DigitalUM :: Browsing by Subject "Extracellular vesicles"

Browsing by Subject "Extracellular vesicles"

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A Size-Exclusion Chromatography-Based Procedure for Isolating Extracellular Vesicle Subsets from Porcine Seminal Plasma
(Humana Press, 2024-04-10) Martínez Díaz, Pablo; Ana Parra; Christian M Sanchez-López; Antonio Marcilla; Bucci, Diego; Roca Aleu, Jorge; Barranco Cascales, Isabel; Medicina y Cirugía Animal; Facultad de Veterinaria
Extracellular vesicles (EVs), membrane nanoparticles (30-to-1000 nm diameter) secreted and released by most of the body functional cells, have emerged as powerful cell-to-cell messengers transferring their bioactive cargo (proteins, lipids, and nucleic acids) from donor to recipient cells. The promising potential utility of EVs as both noninvasive biomarkers and therapeutic carriers for several pathologies, including some types of cancers, has attracted increasing scientific interest. EVs can be found in all body biofluids, including seminal plasma, a complex fluid consisting mainly of a mixture of secretions of the epididymis and accessory sex glands. Seminal EVs are involved in modulating both sperm physiological processes and immune environment of the internal female genital tract, thus playing an essential indirect role in fertilization and embryo development. Seminal plasma, alike other biofluids, contains a heterogenous population of EV-subsets. However, the lack of consensus on the most accurate procedure for isolating EV-subsets has led to a poor definition of their composition/function. Currently, size exclusion chromatography (SEC), a size-selective separation method, is one of the most promising EV-isolation procedures, allowing the isolation of EVs from biological fluids in a purer, easier, cheaper, and more scalable way compared to other alternative isolation procedures. This chapter reports a SEC-based protocol, combined with differential centrifugation and ultrafiltration, to isolate two subsets of seminal EVs differing in size (large and small EVs) in the ejaculate of pigs, a livestock species of great productive interest and an outstanding animal model for human reproduction.
Open Access
Caracterización de exosomas producidos por células oviductales in vivo e in vitro, en la especie bovina
(Facultad de Veterinaria y el Servicio de Publicaciones de la Universidad de Murcia, 2023) Toledo Guardiola, Santa María; Matás Parra, Carmen; Rueda Gomariz, Almudena
Las vesículas extracelulares (VEs), exosomas y micro vesículas son un tipo de estructuras heterogéneas pre-sentes en la mayoría de los fluidos orgánicos incluyendo el fluido oviductal. Las VEs contienen varios compuestos derivados de la célula original, como proteínas, lípidos, ARNm, miARN y ADN. Las VEs en el oviducto son producidas por las células epiteliales y entre sus funciones se encuentran: la interacción con los espermatozoides, mantener la viabilidad de éstos, participar en la maduración de los ovocitos y en el proceso de fecundación. Durante la fecundación in vitro y con el fin de mejorarla imitando las condiciones in vivo, numerosos investigadores han utilizado cultivos de células del epitelio oviductal bovino (CEOB) con notables mejoras. Estas células producen, entre otros componentes VEs, por ello, en este trabajo hemos planteado un estudio com-parativo de VEs presentes en el fluido oviductal (FO) bovino recogido en momentos próximos a la ovulación (in vivo) y de aquellas VEs producidas en cultivos de CEOB a los 7 días de cultivo (in vitro) comparando el tamaño, la distribución de la población y la concentración de proteína en ambos tipos. Las VEs se identificaron mediante microscopía electrónica, su tamaño mediante dispersión de luz láser y la concentración de proteínas mediante el método Bradford. Los resultados mostraron que el tamaño de las VEs fue similar entre ambos grupos experimentales. Por otro lado, sí que se observaron diferencias en cuanto a la concentración de proteínas. Las VEs obtenidas in vivocontenían mayor cantidad de proteína en su cargo que en las VEs obtenidas in vitro.En cuanto a identificación de las VEs mediante microscopía electrónica de transmisión, solo pudieron ser observadas aquellas obtenidas in vivo. Este hecho podría deberse al lugar de dónde han sido recogidas, al mét-odo de cultivo de células epiteliales oviductales bovinas o la escasez en su producción.
