certainly, these findings warrant further exploration in larger studies, which will have significant implications not only for biomarker studies in transplantation but also for the broad field of EV-based biomarker discovery.

Introduction: Extracellular vesicles (EVs) contain protein and nucleic cargo that has been shown to be reflective of physiological and pathological states, or their originating cells, in many diseases including cancer. Specifically, these EV-associated biomarkers have been shown to fluctuate with the change in pathological processes. As a result, characterization of blood-based circulating EVs have been widely investigated for diagnostic applications such as disease monitoring and treatment selection. The heterogeneity of blood-based EVs present significant challenges to current rapid isolation methods due to enrichment with unwanted sub-populations, e.g. apoptotic bodies, or contamination with lipids, e.g. APOB, for both capture-based chemistry-based isolation methods. We have developed a novel lab-on-a-chip technology for isolation and on-chip characterization of EVs from blood-based matrices using an AC electrokinetics (ACE) methodology. Methods: Blood samples were collected from 10 healthy volunteers and 10 donors with known cancer diagnosis into K2EDTA tubes under IRB approved protocols. Plasma was processed from the blood and stored at -80C. 120 µL of thawed plasma was applied to the microelectrode array flow cell (ExoVerita Flex, Biological Dynamics, San Diego, CA) and the EVs were isolated on the microelectrodes. Following isolation, the flow cell was washed, the isolated material was released from the array and eluted from the flow cell. For comparison, isolation of EVs from the same amount of plasma was performed using a chemistry-based method (ExoQuick Ultra, System Biosciences, Palo Alto, CA) according to manufacturer's instructions. Following isolation, the eluates were evaluated for the presence of EV-associated biomarkers using Western blotting capillary electrophoresis and mass spectrometry (Orbitrap Fusion Lumos, TFS). Nanoparticle tracking analysis (qNano Gold, Izon Science, New Zealand) was performed to determine EV concentration and size distribution. Quantitative qRT-PCR analysis of the purified plasma Evs was performed to confirm presence of EV-bound mRNA. Results: Western Blotting demonstrated that eluates from both ACE-based methodology and chemistry-based isolation contain the expected CD9 and CD63. The ACE-isolated EVs showed a single prominent band for CD63, whereas chemistry-based isolated EVs display multiple smaller CD63-reactive species, which was suggestive of proteolysis. Mass spectrometry analysis confirmed presence of CD9 and CD81 in EVs isolated by ACE method; but not in EVs purified by chemistry-based method. APOB contamination was present in chemistry-based EVs, but not in ACE-isolated EVs. For RT-PCR experiments, EVs were purified from 5 subjects with lung cancer and 3 subjects with melanoma patient plasma using ACE method. Quantitative PCR confirmed presence of mRNA, via successful amplification of the housekeeping genes PGK1 and β-actin, in all ACE-isolated EVs. Conclusions: The novel ACE-based platform successfully demonstrated direct isolation of EVs from plasma while preserving the integrity of protein and mRNA biomarkers. The compatibility of the eluted EVs with multiple downstream technologies, such as mass spectrometry, Western blotting and qRT-PCR, may enable novel biomarker discovery and use of EV-associated biomarkers in diagnostic assays. Citation Format: Rajaram Krishnan, Jean Lewis, David Searson, Orlando Perrera, Alfred Kinana, Heath Balcer, Iryna Clark, Juan Pablo Hinestrosa. Characterization of circulating extracellular vesicles isolated from plasma of cancer subjects using novel AC electrokinetics platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2850.

