Roca Aleu, Jorge

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Roca Aleu, Jorge

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Roca Aleu, Jorge

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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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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.
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Measurement of activity and concentration of paraoxonase 1 (PON-1) in seminal plasma and identification of PON-2 in the sperm of boar ejaculates
(Wiley, 2015-01) Barranco Cascales, Isabel; Roca Aleu, Jorge; Tvarijonaviciute, Asta; Rubér, Marie; Vicente Carrillo, Alejandro; Atikuzzaman, Mohammad; Cerón Madrigal, José Joaquín; Martínez Navarro, Emilio; Rodríguez Martínez, Heriberto; Martínez García, Emilio; Medicina y Cirugía Animal
This study revealed and characterised the presence of the antioxidant enzymes paraoxonase (PON) type 1 (PON-1, extracellular) and type 2 (PON-2, intracellular) in boar semen. To evaluate PON-1, an entire ejaculate from each of ten boars was collected and the seminal plasma was harvested after double centrifugation (1,500g for 10 min). Seminal plasma was analysed for concentration as well as enzymatic activity of PON-1 and total cholesterol levels. Seminal-plasma PON-1 concentration ranged from 0.961 to 1.670 ng/ml while its enzymatic activity ranged from 0.056 to 0.400 IU/ml, which represent individual variance. Seminal-plasma PON-1 concentration and enzymatic activity were negatively correlated (r = -0.763; P < 0.01). The activity of seminal-plasma PON-1 negatively correlated with ejaculate volume (r = -0.726, P < 0.05), but positively correlated with sperm concentration (r = 0.654, P < 0.05). Total seminal-plasma cholesterol concentration positively correlated with PON-1 activity (r = 0.773; P < 0.01), but negatively correlated with PON-1 concentration (r = -0.709; P < 0.05). The presence of intracellular PON-2 was determined via immunocytochemistry in spermatozoa derived from artificial insemination. PON-2 localised to the post-acrosomal area of the sperm head and principal piece of the tail in membrane-intact spermatozoa. In summary, PON is present in boar semen, with PON-1 at low levels in seminal plasma and PON-2 within the spermatozoa. Further studies are needed to characterise the relationship between antioxidant PONs with sperm and other seminal-plasma parameters.
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.
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Season of ejaculate collection influences the freezability of boar spermatozoa
(Elsevier, 2013-12-03) Barranco Cascales, Isabel; Ortega, María D.; Martínez Alborcia, María J.; Vázquez, Juan M.; Martínez García, Emilio; Roca Aleu, Jorge; Medicina y Cirugía Animal
The aim of this retrospective study was to evaluate whether the season of ejaculate collection influences the freezability of porcine sperm. A total of 434 ejaculates were collected from boars of six different breeds over three years (2008-2011) and throughout the four seasons of the year identified in the northern hemisphere (winter, spring, summer and autumn). The ejaculates were cryopreserved using a standard 0.5 mL straw freezing protocol. Sperm quality was assessed before (fresh semen samples kept 24h at 17°C) and after freezing and thawing (at 30 and 150 min post-thawing in semen samples kept in a water bath at 37 °C), according to the percentages of total motility, as assessed by the CASA system, and viability, as assessed by flow cytometry after staining with SYBR-14, PI and PE-PNA. The data, in percentages, on sperm motility and viability after freezing and thawing were obtained at each evaluation time (recovered) and were normalized to the values before freezing (normalized). The season of ejaculate collection influenced (P<0.01) sperm quality before freezing and after thawing (recovered and normalized), irrespective of the breed of boar. Sperm quality was lower in summer, both in terms of motility and viability, and in autumn, in terms of motility, than in winter and spring. Seasonality in the normalized data indicates that the season of ejaculate collection influences sperm freezability, regardless of the season's influence on sperm quality before freezing. Consequently, the spermatozoa from ejaculates collected during summer and, to a lesser extent, also in autumn, are more sensitive to cryopreservation than those from ejaculates collected during winter and spring.
Open Access
Extracellular vesicles in seminal plasma: A safe and relevant tool to improve fertility in livestock?
(2022-09) Heriberto Rodriguez-Martinez; Roca Aleu, Jordi; Medicina y Cirugía Animal
Seminal plasma (SP) is not a pre-requisite for pregnancy. Yet, this heterogeneous, composite SP has proven relevant for fertility, as mediator for cell-to-cell communication between producing cells, spermatozoa and the female internal genital tract, regulating complex reproductive processes. Bearing hormones, proteins, cytokines as well as nuclei acids in nano-sized lipid bilayer seminal extracellular vesicles (sEVs), the SP concerts signaling to the female. Signals influence timing of ovulation, sperm transport and, particularly, enable the female immune system to balance her cryptic choice to engage into pregnancy or reject an eventual fertilization. This essay, focusing on livestock in general and pigs in particular, discusses the intrinsic roles of sEVs with regards to reproductive homeostasis, while binding and internalizing their cargo in spermatozoa and female tract epithelia to regulate their functional activity. Since prior studies had inconclusive results using bulk SP or single SP-contained free molecules, argumentation is hereby provided to increase the current incipient research on livestock sEVs, where fragile molecules relevant for fertility are shielded from degradation during handling. Seminal EVs isolated from SP can be used for andrological diagnosis and perhaps to select breeders with optimal fertility. Moreover, sEVs can be laboratory-uploaded with specific molecules or even engineered as lipid nanodroplets used as additives for extenders to improve fertility after artificial insemination (AI) or reproductive biotechnologies.
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Is boar sperm freezability more intrinsically linked to spermatozoa than to the surrounding seminal plasma?
(Elsevier, 2018-08) Li, Junwei; Roca Aleu, Jorge; Pérez Patiño, Cristina; Barranco Cascales, Isabel; Martínez García, Emilio; Rodríguez Martínez, Heriberto; Parrilla Riera, Inmaculada; Medicina y Cirugía Animal
This study aimed to elucidate the effect of seminal plasma (SP) from post-SRF on boar sperm freezability and, in addition, to determine the relevance of sperm itself to sustain cryopreservation, regardless of the SP surrounding them. Twelve ejaculates from three boars were manually collected in fractions/portions, P1: the first 10 mL of the SRF, P2: the rest of the SRF and the post-SRF. Immediately, samples were centrifuged to separate spermatozoa from the surrounding SP. Spermatozoa from P1 and P2 were then incubated with its own SP or that from post-SRF, diluted in BTS (1:1, v/v) at 17 °C overnight before being frozen in 0.5 mL straws using a standard protocol. Sperm motility (total and progressive) deteriorated (P < 0.05) when P1- or P2-sperm when incubated overnight in SP from post-SRF, while sperm viability differed between P1 and P2 (P < 0.05) regardless of the SP they were incubated in. Post-thaw sperm quality and functionality differed between P1 and P2, regardless of the SP used for overnight pre-freezing incubation. Post-thaw motility (P < 0.05) and viability (P < 0.01), as well as plasma membrane fluidity (P < 0.05) or lipid peroxidation values (P < 0.01) were best in P1 sperm compared to those of P2. The protein profile of sperm from P1 and P2, analyzed by 2D-PAGE, showed qualitative differences, which suggest that sperm rather than SP would explain differences in sperm freezability between ejaculate fractions/portions. Use of P1 fraction spermatozoa seems thus optimal for cryopreservation.
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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
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.