Embargo
Cell-specific extracellular vesicles and their miRNA cargo released Into the organ preservations solution during cold ischemia storage as biomarkers for liver transplant outcomes
(Lippincott, Williams & Wilkins, 2024-10) Vidal-Correoso, Daniel; Mateo, Sandra V.; Muñoz-Morales, Ana M.; Lucas-Ruiz, Fernando; Jover-Aguilar, Marta; Alconchel, Felipe; Martinez-Alarcon, Laura; Sanchez-Redondo, Sara; Santos, Vanesa; Lopez-Lopez, Victor; Rios-Zambudio, Antonio; Cascales, Pedro; Pons Miñano, José Antonio; Ramirez, Pablo; Pelegrin, Pablo; Peinado, Hector; Baroja-Mazo, Alberto; Medicina
Background. Liver transplantation (LT) is crucial for end-stage liver disease patients, but organ shortages persist. Donation after circulatory death (DCD) aims to broaden the donor pool but presents challenges. Complications like acute rejection, hepatic artery thrombosis, and biliary issues still impact posttransplant prognosis. Biomarkers, including extracellular vesicles (EVs) and microRNAs (miRNAs), show promise in understanding and monitoring posttransplant events. This study explores the role of EVs and their miRNA cargo in LT, including their potential as diagnostic tools. Methods. EVs from intrahepatic end-ischemic organ preservation solution (eiOPS) in 79 donated livers were detected using different techniques (nanosight tracking analysis, transmission electron microscopy, and flow cytometry). EV-derived miRNAs were identified by quantitative real time-polymerase chain reaction. Bioinformatics analysis was performed using the R platform. Results. Differentsized and origin-specific EVs were found in eiOPS, with significantly higher concentrations in DCD compared with donation after brain death organs. Additionally, several EV-associated miRNAs, including let-7d-5p, miR-28-5p, miR-200a-3p, miR- 200b-3p, miR-200c-3p, and miR-429, were overexpressed in DCD-derived eiOPS. These miRNAs also exhibited differential expression patterns in liver tissue biopsies. Pathway analysis revealed enrichment in signaling pathways involved in extracellular matrix organization and various cellular processes. Moreover, specific EVs and miRNAs correlated with clinical outcomes, including survival and early allograft dysfunction. A predictive model combining biomarkers and clinical variables showed promise in acute rejection detection after LT. Conclusions. These findings provide new insights into the use of EVs and miRNAs as biomarkers and their possible influence on posttransplantation outcomes, potentially contributing to improved diagnostic approaches and personalized treatment strategies in LT.
Open Access
Cryogenic electron microscopy reveals morphologically distinct subtypes of extracellular vesicles among porcine ejaculate fractions
(Nature Research, 2024-07-13) Parra, Ana; Barranco, Isabel; Martínez Díaz, Pablo; González, Esperanza; Albóniga, Oihane E.; Cabrera, Diana; Falcón Pérez, Juan M.; Roca, Jordi; Medicina y Cirugía Animal
Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin.
