Avilés Sánchez, Manuel

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Avilés Sánchez, Manuel

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Avilés Sánchez, Manuel

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Universidad de Murcia. Departamento de Biología Celular e Histología

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Open Access
Protein identification of spermatozoa and seminal plasma in bottlenose dolphin (Tursiops truncatus)
(2021-07-16) Fuentes-Albero, Mari Carmen; González-Brusi, Leopoldo; Luongo, Chiara; Abril-Sánchez, Silvia; Ros-Santaella, José Luis; Pintus, Eliana; Ruiz-Díaz, Sara; Barros-García, Carlos; Sánchez-Calabuig, María Jesús; García-Párraga, Daniel; García-Vázquez, Francisco Alberto; Cots Rodríguez, Paula; Izquierdo Rico, María José; Avilés Sánchez, Manuel; Biología Celular e Histología
Proteins play an important role in many reproductive functions such as sperm maturation, sperm transit in the female genital tract or sperm-oocyte interaction. However, in general, little information concerning reproductive features is available in the case of aquatic animals. The present study aims to characterize the proteome of both spermatozoa and seminal plasma of bottlenose dolphins (Tursiops truncatus) as a model organism for cetaceans. Ejaculate samples were obtained from two trained dolphins housed in an aquarium. Spermatozoa and seminal plasma were analyzed by means of proteomic analyses using an LC-MS/MS, and a list with the gene symbols corresponding to each protein was submitted to the DAVID database. Of the 419 proteins identified in spermatozoa and 303 in seminal plasma, 111 proteins were shared by both. Furthermore, 70 proteins were identified as involved in reproductive processes, 39 in spermatozoa, and 31 in seminal plasma. The five most abundant proteins were also identified in these samples: AKAP3, ODF2, TUBB, GSTM3, ROPN1 for spermatozoa and CST11, LTF, ALB, HSP90B1, PIGR for seminal plasma. In conclusion, this study provides the first characterization of the proteome in cetacean sperm and seminal plasma, opening the way to future research into new biomarkers, the analysis of conservation capacity or possible additional applications in the field of assisted reproductive technologies.
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Calreticulin from suboolemmal vesicles affects membrane regulation of polyspermy
(2014-02-05) Saavedra MD; Coy, Pilar; Betancourt, M; González-Marquez, H; Mondéjar Corbalán, Irene; Jíménez Movilla, María; Avilés Sánchez, Manuel; Romar Andrés, Raquel; Fisiología; Facultades de la UMU
This study was designed to determine whether calreticulin (CRT), a chaperone protein, is present in in vitro-matured (IVM) pig oocytes and to study its potential role in the block to polyspermy. Western blot analysis, using an anti-CRT antibody, of oocyte lysate showed an immunoreactive band of w60 kDa. Simultaneous labeling of IVM oocytes with anti-CRT antibody and peanut agglutinin lectin (PNA lectin, a porcine cortical granules (CG)-specific binding lectin) revealed localization of CRT in the subplasmalemmal region with a 27.7% colocalization with PNA staining. After IVF, PNA labeling was not observed and anti-CRT labeling decreased significantly in zygotes and disappeared in two-cell embryos. Western blot analysis of oocyte exudate obtained from zona pellucida (ZP)-free oocytes activated with calcium ionophore confirmed the presence of a band that reacted with an anti-CRT antibody. Anti-CRT antibody and PNA labeling were not observed in activated oocytes despite being detectable in non-activated oocytes. The presence of CRT in vesicles located under the oolemma was demonstrated using immunogold cytochemistry at the ultrastructural level. To study the role of CRT in fertilization, ZP-enclosed and ZP-free oocytes were incubated with exogenous CRT and then inseminated. Whereas ZP-free oocytes showed fewer penetrating sperm and lower polyspermy rates than untreated oocytes, the opposite effect was observed in ZP-enclosed oocytes. In conclusion, CRT is confined to subplasmalemmal vesicles partially overlapping with CG contents. Its exocytosis after the oocyte activation seems to participate in the membrane block to polyspermy in pigs but is not involved in the ZP block.