Reversion of thermic-shock effect on ram spermatozoa by adsorption of seminal plasma proteins revealed by partition in aqueous two-phase systems

Density-based lipoprotein depletion improves extracellular vesicle isolation and functional analysis

Quantitative proteomic studies are contributing greatly to the understanding of the spermatozoon through the provision of detailed information on the proteins spermatozoa acquire and shed in the acquisition of fertility. Extracellular vesicles (EVs) are thought to aid in the delivery of proteins to spermatozoa in the male reproductive tract. The aim of this study is to isolate, identify and quantify EV proteins isolated from ram seminal plasma. Ram sperm plasma membrane proteins are also isolated using nitrogen cavitation and identified to better understand the interplay of proteins between the sperm membrane and extracellular environment. The categorization of proteins enriched in the EV population according to their function revealed three main groupings: vesicle biogenesis, metabolism, and membrane adhesion and remodeling. The latter group contains many reproduction-specific proteins that show demonstrable links to sperm fertility. Many of these membrane-bound proteins show testicular expression and are shed from the sperm surface during epididymal maturation (e.g., testis expressed 101; TEX101 and lymphocyte Antigen 6 Family Member K; LY6K). Their association with seminal EVs suggests that EVs may not only deliver protein cargo to spermatozoa but also assist in the removal of proteins from the sperm membrane.

Extracellular vesicles (EVs) are crucial mediators of intercellular communication, transporting various macromolecules between cells. They are increasingly recognized for their roles in cancer progression, immune modulation, and therapeutic resistance. However, standard EV isolation methods often struggle to preserve EV heterogeneity and functional integrity. In this study, we used hollow-fiber flow field-flow fractionation (HF5) to isolate and characterize plasma-derived EVs from just 60μL of plasma. HF5 is a cutting-edge, disposable microfluidic technique designed for advanced EV fractionation. EVs were from patients with polycythemia vera (PV), a rare hematological malignancy. We evaluated EVs isolated using HF5 against those obtained by size-exclusion chromatography (SEC), assessing their physico-chemical characteristics, surface marker expression and functionality in terms of up taking and inflammatory potential. EVs isolated through HF5 closely resembled SEC-derived EVs in size, morphology, and classical EV markers, including platelet-specific proteins. HF5 consistently yielded purer EV preparations with reduced aggregation and greater reproducibility. Notably, HF5 achieved this using 8.3 times less plasma than SEC. HF5 EVs, along with inflammatory potential, showed superior cell up take to the SEC counterparts. Chemical analysis showed that HF5-EVs contained a higher protein concentration, while SEC-EVs had more aggregated material. HF5 integrates EV isolation and characterization, enhancing efficiency, and preserving sample integrity and functionality. Its minimal sample requirement and reproducibility make it particularly suitable for clinical and translational research. Our study demonstrated HF5 as a powerful better alternative to conventional EV isolation methods, with strong potential for standardized applications in biomarker discovery and cancer research.

Extracellular Vesicles: How a Circulating Biomarker Can Double As a Regulator of Blood Pressure.