Open Access
Extracellular vesicles derived from different brain tissue cells: A potential therapeutic measure for hypoxic-ischemic brain injury in immature brains
(Universidad de Murcia, Departamento de Histología e Histopatología, 2025) Guan Yitong; Yang Lijun; Cui Hong; Biología Celular e Histología
Neonatal hypoxic-ischemic encephalopathy/ neonatal hypoxic-ischemic brain damage and neonatal acute ischemic stroke are common causes of hypoxic-ischemic brain injury (HIBI) in the neonatal period, which may lead to permanent neurological sequelae. It is difficult to distinguish the two in the early stage. As a timely brain protection measure, hypothermia is still the standard treatment, but its efficacy in the treatment of immature brain injury is still controversial. The underlying pathophysiological mechanisms and effective treatment strategies of neonatal HIBI have been an active area of research. Extracellular vesicles (EVs), a class of nanoscale membranous structures, play a critical role in intercellular communication by facilitating the transfer of bioactive molecules or engaging in receptor-mediated interactions. Recent studies have demonstrated that various cell types within brain tissue, including neurons, astrocytes, microglia, endothelial cells, and stem cells, secrete substantial amounts of EVs. These vesicles carry diverse cargo, such as microRNAs, DNA, and proteins, which exert regulatory effects on recipient cells within the brain, thereby mediating neuroprotective effects. These effects include enhancing synaptic plasticity, modulating neuroinflammation, promoting angiogenesis, and regulating cellular autophagy, collectively contributing to neuroprotection. This review aims to summarize the functional characteristics of EVs derived from different cell types within the brain and to highlight recent advancements in this field. By providing insights into the role of EVs in HIBI, it seeks to provide novel insights and references for understanding the pathogenesis of neonatal HIBI and exploring innovative therapeutic approaches
Open Access
Extracellular vesicles derived from mesenchymal stem cells: A platform that can be engineered
(Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2021) Qin, Bo; Zhang, Qi; Chen, Dan; Yu, Hai-yang; Luo, Ai-xiang; Suo, Liang-peng; Cai, Yan; Cai, De-yang; Luo, Jia; Huang, Ju-fang; Xiong, Kun
y. Mesenchymal stem cells play an important role in tissue damage and repair. This role is mainly due to a paracrine mechanism, and extracellular vesicles (EVs) are an important part of the paracrine function. EVs play a vital role in many aspects of cell homeostasis, physiology, and pathology, and EVs can be used as clinical biomarkers, vaccines, or drug delivery vehicles. A large number of studies have shown that EVs derived from mesenchymal stem cells (MSC-EVs) play an important role in the treatment of various diseases. However, the problems of low production, low retention rate, and poor targeting of MSC-EVs are obstacles to current clinical applications. The engineering transformation of MSC-EVs can make up for those shortcomings, thereby improving treatment efficiency. This review summarizes the latest research progress of MSC-EV direct and indirect engineering transformation from the aspects of improving MSC-EV retention rate, yield, targeting, and MSC-EV visualization research, and proposes some feasible MSC-EV engineering methods of transformation.
Restricted
Extracellular vesicles in seminal fluid and effects on male reproduction. An overview in farm animals and pets
(2022-11) Roca Aleu, Jorge; Rodríguez Martínez, Heriberto; Padilla, Lorena; Lucas, Xiomara; Barranco Cascales, Isabel; Medicina y Cirugía Animal
Extracellular vesicles (EVs) are lipid bilayer nanovesicles released by most functional cells to body fluids, containing bioactive molecules, mainly proteins, lipids, and nucleic acids having actions at target cells. The EVs have essential functions in cell-to-cell communication by regulating different biological processes in target cells. Fluids from the male reproductive tract, including seminal plasma, contain many extracellular vesicles (sEVs), which have been evaluated to a lesser extent than those of other body fluids, particularly in farm animals and pets. Results from the few studies that have been conducted indicated epithelial cells of the testis, epididymis, ampulla of ductus deferens and many accessory sex glands release sEVs mainly via apocrine mechanisms. The sEVs are morphologically heterogeneous and bind to functional cells of the male reproductive tract, spermatozoa, and cells of the functional tissues of the female reproductive tract after mating or insemination. The sEVs encapsulate proteins and miRNAs that modulate sperm functions and male fertility. The sEVs, therefore, could be important as reproductive biomarkers in breeding sires. Many of the current findings regarding sEV functions, however, need experimental confirmation. Further studies are particularly needed to characterize both membranes and contents of sEVs, as well as the interaction between sEVs and target cells (spermatozoa and functional cells of the internal female reproductive tract). A priority for conducting these studies is development of methods that can be standardized and that are scalable, cost-effective and time-saving for isolation of different subtypes of EVs present in the entire population of sEVs.