Open Access
Characterization of the lectin binding pattern in human spermatozoa after swim-up selection
(F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología, 2012) Gómez-Torres, María José; Girela, Jose Luis; Murcia, Verónica; Fernández-Colom, Pedro José; Romeu, Alberto; Juan, Joaquín de; Avilés Sánchez, Manuel
Capacitation is characterized by a hyperactivated pattern of sperm motility. The acquisition of highly motility is present in the early stages of capacitation. Sperm progressive motility is one of the most important parameters for determining the suitability of semen for processing. However, previous studies have shown that some sperm showing good motility have membrane damage. The aim of our study was to characterize the lectin staining pattern on the sperm plasma membrane of unselected and selected human sperm of normozoospermic donors. Sperm selection was performed by the swim-up technique. Fourteen samples from healthy consenting donors classified as normozoospermic according to the World Health Organization were used. We observed changes in the distribution of the carbohydrate residues after the swim-up selection. With Triticum vulgaris, the most abundant pattern was dotted labeling all over the head plasma membrane in the unselected sperm. However, this lectin was distributed homogenously over the acrosomal region after selection. With Arachis hypogaea, the most abundant pattern in fresh sperm was a highly stained acrosomal region. In the highly motility sperm population, the most frequent pattern was dotted fluorescence on the acrosomal region and a highly stained equatorial segment. Meanwhile, with the Aleuria aurantia and Canavalia ensiformis lectins, the most representative patterns were the same before and after the swim-up selection. Our data indicate that modifications which occur in carbohydrate residues during swim-up selection could be important for the regulation of progressive motility and prepare the sperm for capacitation.
Open Access
Composition of marsupial zona pellucida: a molecular and phylogenetic approach
(2017) Chevret, P.; Holt, W.V.; Esteban-Dían, D.; López-Béjar, M.; Martínez-Nevado, E.; Nilsson, M.A.; Ballesta, J.; Izquierdo Rico, María José; Avilés Sánchez, Manuel; Moros Nicolás, Carla; Biología Celular e Histología
The zona pellucida (ZP) is an extracellular matrix that surrounds mammalian oocytes. In eutherians it is formed from three or four proteins (ZP1, ZP2, ZP3, ZP4). In the few marsupials that have been studied, however, only three of these have been characterised (ZP2, ZP3, ZP4). Nevertheless, the composition in marsupials may be more complex, since a duplication of the ZP3 gene was recently described in one species. The aim of this work was to elucidate the ZP composition in marsupials and relate it to the evolution of the ZP gene family. For that, an in silico and molecular analysis was undertaken, focusing on two South American species (gray short-tailed opossum and common opossum) and five Australian species (brushtail possum, koala, Bennett’s wallaby, Tammar wallaby and Tasmanian devil). This analysis identified the presence of ZP1 mRNA and mRNA from two or three paralogues of ZP3 in marsupials. Furthermore, evidence for ZP1 and ZP4 pseudogenes in the South American subfamily Didelphinae and for ZP3 pseudogenes in two marsupials is provided. In conclusion, two different composition models are proposed for marsupials: a model with four proteins (ZP1, ZP2 and ZP3 (two copies)) for the South American species and a model with six proteins (ZP1, ZP2, ZP3 (three copies) and ZP4) for the Australasian species
Open Access
Comunicaciones Póster.-Identification of lactadherin in the porcine oviductal secretion
(2020-06-03) Acuña, O.S.; Jara, L.; Soriano-Úbeda, C.; Algarra-Oñate, B.; López Albors, Octavio Miguel; Guillén Martínez, Ascensión; Izquierdo Rico, María José; Avilés Sánchez, Manuel; Moros Nicolás, Carla; Facultades, Departamentos, Servicios y Escuelas::Departamentos de la UMU::Biología Celular e Histología; Facultades, Departamentos, Servicios y Escuelas::Departamentos de la UMU::Fisiología; Facultades, Departamentos, Servicios y Escuelas::Departamentos de la UMU::Anatomía y Anatomía Patológica Comparada
Open Access
New Insights into the Mammalian Egg Zona Pellucida
(2021) Chevret, Pascale; Algarra, Blanca; González-Brusi, Leopoldo; Cots Rodríguez, Paula; Izquierdo Rico, María José; Avilés Sánchez, Manuel; Jíménez Movilla, María; Moros Nicolás, Carla; Biología Celular e Histología