Study question How does the woman’s reproductive age affect the ability of follicular fluid (FF) extracellular vesicles (EV) to change the motility and hyperactivation of spermatozoa and follicular fluid microRNA profiles? Summary answer The effect of FF EVs on sperm motility and hyperactivation decreases with a woman's age. MiR-134-5p, miR-21-5p expression levels increase in older age group. What is known already Aging reduces human fertility. FF is an important factor in attracting and activating spermatozoa in the oviduct during oocyte fertilization, enhancing the process of sperm capacitation and acrosomal reaction in various mammalian species, including humans. EVs from seminal plasma and oviduct fluid carry proteins and miRNAs playing a vital role in a multi-step process including sperm motility, capacitation, acrosomal reaction, further fertilization. Results of our previous experiments showed that FF EVs from young women significantly improve the indices of sperm motility and hyperactivation. Several studies have isolated some age associated differentially expressed miRNAs (hsa-miR-424, hsa-miR-21-5p, hsa-miR-134, hsa-miR-190b, hsa-miR-99b -3p). Study design, size, duration FF EVs were obtained by sequential centrifugation at different rotational speeds, frozen at -80 °C. The sperm fraction was isolated, washed by differential centrifugation in a density gradient, suspended in the saline to a concentration of 106/ml and incubated with EVs (1:2) at 37 °C in CO2-incubator for 1 h. Samples were centrifuged and fixed in 2.5% glutaraldehyde in 0.1 M buffer for TEM. Sperm motility were assessed using CASA. MicroRNAs isolation (miRNeasy Serum/Plasma Kit (Qiagen)). Participants/materials, setting, methods All study participants signed a voluntary informed consent for the use of biological samples for research purposes. Sperm samples were isolated from seminal fluid (n = 18) aged 28-36 years without severe pathozoospermia. FF EVs were obtained from young (n = 4) aged 27-31 (EVs+) and older patients (n = 4) aged 41-46 with several IVF attempts (EVs-). The methods used in this work include sperm and EVs isolation, incubation, transmission electron microscopy, RT-PCR, statistical data analysis, computer-assisted semen analysis (CASA). Main results and the role of chance Our results showed that sperm incubation with EVs+ led to a significant increase in the number of progressively motile spermatozoa (paired Student's t test, p < 0.001), indicators of general mobility (p = 0.05) and hyperactivation of spermatozoa after 1 hour. Sperm incubation with EVs- slightly changed these parameters compared to the control (p = 0.171). Results from TEM showed that EVs- bind to spermatozoa worse than EVs+ after incubation. EVs- bind predominantly to the lateral membrane of the spermatozoon, rather than to the acrosomal region, compare to EVs+, which may reflect changes in the functional composition of FF EVs in older patients group and target areas of interaction with spermatozoa. This hypothesis is also supported by slight improvements in progressive sperm motility after incubation. Age-related changes are reflected in energy, metabolic and other important biological processes, which leads to a change in the composition and functions of FF (decrease in progesterone, glucose, increase in lactate and pyruvate, increase in reactive oxygen species) and affect the functional role of FF EVs. MiR-134-5p (p = 0.008), miR-21-5p (p = 0.008) expression levels were statistically significantly higher in EVs- group compared with EV + group with a successful first attempt. The expression of other microRNAs analyzed did not differ between groups. Limitations, reasons for caution Small amount of data. This study does not characterize isolated FF EVs in any way (specific markers, EVs concentration, size). To discuss the possible mechanisms underlying the effects of FF EVs of different age groups on sperm characteristics, it is necessary to perform metabolomics analyses of FF EVs. Wider implications of the findings Further studies of the influence of FF EVs on sperm motility may help to improve of ART outcomes, associated with male and female infertility by improving sperm morphofunctional characteristics and increasing their fertilizing ability. The solutions to this problem could be the use of FF EVs from young fertile donors. Trial registration number not applicable

Extracellular vesicles (EVs) are highly sought after as a source of biomarkers for disease detection and monitoring. Tumor EV isolation, processing, and evaluation from biofluids is convoluted by EV heterogeneity and biological contaminants and is limited by technical processing efficacy. This study rigorously compares common bulk EV isolation workflows (size exclusion chromatography, SEC; membrane affinity, MA) alongside downstream RNA extraction protocols to investigate molecular analyte recovery. EV integrity and recovery is evaluated using a variety of technologies to quantify total intact EVs, total and surface proteins, and RNA purity and recovery. A comprehensive evaluation of each analyte is performed, with a specific emphasis on maintaining user (n = 2), biological (n = 3), and technical replicates (n≥3) under in vitro conditions. Subsequent study of tumor EV spike-in into healthy donor plasma samples is performed to further validate biofluid-derived EV purity and isolation for clinical application. Results show that EV surface integrity is considerably preserved in eluates from SEC-derived EVs, but RNA recovery and purity, as well as bulk protein isolation, is significantly improved in MA-isolated EVs. This study concludes that EV isolation and RNA extraction pipelines govern recovered analyte integrity, necessitating careful selection of processing modality to enhance recovery of the analyte of interest.