Open Access
Extracellular vesicles isolated from porcine seminal plasma exhibit different tetraspanin expression profiles
(2019-08-09) Barranco, Isabel; Padilla, Lorena; Parrilla, Inmaculada; Alvarez-Barrientos, Alberto; Pérez-Patiño, Cristina; Peña, Fernando; Martínez, Emilio A; Rodriguez-Martínez, Heriberto; Roca, Jordi; Roca, Jordi; Medicina y Cirugía Animal
Seminal extracellular vesicles (EVs) include exosomes (ø 40-120 nm) and microvesicles (MVs, ø 120-1000 nm), which would be involved in multiple functional reproductive roles. The study aimed to establish which EV subtypes are present in pig semen, using a high-resolution flow cytometer to explore differences in their tetraspanin expression profile. The EVs were isolated from 12 pig ejaculates using serial ultracentrifugation and characterized by dynamic light scattering and electron microscopy for size and morphology as well as for tetraspanin expression using flow cytometry with Carboxyfluorescein succinimidyl ester (CFSE) and antibodies against CD9, CD63 and CD81. Pig semen contained a heterogeneous EV-population regarding size and morphology. Flow cytometric analysis demonstrated that the proportion of EVs expressing CD63 and CD9 was higher in MVs (P < 0.001 and P < 0.05, respectively) than in exosomes, while the opposite was true for CD81; higher (P < 0.001) in exosomes than in MVs. In conclusion, (1) the new generation of flow cytometers are able to accurately identify EVs and to gate them in two size-different populations named exosomes and MVs. (2) Tetraspanins CD9, CD63 and CD81 are present in both seminal EVs, albeit with exosomes and MVs differing in expression profiles, suggesting dissimilar cargo and binding affinity.
Open Access
Extracellular vesicles would be involved in the release and delivery of seminal TGF-β isoforms in pigs
(Frontiers Media, 2023-02-10) Padilla, Lorena; Barranco, Isabel; Parra, Ana; Parrilla, Inmaculada; Rodríguez Martínez, Heriberto; Lucas, Xiomara; Roca, Jordi; Martínez Hernández, Jesús; Pastor García, Luis Miguel; Medicina y Cirugía Animal
Introduction: pig seminal plasma (SP) is rich in active forms of all three isoforms (1-3) of transforming growth factor β (TGF-β), a chemokine modulatory of the immune environment in the female genital tract once semen is delivered during mating or artificial insemination (AI). The present study aimed to examine how TGF-βs are secreted by the epithelium of the male reproductive tract and how they are transported in semen, emphasizing the interplay with seminal extracellular vesicles (sEVs). Methods: Source of TGF-βs was examined by immunohistochemistry in testis, epididymis, and accessory sex glands, by immunocytochemistry in ejaculated spermatozoa, and by Luminex xMAP® technology in SP and sEVs retrieved from healthy, fertile male pigs used as breeders in AI programs. Results: All three TGF-β isoforms were expressed in all reproductive tissues explored and would be released into ductal lumen either in soluble form or associated with sEVs. Ejaculated spermatozoa expressed all three TGF-β isoforms, both inside and outside, probably the outer one associated with membrane-bound sEVs. The results confirmed that pig SP contains all three TGF-β isoforms and demonstrated that a substantial portion of them is associated with sEVs. Discussion: Seminal EVs would be involved in the cellular secretion of the active forms of seminal TGF-β isoforms and in their safe transport from the male to the female reproductive tract.
Open Access
Immunophenotype profile by flow cytometry reveals different subtypes of extracellular vesicles in porcine seminal plasma
(2024-01-23) Barranco, Isabel; Alvarez-Barrientos, Alberto; Parra, Ana; Martinez-Diaz, Pablo; Lucas, Xiomara; Roca, Jordi; Medicina y Cirugía Animal
Background: Porcine seminal plasma (SP) is endowed with a heterogeneous population of extracellular vesicles (sEVs). This study evaluated the immunophenotypic profile by high-sensitivity flow cytometry of eight sEV subpopulations isolated according to their size (small [S-sEVs] and large [L-sEVs]) from four different SP sources, namely three ejaculate fractions (the first 10 mL of the sperm rich fraction [SRF-P1], the remaining SRF [SRF-P2], and the post-SRF [PSRF]) and entire ejaculate (EE). Methods: Seminal EVs were isolated using a size exclusion chromatography-based protocol from six SP pools (five ejaculates/pool) of each SP source and characterized using complementary approaches including total protein (BCA™assay), particle size distribution (dynamic light scattering), morphology (transmission electron microscopy), and purity (albumin by Western blot). Expression of CD9, CD63, CD81, CD44 and HSP90β was analyzed in all sEV subpopulations by high-sensitivity flow cytometry according to MIFlowCyt-EV guidelines, including an accurate calibration, controls, and discrimination by CFSE-labelling. Results: Each sEV subpopulation exhibited a specific immunophenotypic profile. The percentage of sEVs positive for CD9, CD63, CD81 and HSP90β differed between S- and L-sEVs (P < 0.0001). Specifically, the percentage of sEVs positive for CD9 and CD63 was higher and that for CD81 was lower in S- than L-sEVs in the four SP sources. However, the percentage of HSP90β-positive sEVs was lower in S-sEVs than L-sEVs in the SRF-P1 and EE samples. The percentage of sEVs positive for CD9, CD63, and CD44 also differed among the four SP sources (P < 0.0001), being highest in PSRF samples. Notably, virtually all sEV subpopulations expressed CD44 (range: 88.04-98.50%). Conclusions: This study demonstrated the utility of high-sensitivity flow cytometry for sEV immunophenotyping, allowing the identification of distinct sEV subpopulations that may have different cellular origin, cargo, functions, and target cells.
