Martínez Díaz, Pablo

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Martínez Díaz, Pablo

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Martínez Díaz, Pablo

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Universidad de Murcia. Departamento de Medicina y Cirugía Animal

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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
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
Updating research on extracellular vesicles of the male reproductive tract in farm animals: a systematic review
(MDPI, 2024-10-31) Martínez Díaz, Pablo; Parra, Ana; Montesdeoca, Marina; Barranco Cascales, Isabel; Roca Aleu, Jorge; Medicina y Cirugía Animal
This systematic review examined research studies on extracellular vesicles (EVs) of the male reproductive tract in livestock species to summarize the research topics and methodologies used, key findings, and future directions. PubMed and Scopus were searched for time ranges up to 1 September 2024, and 1383 articles were identified. The application of screening and eligibility criteria resulted in the selection of 79 articles focusing on male reproductive EVs in livestock. Porcine and bovine male reproductive EVs were the most studied. A variety of EV isolation techniques were used, with ultracentrifugation being the most common. Characterization of male reproductive EVs in livestock was a weak point, with only 24.05% of the articles characterizing EVs according to MISEV guidelines. Inadequate characterization of EVs compromises the reliability of results. The results of 19 articles that provided a good characterization of EVs showed that male reproductive EVs from livestock species are phenotypically and compositionally heterogeneous. These papers also showed that these EVs would be involved in the regulation of sperm functionality. Research on male reproductive EVs in livestock species remains scarce, and further research is needed, which should include appropriate characterization of EVs and aim to find efficient methods to isolate them and assess their involvement in the functionality of spermatozoa and the cells of the female genital tract.
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.
Embargo
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
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 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.
Restricted
Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle
(CSIRO Publishing, 2024-05-07) Toledo-Guardiola, S.M.; Martínez Díaz, Pablo; Martínez-Núñez, R.; Navarro-Serna, S.; Soriano-Úbeda, C.; Romero Aguirregomezcorta, Jon; Matas Parra, Carmen; Anatomía y Anatomía Patológica Comparada; Facultad de Veterinaria
Context: Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims: To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods: poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results: poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P < 0.05). Conclusions: poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm–oocyte interaction. Implications: The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm–oocyte interaction.