Mammalian oocytes are surrounded by an extracellular coat called the zona pellucida (ZP), which, from an evolutionary point of view, is the most ancient of the coats that envelope vertebrate oocytes and conceptuses. This matrix separates the oocyte from cumulus cells and is responsible for species-specific recognition between gametes, preventing polyspermy and protecting the preimplantation embryo. The ZP is a dynamic structure that shows different properties before and after fertilization. Until very recently, mammalian ZP was believed to be composed of only three glycoproteins, ZP1, ZP2 and ZP3, as first described in mouse. However, studies have revealed that this composition is not necessarily applicable to other mammals. Such differences can be explained by an analysis of the molecular evolution of the ZP gene family, during which ZP genes have suffered pseudogenization and duplication events that have resulted in differing models of ZP protein composition. The many discoveries made in recent years related to ZP composition and evolution suggest that a compilation would be useful. Moreover, this review analyses ZP biosynthesis, the role of each ZP protein in different mammalian species and how these proteins may interact among themselves and with other proteins present in the oviductal lumen.
Open Access
Boar sperm motility is modulated by CCK at a low concentration of bicarbonate under capacitation conditions
(2023) López-Úbeda, R.; Luongo, C.; Sòria-Monzó, P.; Abril-Sánchez, S.; Grudzinska, P.; García-Vázquez, F.A.; Izquierdo Rico, María José; Avilés Sánchez, Manuel; Moros Nicolás, Carla; Biología Celular e Histología
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The human is an exception to the evolutionarily-conserved phenomenon of pre-fertilization zona pellucida resistance to proteolysis induced by oviductal fluid
(2014-01-04) Mondéjar Corbalán, Irene; Avilés Sánchez, Manuel; Coy Fuster, Pilar; Coy Fuster, Pilar; Fisiología
study question: Is zona pellucida (ZP) resistance to proteolysis, induced by oviductal fluid (OF), a mechanism common to species other than the pig and cow? summary answer: ZP resistance to proteolysis induced by OF was observed in the mouse, rat, hamster, rabbit, sheep, goat, pig and cow, but not in humans. what is known already: Oviductal ZP resistance to proteolysis occurs in the pig and cow where it influences the incidence of fertilization and polyspermy. The effect is observed after incubation of ZP in OFs from pig (pOF), cow (cOF), rabbit (rOF) and sheep (sOF). study design, size, duration: Oocytes from nine different species, including ungulates, rodents, lagomorphs and primates were incubated in rOF, sOF, gOF, cOF, pOF and human oviductal fluid (hOF). ZP digestion times for the matured oocytes of these nine species, without any treatment or incubated in 5 (mouse, rat, hamster, rabbit, cow, ewe and goat) or 6 (pig and humans) of the OFs collected were compared using three replicates per treatment and at least three oocytes per replicate. materials, setting, methods: In vivo matured oocytes from rat, hamster, mouse, rabbit and humans, in vitro matured oocytes from cow, goat, ewe and pig and rOF, cOF, gOF, sOF, pOF and human (hOF) were collected and processed for the study. Oocytes from each species were incubated in the different OFs for 30 min. The resistance of the ZP of the oocytes to enzymatic digestion in a pronase solution (0.5% in PBS) was measured and registered as ZP digestion time. main results and the role of chance: rOF increased ZP resistance to proteolytic digestion in the range of between 96 and 720 h for any of the species tested, whereas the corresponding increase in human ZP was only 1 min. OFs from the remaining species also had a significant effect, with variations among the cross-species experiments (P , 0.05). hOF, which was only tested on human and porcine oocytes, had no effect on ZP chemical hardening. Measurements of ZP digestion times are not of extreme accuracy and errors of a few seconds can be assumed in the experimental data. However, when differences are in the range of hours among treatments, variations measured in seconds do not alter the robustness of the findings. limitations, reasons for caution: Human oocytes and OF were of limited access, compared with oocytes from species collected in slaughterhouses. OFs from mouse, rat and hamster were not tested due to the small size of the genital tract in these species and the small volume of fluid available. wider implications of the findings: Since oviductal modification of ZP resistance to proteolytic digestion has been demonstrated to influence fertilization and this pre-fertilization mechanism is considered to contribute to the control of polyspermy, the apparent absence of this mechanism in humans suggests that the regulation of polyspermy depends mainly on other mechanisms, most probably of cortical granule origin. Investigation into a possible relationship between the lack of oviductal ZP hardening in human oocytes and the existence of tubal ectopic pregnancies in this species is proposed.