Open Access
Isolation and characterization of extracellular vesicle subsets in donkey seminal plasma
(Elsevier, 2025-05-22) Catalán, Jaime; Martínez Díaz, Pablo; Parra, Ana; Bonet, Sergi; Yeste, Marc; Roca, Jordi; Barranco Cascales, Isabel; Miró, Jordi; Medicina y Cirugía Animal; Facultad de Veterinaria
Seminal plasma (SP), a fluid composed of secretions from the male genital tract, is rich in seminal extracellular vesicles (sEVs), nano-sized particles surrounded by a lipid bilayer membrane and loaded with functionally active molecules. Seminal EVs are secreted by functional cells of the male genital tract and play a key role in modulating reproductive processes, including sperm function and immune response in the female genital tract. The aim of this study was to isolate and characterize sEVs from donkey SP for the first time. Nine SP samples were collected from nine healthy and reproductive active donkeys. The SP samples were randomly pooled to create three pools (three SP samples per pool). The SP pools were subjected to differential centrifugation and size-exclusion chromatography to separately isolate two subsets of sEVs: small (S-) and large (L-). Orthogonal characterization of sEV samples was performed according to MISEV 2023 guidelines, including morphology (by cryogenic electron microscopy), concentration (by total protein concentration and total and CFSE positive particles by flow cytometry [FC]), particle size distribution (by dynamic light scattering), purity (by albumin assessment by FC), and specific EV protein markers (tetraspanins CD9, CD63, and CD81, and HSP70 by FC). The results showed that donkey SP is highly enriched in sEVs. Size differences were found between both sEV subsets, with S-sEVs being smaller (∼160 nm) and L-sEVs larger (∼290 nm). Both sEV subsets were positive for the four EV protein markers. However, the percentage of CD81-positive events was higher in S-sEV samples than in L-sEV samples (P < 0.05). This study is the first to isolate and characterize sEVs in donkey SP, demonstrating their heterogeneity and suggesting differences in biogenesis and function between S-sEVs and L-sEVs.
