Numerical modelling of oocytes partially covered by magnetic nanoparticles in external magnetic fields

Nanorep® is a novel device based on the magnetic tweezer concept that has been developed for precise, fast, and non-aggressive oocytes and embryos manipulation. To operate the reproductive cells, they are first immersed in a standard host medium with ferrite containing nanoparticles. The magnetic particles are attached to the external matrix of the oocytes/embryos thanks to a linked protein making possible to control them by using external magnetic fields. Electron microscope photographs show magnetic particle aggregates around the oocyte. The total mass and distribution of the attached magnetic particles per oocyte show a wide range of variability, which strongly affects their magnetic response. Despite the uncertainties on the attachment of magnetic nanoparticles, the set formed by the oocyte and the nanoparticles can be caught and transported by using standard neodymium magnets. Here we present an initial study of the interaction between the set oocyte/nanoparticles and the external magnetic field in aqueous environment. We are going to discuss several relevant topics of the model such us the characterization of magnetic field produced by cylindrical neodymium magnets, the model of the ferrite magnetization as an effective spherical dipole or a magnetized spherical shell, the measurement processes to obtain the temporal evolution of the particle position and the different observed dynamic behaviours

Comparative Study of Semen Parameters and Hormone Profile in Small-Spotted Catshark (Scyliorhinus canicula): Aquarium-Housed vs. Wild-Captured

[EN] Comprehensive knowledge of chondrichthyan reproductive biology is crucial for the development of reproductive technologies. For that reason, a male reproductive evaluation was performed on the basis of a comparison of samples collected from wild-captured and aquarium-housed small-spotted catshark (Scyliorhinus canicula). Semen quality, sperm morphometry, and reproductive hormones were assessed. The results demonstrate good in vitro semen quality in aquarium-housed sharks, although there was lower plasma testosterone. Several chondrichthyan species are threatened, and we must increase our knowledge of their reproductive biology in order to establish assisted reproductive protocols for ex situ or in situ endangered species. The small-spotted catshark (Scyliorhinus canicula) is one of the most abundant shark species of the Mediterranean coast and is easy to maintain in aquaria; therefore, it is considered an ideal reproductive model. This study aimed to compare S. canicula male reproductive function in aquarium-housed (n = 7) and wild-captured animals, recently dead (n = 17). Aquarium-housed animals had lower semen volume (p = 0.005) and total sperm number (p = 0.006) than wild-captured animals, but similar sperm concentrations. In terms of sperm parameters, aquarium-housed sharks showed higher total sperm motility (p = 0.004), but no differences were observed regarding sperm viability, mitochondrial membrane potential, or membrane integrity. A morphometric study pointed to a significantly longer head (p = 0.005) and acrosome (p = 0.001) in wild-captured animals. The results of the spermatozoa morphological study of S. canicula were consistent with previous results obtained in other chondrichthyan species. With regard to sex hormones, testosterone levels were significantly lower in aquarium-housed animals (p & LE; 0.001), while similar levels of 17 beta-estradiol and progesterone were found. In short, the present study provides evidence of good in vitro semen quality in S. canicula housed in an aquarium, underlining their excellent potential for application in reproductive technologies for this and other chondrichthyan species.Muñoz-Baquero, M.; Marco-Jiménez, F.; Garcia-Domínguez, X.; Ros-Santaella, JL.; Pintus, E.; Jiménez-Movilla, M.; García-Párraga, D.... (2021). Comparative Study of Semen Parameters and Hormone Profile in Small-Spotted Catshark (Scyliorhinus canicula): Aquarium-Housed vs. Wild-Captured. Animals. 11(10):1-14. https://doi.org/10.3390/ani11102884114111

Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads

The egg is a spherical cell encapsulated by the zona pellucida (ZP) which forms a filamentous matrix composed of several glycoproteins that mediate gamete recognition at fertilization. Studies on molecular mechanisms of sperm-egg binding are limited in many mammalian species by the scarcity of eggs, by ethical concerns in harvesting eggs, and by the high cost of producing genetically modified animals. To address these limitations, we have reproduced a three-dimensional (3D) model mimicking the oocyte’s shape, by means of magnetic sepharose beads coated with recombinant ZP glycoproteins (BZP) and cumulus cells. Three preparations composed of either ZP2 (C and N-termini; BZP2), ZP3 (BZP3) or ZP4 (BZP4) were obtained and characterized by protein SDS-PAGE, immunoblot and imaging with confocal and electron microscopy. The functionality of the model was validated by adhesion of cumulus cells, the ability of the glycoprotein-beads to support spermatozoa binding and induce acrosome exocytosis. Thus, our findings document that ZP-beads provide a novel 3D tool to investigate the role of specific proteins on egg-sperm interactions becoming a relevant tool as a diagnostic predictor of mammalian sperm function once transferred to the industry.Supported by Fundación Seneca-Agencia de Ciencia y Tecnología de la Región de Murcia “Ayudas a la realización de proyectos para el desarrollo de investigación científica y técnica por grupos competitivos 2018” (20887/PI/18), MINECO and FEDER (AGL2015 70159 P), Groups and Units of Scientific Excellence of the Region of Murcia (20040/GERM/16) and the AGL2015–66341‐R from the Ministry of Economy and Competitiveness (Spain)

Estímulo del trabajo autónomo en el aprendizaje práctico de la Histología: una experiencia transversal en Ciencias de la salud

El estudio microscópico de tejidos y órganos constituye un aspecto muy importante del aprendizaje de la histología, pero no siempre se dispone de suficientes colecciones de muestras, sobre todo humanas, para que el alumnado pueda disponer de ellas de forma presencial durante las prácticas. Por ello, el portal virtual digital slidebox (DSB) permite al profesorado generar una colección de preparaciones histológicas virtuales, de manera que los estudiantes tienen acceso "online" a estas muestras y pueden visualizarlas a la misma escala que ofrece la observación directa del microscopio óptico, e incluso mayor. Para la realización de cada práctica en sus horas presenciales el alumno dispone previamente de un guión con los objetivos que debe cumplir al realizar la observación de cada preparación histológica. Al final de cada práctica presencial los objetivos son explicados por el alumno, y evaluados por el profesor, realizando una puesta en común. Una vez realizada la práctica, y en horario no presencial, el alumnado tiene la posibilidad de completar este trabajo usando el portal virtual DSB. Con las imágenes digitales captadas el alumno realiza su portafolio de prácticas incorporando dichas imágenes y rotulándolas. Al final del curso se realiza un examen práctico global y la evaluación final del portafolio

TMEM95 is a sperm membrane protein essential for mammalian fertilization.

The fusion of gamete membranes during fertilization is an essential process for sexual reproduction. Despite its importance, only three proteins are known to be indispensable for sperm-egg membrane fusion: the sperm proteins IZUMO1 and SPACA6, and the egg protein JUNO. Here we demonstrate that another sperm protein, TMEM95, is necessary for sperm-egg interaction. TMEM95 ablation in mice caused complete male-specific infertility. Sperm lacking this protein were morphologically normal exhibited normal motility, and could penetrate the zona pellucida and bind to the oolemma. However, once bound to the oolemma, TMEM95-deficient sperm were unable to fuse with the egg membrane or penetrate into the ooplasm, and fertilization could only be achieved by mechanical injection of one sperm into the ooplasm, thereby bypassing membrane fusion. These data demonstrate that TMEM95 is essential for mammalian fertilization. © 2020, Lamas-Toranzo et al

ODS en las aulas de ingeniería y arquitectura: tomar conciencia y transmitir

Memoria ID-014. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2021-2022

Análisis de la composición, estructura y función de las glicoproteínas de la zona pelúcida con especial referencia a la especie humana