Extracellular vesicles (EV) are important mediators of intercellular communication because they transfer microRNA (miRNA) that are able to repress translation of mRNA. Their presence in seminal plasma suggests a role in sperm fertility. It is known that bull seminal plasma contains fertility-associated proteins that are predictive of high and low fertility (Killian et al. 1993 Biol. Reprod. 49, 1202-1207). In addition, a difference in miRNA content between high and low spermatozoa motility has been observed in bulls, highlighting a potential role of EV on fertility (Capra et al. 2017 BMC Genom. 18, 14). We hypothesised that co-incubation of sperm of low-fertility bulls with EV isolated from the seminal plasma of high-fertility bulls could improve their fertility. Before testing this hypothesis, a preliminary study was carried out to investigate the presence and type of EV in bovine seminal plasma and their interaction with spermatozoa. Ejaculates of eight Holstein bulls collected weekly by artificial vagina were centrifuged at 1600×g for 10min to pellet spermatozoa and then centrifuged again at 2400×g for 30min to eliminate cell debris and large vesicles. After centrifugation, supernatants were collected and filtered twice (0.45 and 0.22µm) and stored at −80°C. A double ultracentrifugation at 100 000×g for 1h was performed, and pellets resuspended in a small volume of Tris buffer were kept at −80°C until used. Three ejaculates of the same bull were pooled to detect the concentration and size of EV by Nanosight Instruments. To trace the interaction with spermatozoa by fluorescence microscopy, EV were labeled with PKH26 dye and a dose-response curve in three replicates was performed. A suspension of 1×106 spermmL−1 was co-incubated with 200 or 400×106 EV labelled with pKH26 for 30, 60, 90, 120, 150, and 180min at 38.5°C. The end point of incubation was at 24h. Internalisation of EV was assessed using confocal microscopy. Our results showed that the size of EV ranged from 145.1 to 187.7nm, with an average of 166±29nm. For all seminal plasma samples, the number of EV ranged from 3.62 to 6.08×1013 particlesmL−1, with an average of 4.37±0.61×1013. Based on size, these EV can be categorised as shedding vesicles. Confocal microscopy was set to take fluorescent images at different planes scanned every 0.12µm from top to bottom of the spermatozoa. Our results showed that no fluorescence signal was detectable after co-incubation with 200×106 EV. At the concentration of 400×106 EV, up to 60min no signal was detectable, whereas at 90min spermatozoa showed a fine granular fluorescent pattern within the intermediate portion. At 120min, the signal was within the acrosome, and at 180min the spermatozoa were stained for the whole length, supposing a distribution of incorporated EV throughout all the cell. At 24h, the fluorescence signal decreased. In conclusion, this is the first study to demonstrate that bull spermatozoa incorporate EV from bull semen. We hypothesise that a transfer of molecules, such as miRNA and other noncoding RNA molecules, from EV to spermatozoa is probably involved in sperm fertility.

Extracellular vesicles (EVs) are membrane-bound particles that engage in inflammatory reactions by mediating cell–cell interactions. Previously, EVs have been isolated from the bronchoalveolar lavage fluid (BALF) of humans and rodents. The aim of this study was to investigate the number and size distribution of EVs in the BALF of asthmatic horses (EA, n = 35) and healthy horses (n = 19). Saline was injected during bronchoscopy to the right lung followed by manual aspiration. The retrieved BALF was centrifuged twice to remove cells and biological debris. The supernatant was concentrated and EVs were isolated using size-exclusion chromatography. Sample fractions were measured with nanoparticle tracking analysis (NTA) for particle number and size, and transmission electron microscopy and confocal laser scanning microscopy were used to visualize EVs. The described method was able to isolate and preserve EVs. The mean EV size was 247 ± 35 nm (SD) in the EA horses and 261 ± 47 nm in the controls by NTA. The mean concentration of EVs was 1.38 × 1012 ± 1.42 × 1012 particles/mL in the EA horses and 1.33 × 1012 ± 1.07 × 1012 particles/mL in the controls with no statistically significant differences between the groups. With Western blotting and microscopy, these particles were documented to associate with EV protein markers (CD63, TSG101, HSP70, EMMPRIN, and actin) and hyaluronan. Equine BALF is rich in EVs of various sizes, and the described protocol is usable for isolating EVs. In the future, the role of EVs can be studied in horses with airway inflammation.