Open Access
Pravastatin reduces plasma levels of extracellular vesicles in pregnancies at high risk of term preeclampsia
(Frontiers Media, 2023-06-22) Santoyo, Jean Michell; Paco-Matallana, Catalina de; Delgado, Juan Luis; Cuevas, Santiago; Llinás Más, María Teresa; Hernández, Isabel; Avilés Plaza, Francisco Valeriano; Hernández Caselles, Trinidad; Noguera Velasco, José Antonio; Fisiología
Open Access
Progress of research on engineered extracellular vesicles from different sources for disease treatment
(Universidad de Murcia, Departamento de Histología e Histopatología, 2025) Kang Jinhui; Wen Jun; Chen Huifang; Zhu Cui; Bai Yinshan; Biología Celular e Histología
Extracellular vesicles (EVs) are lipid bilayer nanoparticles that encapsulate proteins, lipids, nucleic acids, and small molecules and display low immunogenicity, high stability, and cross-species transmission. By applying engineering technologies, the surface of EVs can be modified and loaded with cargo with therapeutic properties. Thus, engineered EVs can play important roles in preventing and treating various diseases. However, many challenges and uncertainties are faced in the clinical translation of EVs. In this review, we comprehensively analyzed the types of EVs derived from animal cells, plant cells, and microorganisms and summarized their biological properties and potential for engineering modifications. Furthermore, we also explored their therapeutic potential and discussed recent advancements in relevant clinical trials, aiming to provide scientific guidance for future research on the engineering of EVs and precision treatment of clinical diseases
Open Access
Protective role of extracellular vesicles against oxidative DNA damage
(BioMed Central, 2025-03-13) Ribas Maynou, Jordi; Parra, Ana; Martínez Díaz, Pablo; Peres Rubio, Camila; Lucas Arjona, Xiomara; Yeste, Marc; Roca, Jordi; Barranco Cascales, Isabel; Medicina y Cirugía Animal; Facultad de Veterinaria
Background: Oxidative stress, a source of genotoxic damage, is often the underlying mechanism in many functional cell disorders. Extracellular vesicles (EVs) have been shown to be key regulators of cellular processes and may be involved in maintaining cellular redox balance. Herein, we aimed to develop a method to assess the effects of EVs on DNA oxidation using porcine seminal plasma extracellular vesicles (sEVs). Results: The technique was set using a cell-free plasmid DNA to avoid the bias generated by the uptake of sEVs by sperm cells, employing increasing concentrations of hydrogen peroxide (H2O2) that generate DNA single-strand breaks (SSBs). Because SSBs contain a free 3'-OH end that allow the extension through quantitative PCR, such extension -and therefore the SYBR intensity- showed to be proportional to the amount of SSB. In the next stage, H2O2 was co-incubated with two size-differentiated subpopulations (small and large) of permeabilized and non-permeabilized sEVs to assess whether the intravesicular content (IC) or the surface of sEVs protects the DNA from oxidative damage. Results obtained showed that the surface of small sEVs reduced the incidence of DNA SSBs (P < 0.05), whereas that of large sEVs had no impact on the generation of SSBs (P > 0.05). The IC showed no protective effect against DNA oxidation (P > 0.05). Conclusions: Our results suggest that the surface of small sEVs, including the peripheral corona layer, may exert a protective function against alterations that are originated by oxidative mechanisms. In addition, our in vitro study opens path to detect, localize and quantify the protective effects against oxidation of extracellular vesicles derived from different fluids, elucidating their function in physiopathological states.
Open Access
Proteomic profiling of porcine seminal extracellular vesicles reveals potential in vivo fertility biomarkers
(Wiley, 2025-07-04) Barranco Cascales, Isabel; Martínez Díaz, Pablo; Parra, Ana; Martínez-Alborcia, María José; Lucas Arjona, Xiomara; Rodríguez-Martínez, Heriberto; Roca, Jordi; Medicina y Cirugía Animal; Facultad de Veterinaria
Background: Predicting male fertility in farm animals remains a challenge. Seminal plasma (SP) contains a high amount of heterogeneous seminal extracellular vesicles (sEVs), believed involved in reproductive processes and maybe key to understanding male fertility. Aims: To identify the sEV proteins that are differentially expressed between more and less fertile boars and that could be candidates for fertility biomarkers in boars used in artificial insemination (AI) programs. Materials and methods: Small (S) and large (L) sEV subsets from SP samples of AI boars with differences in fertility: high (H) or low (L) farrowing rate (FR) and large (L) or small (S) litter size (LS). The S- and L-sEV subsets were isolated by size exclusion chromatography and characterized according to the Minimal Information for Studies of Extracellular Vesicles (MISEV2023) guidelines. Proteomic analyses (three biological replicates per fertility group and sEV subset) were performed using a Bruker timsTOF fleX™ instrument with data-independent acquisition parallel accumulation-serial fragmentation (diaPASEF) technology. Results: A total of 470 and 726 proteins were quantified in S-sEVs and 1801 and 1834 proteins in L-sEVs from FR and LS boars, respectively. Differentially expressed sEV proteins (log2fold change ≥±1, p ≤ 0.05 and effect size d of Cohen >2.0) were found between the fertility groups: seven in S-sEVs and 52 in L-sEVs between H-FR and L-FR boars, and 47 in S-sEVs and 52 in L-sEVs between L-LS and S-LS boars. Many of these differentially expressed sEV proteins are involved in reproductive processes, particularly in sperm function and sperm-zona pellucida binding, but also in embryo development and implantation. Conclusions: The sEV proteome differs between more and less fertile boars, with many of the differentially expressed proteins known as involved in reproductive processes. This would suggest that sEVs may be involved in male fertility and that some of the differentially expressed sEV proteins could be potential fertility markers for AI boars.
