Barranco Cascales, Isabel

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Barranco Cascales, Isabel

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Barranco Cascales, Isabel

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Universidad de Murcia. Departamento de Medicina y Cirugía Animal

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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.
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The seminal plasma of the boar is rich in cytokines, with significant individual and intra-ejaculate variation
(Wiley, 2015-12) Barranco Cascales, Isabel; Rubér, Marie; Pérez Patiño, Cristina; Atikuzzaman, Mohammad; Martínez García, Emilio; Roca Aleu, Jorge; Rodríguez Martínez, Heriberto; Medicina y Cirugía Animal
Problem: The boar, as human, sequentially ejaculates sperm-rich and sperm-poor fractions. Seminal plasma (SP) spermadhesins (PSP-I/PSP-II) induce a primary endometrial inflammatory response in female sows, similar to that elicited by semen deposition in other species, including human. However, the SP is also known to mitigate such response, making it transient to allow for embryo entry to a cleansed endometrium. Although cytokine involvement has been claimed, the exploration of cytokines in different SP fractions is scarce. This study determines Th1, Th2, Th17 and Th3 cytokine profiles in specific ejaculate SP fractions from boars of proven fertility. Methods: SP samples from the sperm-rich fraction (SRF) and the sperm-poor post-SRF fraction (post-SRF) of manually collected ejaculates from eight boars (four ejaculates per boar) were analysed by commercial multiplex bead assay kits (Milliplex MAP, Millipore, USA) for interferon-γ, interferon gamma-induced protein 10, macrophage-derived chemokine, growth-regulated oncogene, granulocyte-macrophage colony-stimulating factor, monocyte chemo-attractant protein-1, interleukins (IL)-6, IL-8, IL-10, IL-15, IL-17 and transforming growth factor (TGF)-β1-β3. Results: Cytokine concentrations differed between the ejaculate fractions among boars, being highest in the post-SRF. Conclusion: Boar SP is rich in Th1, Th2, Th17 and Th3 cytokines, with lowest concentrations in the sperm-peak-containing fraction, indicating its main immune influence might reside in the larger, protein-rich sperm-poor post-SRF.
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The activity of paraoxonase type 1 (PON-1) in boar seminal plasma and its relationship with sperm quality, functionality, and in vivo fertility
(Wiley, 2015-01-19) Tvarijonaviciute, Asta; Pérez-Patiño, Cristina; Alkmin, Diego V.; Cerón, José J.; Martínez, Emilio A.; Rodríguez-Martínez, Heriberto; Roca, Jordi; Barranco Cascales, Isabel; Medicina y Cirugía Animal
Paraoxonase 1 (PON-1) is a hydrolytic enzyme present in body fluids, capable of protecting cells against oxidative stress. The hypothesis was hereby to test that PON-1, present in seminal plasma (SP), acts protecting boar spermatozoa when showing a reasonable high activity in the ejaculate. SP-PON-1 activity differed (p < 0.001) among boars (from 0.10 to 0.29 IU/mL). Intra-boar variability was also observed (p < 0.05), but only in two of the 15 boars. SP-PON-1 activity differed among ejaculate portions, showing the spermatozoa-peak portion of spermatozoa-rich ejaculate fraction the highest levels (0.35 ± 0.03 IU/mL, ranging from 0.12 to 0.69) and the post-sperm ejaculate fraction the lowest levels (0.12 ± 0.01 IU/mL, ranging from 0.03 to 0.21). SP-PON-1 activity was positively correlated with the percentage of spermatozoa with rapid and progressive movement (p < 0.01) and negatively correlated with the generation of intracellular reactive oxygen species (p < 0.01) in semen samples after 72 h of liquid storage. SP-PON-1 activity was highest (p < 0.01) in boars with highest farrowing rates. In conclusion, SP-PON-1 activity differed among boars and ejaculate fractions/portions. SP-PON-1 activity was positively correlated with sperm quality and functionality of liquid-stored semen samples and it evidenced a positive association with in vivo fertility.