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Expression of IZUMO1 and JUNO in the gonads of domestic cats (Felis catus)
(Elsevier, 2024-05) Sanguansook, P; Rodprasert, W; Sawangmake, C; Ferrán, JL; Soria-Monzó, P; Chatdarong, K; Gimeno Arias, Lourdes; Izquierdo Rico, María José; Avilés Sánchez, Manuel; Biología Celular e Histología
Because of the time-consuming nature of surgical neutering and the rapid rate of reproduction among domestic cats, it is crucial to investigate alternative, nonsurgical methods of contraception for this species. Sperm protein IZUMO1 and its oocyte receptor JUNO have been proposed as potential targets for nonsurgical contraceptives. This study aimed to demonstrate (1) the protein coding sequence of feline IZUMO1 and JUNO, (2) gene expression in specific organs by measuring mRNA levels in different visceral tissues, and (3) the expression of IZUMO1 and JUNO during sperm maturation and folliculogenesis, respectively. Amplification for sequencing of feline IZUMO1 and JUNO was performed using the RT-PCR method. Levels of gene expression in different tissues were evaluated using real-time PCR. In situ hybridization was performed to localize JUNO mRNA in ovarian tissues. The complete coding sequences of IZUMO1 and JUNO were obtained and analyzed. A comparison between protein orthologs demonstrated the conservation of IZUMO1 and JUNO in Felidae. The real-time PCR results from various visceral organs indicated that IZUMO1 was significantly higher in the testis than in other organs, whereas JUNO was significantly higher in the ovary than in other organs. Expression of IZUMO1 was found to be higher in the testes than in the caput, corpus, and cauda of epididymides. In situ hybridization revealed that JUNO mRNA was in the ooplasm and nucleus of the primordial, primary, secondary, and antral follicles. Importantly, this was the first study to demonstrate the IZUMO1 and JUNO genes in the testis and ovary of cats. The results are useful for future research related to these genes and for developing contraceptives against these targets.
Open Access
ZP4 confers structural properties to the zona pellucida essential for embryo development
(2019-10-22) Lamas-Toranzo, Ismael; Fonseca Balvís, Noelia; Querejeta-Fernández, Ana; González-Brusi, Leopoldo; Lorenzo, Pedro L.; García-Rebollar, Pilar; Bermejo-Álvarez, Pablo; Izquierdo Rico, María José; Avilés Sánchez, Manuel; Biología Celular e Histología
Zona pellucida (ZP), the extracellular matrix sheltering mammalian oocytes and embryos, is composed by 3 to 4 proteins. The roles of the three proteins present in mice have been elucidated by KO models, but the function of the fourth component (ZP4), present in all other eutherian mammals studied so far, has remained elusive. Herein, we report that ZP4 ablation impairs fertility in female rabbits. Ovulation, fertilization and in vitro development to blastocyst were not affected by ZP4 ablation. However, in vivo development is severely impaired in embryos covered by a ZP4-devoided zona, suggesting a defective ZP protective capacity in the absence of ZP4. ZP4-null ZP was significantly thinner, more permeable, and exhibited a more disorganized and fenestrated structure. The evolutionary conservation of ZP4 in other mammals, including humans, suggests that the structural properties conferred by this protein are required to ensure proper embryo sheltering during in vivo preimplantation development.