Open Access
RNA profiles differ between small and large extracellular vesicle subsets isolated from porcine seminal plasma
(BioMed Central, 2024-12-27) Barranco Cascales, Isabel; Almiñana, Carmen; Parra, Ana; Martínez Díaz, Pablo; Lucas Arjona, Xiomara; Bauersachs, Stefan; Roca Aleu, Jorge; Medicina y Cirugía Animal
Background: Extracellular vesicles (EVs) are essential for cell-to-cell communication because they transport functionally active molecules, including proteins, RNA, and lipids, from secretory cells to nearby or distant target cells. Seminal plasma contains a large number of EVs (sEVs) that are phenotypically heterogeneous. The aim of the present study was to identify the RNA species contained in two subsets of porcine sEVs of different sizes, namely small sEVs (S-sEVs) and large sEVs (L-sEVs). The two subsets of sEVs were isolated from 54 seminal plasma samples by a method combining serial centrifugations, size exclusion chromatography, and ultrafiltration. The sEVs were characterized using an orthogonal approach. Analysis of RNA content and quantification were performed using RNA-seq analysis. Results: The two subsets of sEVs had different size distributions (P < 0.001). They also showed differences in concentration, morphology, and specific protein markers (P < 0.05). A total of 735 RNAs were identified and quantified, which included: (1) mRNAs, rRNAs, snoRNAs, snRNAs, tRNAs, other ncRNAs (termed as "all RNAs"), (2) miRNAs and (3) piRNAs. The distribution pattern of these RNA classes differed between S-sEVs and L-sEVs (P < 0.05). More than half of "all RNAs", miRNAs and piRNAs were found to be differentially abundant between S- and L-sEVs (FDR < 0.1%). Among the differentially abundant RNAs, "all RNAs" were more abundant in L- than in S-sEVs, whereas the most of the miRNAs were more abundant in S- than in L-sEVs. Differentially abundant piRNAs were equally distributed between S- and L-sEVs. Some of the all RNAs and miRNAs found to be differentially abundant between S- and L-sEVs were associated with sperm quality and functionality and male fertility success. Conclusions: Small and large sEVs isolated from porcine seminal plasma show quantitative differences in RNA content. These differences would suggest that each sEV subtype exerts different functional activities in the targeted cells, namely spermatozoa and functional cells of the female reproductive tract.
Open Access
Seminal extracellular vesicles alter porcine in vitro fertilization outcome by modulating sperm metabolism
(Elsevier, 2024-02-26) Barranco, Isabel; Spinaci, Marcella; Nesci, Salvatore; Mateo Otero, Yentel; Baldassarro, Vito Antonio; Algieri, Cristina; Bucci, Diego; Roca, Jordi; Medicina y Cirugía Animal
Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry; albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO2 in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism.