Open Access
Deactivation of the JNK Pathway by GSTP1 is essential to maintain sperm functionality
(Frontiers Media, 2021-02-25) Llavanera, Marc; Mateo-Otero, Yentel; Delgado-Bermúdez, Ariadna; Recuero, Sandra; Olives, Samuel; Barranco Cascales, Isabel; Yeste, Marc; Medicina y Cirugía Animal; Facultad de Veterinaria
Fifty percent of male subfertility diagnosis is idiopathic and is usually associated with genetic abnormalities or protein dysfunction, which are not detectable through the conventional spermiogram. Glutathione S-transferases (GSTs) are antioxidant enzymes essential for preserving sperm function and maintaining fertilizing ability. However, while the role of GSTP1 in cell signaling regulation via the inhibition of c-Jun N-terminal kinases (JNK) has been enlightened in somatic cells, it has never been investigated in mammalian spermatozoa. In this regard, a comprehensive approach through immunoblotting, immunofluorescence, computer-assisted sperm assessment (CASA), and flow cytometry analysis was used to characterize the molecular role of the GSTP1-JNK heterocomplex in sperm physiology, using the pig as a model. Immunological assessments confirmed the presence and localization of GSTP1 in sperm cells. The pharmacological dissociation of the GSTP1-JNK heterocomplex resulted in the activation of JNK, which led to a significant decrease in sperm viability, motility, mitochondrial activity, and plasma membrane stability, as well as to an increase of intracellular superoxides. No effects in intracellular calcium levels and acrosome membrane integrity were observed. In conclusion, the present work has demonstrated, for the first time, the essential role of GSTP1 in deactivating JNK, which is crucial to maintain sperm function and has also set the grounds to understand the relevance of the GSTP1-JNK heterocomplex for the regulation of mammalian sperm physiology.
Open Access
Redox profiling of preovulatory follicular fluid in the donkey is species-specific, and contributes to modulate sperm function
(Nature Research, 2025-02-23) Catalán, Jaime; Padilla, Lorena; Maside, Carolina; Yánez-Ortiz, Iván; Tvarijonaviciute, Asta; Barranco Cascales, Isabel; Bonet, Sergi; Miró, Jordi; Yeste, Marc; Martínez Hernández, Jesús; Medicina y Cirugía Animal; Facultad de Veterinaria
The follicular fluid (FF) is crucial for providing oocytes with an ideal environment that promotes their development and maturation. Not only does this fluid supply nutrients and hormones, but also other components that protect both follicular cells and the oocyte itself from potential harmful factors, such as those inducing oxidative stress (OS). The FF has also been suggested to have beneficial effects on sperm when they reach the oviduct. The objectives of this study were to describe the presence and activity levels of redox biomarkers in the preovulatory follicular fluid (PFF), and to analyze the impact of PFF on the motility, viability and reactive oxygen species (ROS) levels of donkey sperm. For this purpose, ten PFF samples obtained from Catalan jennies and nine ejaculates collected from Catalan donkeys were used. Redox biomarkers -including enzymatic and non-enzymatic antioxidants, and oxidative biomarkers- were analyzed in jennies' PFF. After collection, each semen sample was split into two aliquots of equal volume. The two aliquots were centrifuged to remove the seminal plasma; one pellet was resuspended in Tris Buffered Medium (TBM), and the other was resuspended in TBM supplemented with 20% PFF. Motility parameters (CASA) and other semen quality biomarkers (flow cytometry) were assessed after 0, 60 and 120 min of incubation at 38 °C. Exposure of donkey sperm to PFF reduced intracellular ROS levels and helped maintain sperm motility and viability. These findings suggest that, in the oviduct, the PFF components protect donkey sperm from oxidative stress. Furthermore, knowing better the composition of donkey PFF in terms of antioxidant biomarkers may be used to improve the formulation of media for oocyte maturation and fertilization in this and other species.
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
The triple role of glutathione S-transferases in mammalian male fertility
(2019-12-05) Llavanera, Marc; Mateo Otero, Yentel; Bonet, Sergi; Barranco Cascales, Isabel; Fernández Fuerstes, Beatriz; Yeste, Marc; Medicina y Cirugía Animal
Male idiopathic infertility accounts for 15-25% of reproductive failure. One of the factors that has been linked to this condition is oxidative stress (OS), defined as the imbalance between antioxidants and reactive oxygen species. Amongst the different factors that protect the cell against OS, the members of the glutathione S-transferase (GST) superfamily play an important role. Interestingly, reduction or lack of some GSTs has been associated to infertility in men. Therefore, and to clarify the relationship between GSTs and male fertility, the aim of this work is to describe the role that GSTs play in the male reproductive tract and in sperm physiology. To that end, the present review provides a novel perspective on the triple role of GSTs (detoxification, regulation of cell signalling and fertilisation), and reports their localisation in sperm, seminal plasma and the male reproductive tract. Furthermore, we also tackle the existing correlation between some GST classes and male fertility. Due to the considerable impact of GSTs in human pathology and their tight relationship with fertility, future research should address the specific role of these proteins in male fertility, which could result in new approaches for the diagnosis and/or treatment of male infertility.