En la presente tesis doctoral se han estudiados diferentes aspectos relacionados con la composición glucídica, estructura, formación y origen de la zona pelúcida y las glicoproteínas que la forman, poniendo especial interés en la especie humana. Para este propósito hemos desarrollado tres líneas principales de investigación, estudio ultraestructural de la zona pelúcida y gránulos corticales de ovocitos humanos y de la síntesis de las glicoproteínas de la ZP humana y de ratón, análisis bioquímico y biofísico de las glicoproteínas de la zona pelúcida de hámster y análisis bioquímico y fisiológico de ZP2 y ZP3 recombinantes humanas expresadas en células CHO. In the present thesis, several aspects of the carbohydrate content, structure, formation and origin of the ZP, with special attention to the human ZP, have been analyzed. Therefore, the methods and techniques used in the present thesis included the ultrasctuctural study of human ZP, cortical granules and biosynthetic pathway of the human and mouse ZP, biochemical and cytochemical characterization of the hamster zona pellucid and characterization of recombinant human ZP2 y ZP3 expressed in CHO cells

Proteínas de membrana esenciales para la fecundación. Aplicación en microesferas y modelos tridimensionales

Para generar un individuo nuevo, único y diploide con la capacidad totipotente de desarrollarse como un organismo específico de la especie, dos células haploides altamente diferenciadas de cada progenitor, el óvulo y el espermatozoide, tienen que reconocerse, unirse y fusionarse. Este evento, denominado fecundación, da como resultado la generación de un cigoto e involucra varias etapas en las que diferentes procesos moleculares deben desencadenar la actividad celular de los gametos necesaria para cumplir este proceso. De modo que, una vez que el espermatozoide ha alcanzado al óvulo, el primer contacto para el reconocimiento de gametos está mediado por la matriz extracelular que los rodea, denominada zona pelúcida (ZP). El contacto inicial asegura el reconocimiento de gametos específicos de la especie y desencadena una cascada de reacciones en los espermatozoides necesarios para penetrar a través de ZP y alcanzar así el espacio perivitelino para encontrarse con la membrana del óvulo, conocida como oolema. Para unirse y fusionarse, las membranas de ambos gametos deben adherirse, y las proteínas de la membrana en cada una de las células deben interaccionar adecuadamente entre sí. Finalmente, deben ocurrir una serie de eventos moleculares en la membrana para que las dos células se fusionen y compartan su contenido celular. Todos estos fenómenos requieren la participación de moléculas específicas y la activación de cascadas moleculares que coordinan adecuadamente todos los procesos celulares que implican el reconocimiento y la fusión de gametos

Effects of recombinant OVGP1 protein on <i>in vitro</i> bovine embryo development

Previously, our group demonstrated that recombinant porcine oviductin (pOVGP1) binds to the zona pellucida (ZP) of in vitro-matured (IVM) porcine oocytes with a positive effect on in vitro fertilization (IVF). The fact that pOVGP1 was detected inside IVM oocytes suggested that this protein had a biological role during embryo development. The aim of this study was to evaluate the effects of pOVGP1 on bovine in vitro embryo development. We applied 10 or 50 µg/ml of pOVGP1 during IVF, embryonic in vitro culture (IVC), or both, to evaluate cleavage and embryo development. Blastocyst quality was assessed by analyzing the expression of important developmental genes and the survival rates after vitrification/warming. pOVGP1 was detected in the ZP, perivitelline space, and plasma membrane of blastocysts. No significant differences (P > 0.05) were found in cleavage or blastocyst yield when 10 or 50 µg/ml of pOVGP1 was used during IVF or IVC. However, when 50 µg/ml pOVGP1 was used during IVF + IVC, the number of blastocysts obtained was half that obtained with the control and 10 µg/ml pOVGP1 groups. The survival rates after vitrification/warming of expanded blastocysts cultured with pOVGP1 showed no significant differences between groups (P > 0.05). The use of pOVGP1 during IVF, IVC, or both, increased the relative abundance of mRNA of DSC2, ATF4, AQP3, and DNMT3A, the marker-genes of embryo quality. In conclusion, the use of pOVGP1 during bovine embryo in vitro culture does not affect embryo developmental rates but produces embryos of better quality in terms of the relative abundance of specific genes