Open Access
Seminal extracellular vesicles and their involvement in male (In)fertility: a systematic review
(MDPI, 2023-03-02) Parra, Ana; Padilla, Lorena; Lucas, Xiomara; Rodríguez Martínez, Heriberto; Barranco, Isabel; Roca, Jordi; Medicina y Cirugía Animal
Seminal plasma contains numerous extracellular vesicles (sEVs). Since sEVs are apparently involved in male (in)fertility, this systematic review focused on studies specifically investigating such relationship. Embase, PubMed, and Scopus databases were searched up to 31 December 2022, primarily identifying a total of 1440 articles. After processing for screening and eligibility, 305 studies were selected as they focused on sEVs, and 42 of them were considered eligible because they included the word fertility or a related word such as infertility, subfertility, fertilization, and recurrent pregnancy loss in the title, objective(s), and/or keywords. Only nine of them met the inclusion criteria, namely (a) conducting experiments aimed at associating sEVs with fertility concerns and (b) isolating and adequately characterizing sEVs. Six studies were conducted on humans, two on laboratory animals, and one on livestock. The studies highlighted some sEV molecules, specifically proteins and small non-coding RNAs, that showed differences between fertile and subfertile or infertile males. The content of sEVs was also related to sperm fertilizing capacity, embryo development, and implantation. Bioinformatic analysis revealed that several of the highlighted sEV fertility-related proteins would be cross-linked to each other and involved in biological pathways related to (i) EV release and loading and (ii) plasma membrane organization.
Open Access
Seminal extracellular vesicles influence porcine spermatozoa physiology by modulating key functional parameters
(Elsevier, 2025-09-27) Parra, Ana; Martín-Cano, Francisco E.; Martínez Díaz, Pablo; Panales, Patricia; Lucas Arjona, Xiomara; Roca, Jordi; Barranco Cascales, Isabel; Peña, Fernando J.; Medicina y Cirugía Animal; Facultad de Veterinaria
Seminal plasma (SP) contains a heterogeneous population of extracellular vesicles (EVs) recognized as key modulators of sperm function. However, the specific functional roles of each seminal EV (sEV) subset remain poorly understood. This study aimed to evaluate the interaction of two sized sEV subsets (small [S-sEVs] and large [L-sEVs]) with pig liquid-stored spermatozoa under different pH conditions and their effect on specific sperm functional parameters. Seminal EV subsets were isolated from SP samples using size exclusion chromatography and characterized following the MISEV2023 guidelines. Semen samples were incubated with each sEV subset or without sEVs (control) for 6 h at 37 ºC, 100 % humidity and 5 % CO₂ under different pH conditions (6.5, 7.0, or 7.5). Sperm functional parameters were assessed by flow cytometry (Cytoflex®S and LX, Beckman Coulter), under capacitating and non-capacitating conditions. Confocal microscopy revealed that both sEV subsets bound to and were internalized by spermatozoa as early as 30 min after incubation, regardless of pH. Flow cytometry revealed that both sEVs decreased reactive oxygen species production (P ≤ 0.0001), mitochondrial membrane potential (P ≤ 0.0001) and mitochondrial O₂•⁻ levels (P ≤ 0.01) and increased apoptosis (active caspase-3) in viable spermatozoa (P ≤ 0.0001). However, the influence of sEV on acrosome integrity in viable sperm was time- and condition-dependent (P ≤ 0.05). This study showed that both S- and L-sEVs interact with porcine spermatozoa across a range of physiological pH conditions. This interaction is reflected by decreased oxidative stress and mitochondrial activity, as well as increased apoptosis in spermatozoa.
Open Access
Small and large extracellular vesicles of porcine seminal plasma differ in lipid profile
(MDPI, 2024-07-08) Martínez Diaz, Pablo; Parra, Ana; Sánchez López, Christian M.; Casas, Josefina; Lucas, Xiomara; Marcilla, Antonio; Roca, Jordi; Barranco, Isabel; Medicina y Cirugía Animal
Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs) that remains poorly characterized. This study aimed to characterize the lipidomic profile of two subsets of differently sized sEVs, small (S-) and large (L-), isolated from porcine seminal plasma by size-exclusion chromatography and characterized by an orthogonal approach. High-performance liquid chromatography-high-resolution mass spectrometry was used for lipidomic analysis. A total of 157 lipid species from 14 lipid classes of 4 major categories (sphingolipids, glycerophospholipids, glycerolipids, and sterols) were identified. Qualitative differences were limited to two cholesteryl ester species present only in S-sEVs. L-sEVs had higher levels of all quantified lipid classes due to their larger membrane surface area. The distribution pattern was different, especially for sphingomyelins (more in S-sEVs) and ceramides (more in L-sEVs). In conclusion, this study reveals differences in the lipidomic profile of two subsets of porcine sEVs, suggesting that they differ in biogenesis and functionality.