Open Access
Aldose reductase B1 in pig sperm is related to their function and fertilizing ability
(2022-01-31) Mateo Otero, Yentel; Ribas Maynou, Jordi; Delgado Bermúdez, Ariadna; Llavanera, Marc; Recuero, Sandra; Barranco Cascales, Isabel; Yeste, Marc; Medicina y Cirugía Animal
Aldose reductase B1 (AKR1B1) has been reported to participate in the modulation of male and female reproductive physiology in several mammalian species. In spite of this, whether or not AKR1B1 could be related to sperm quality, functionality and fertilizing ability is yet to be elucidated. The present study, therefore, aimed to investigate: i) the presence of AKR1B1 in epididymal and ejaculated sperm; ii) the relationship between the AKR1B1 present in sperm and the physiology of the male gamete; iii) the liaison between the relative content of AKR1B1 in sperm and their ability to withstand preservation for 72 h; and iv) the potential link between sperm AKR1B1 and in vitro fertility outcomes. Immunoblotting revealed that AKR1B1 is present in both epididymal and ejaculated sperm with a similar relative content. Moreover, the relative levels of AKR1B1 in sperm (36 kDa band) were found to be negatively related to several kinematic parameters and intracellular calcium levels, and positively to the percentage of sperm with distal cytoplasmic droplets after storage. Finally, AKR1B1 amounts in sperm (36 kDa band) were negatively associated to fertilization rate at two days post-fertilization and embryo development at six days post-fertilization. The results of the present work suggest that AKR1B1 in sperm is probably acquired during maturation rather than at ejaculation and could play a role in that process. Moreover, AKR1B1 seems to be related to the sperm resilience to preservation and to their fertilizing capacity, as lower levels of the 36 kDa band (putative inactive form of this protein) result in better reproductive outcomes.
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Boar sperm cryosurvival is better after exposure to seminal plasma from selected fractions than to those from entire ejaculate
(Elsevier, 2014-07-15) Alkmin, Diego V.; Pérez Patino, Cristina; Barranco Cascales, Isabel; Parrilla Riera, Inmaculada; Vázquez, Juan M.; Váquez, Juan M.; Martínez Navarro, Emilio; Rodríguez Martínez, Heriberto; Roca Aleu, Jorge; Medicina y Cirugía Animal
Boar bulk ejaculates are now being collected instead of usual sperm-rich fractions (SRF) for artificial insemination purpose. The present study evaluated the influence of holding boar sperm samples before freezing surrounded in their own seminal plasma (SP), from either fractions/portions or the entire ejaculate, on post-thawing sperm quality and functionality. Ejaculates collected as bulk (BE) or as separate (first 10 mL of SRF [P1] and rest of SRF [P2]) from 10 boars were held 24h at 15-17°C and then frozen. Some bulk ejaculate samples were frozen immediately after collections as Control. In addition, epididymal sperm samples from the same 10 boars were collected post-mortem and extended in SP from P1 (EP1), P2 (EP2) and post SRF (EP3), and also held 24h before freezing for a better understanding of the influence of SP on boar sperm cryopreservation. The sperm quality (motility, evaluated by CASA, and viability, evaluated by flow cytometry) and functionality (flow cytometry assessment of plasma membrane fluidity, mitochondrial membrane potential and intracellular generation of reactive oxygen species [ROS] in viable sperm) were evaluated at 30, 150 and 300 min post-thaw. Post-thawing sperm quality and functionality of P1 and P2 were similar but higher (p < 0.01) than BE samples. Control samples showed higher (p < 0.01) post-thaw sperm quality and functionality than BE samples. Post-thawing sperm quality and functionality of EP1 and EP2 were similar but higher (p < 0.05) than EP3. These results showed that boar sperm from BE are more cryosensitive than those from the SRF, particularly when held 24h before freezing, which would be attributable to the cryonegative effects exerted by the SP from post SRF.
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.