Moros Nicolás, Carla

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Moros Nicolás, Carla

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Moros Nicolás, Carla

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Universidad de Murcia. Departamento de Biología Celular e Histología

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Open Access
Highlight of new agents inducing capacitation-related changes in stallion spermatozoa
(2018) Accogli, Gianluca; Magistrini, Michèle; Le Vern, Yves; Sausset, Alix; Desantis, Salvatore; Goudet, Ghylène; Douet, Cécile; Moros Nicolás, Carla; Biología Celular e Histología
Open Access
Effect of cumulus cell removal and sperm pre‐incubation with progesterone on in vitro fertilization of equine gametes in the presence of oviductal fluid or cells
(2019-05-30) Douet, C; Reigner, F; Goudet, G; Moros Nicolás, Carla; Biología Celular e Histología
In spite of many attempts to establish an in vitro fertilization (IVF) technique in the equine, no efficient conventional IVF technique is available. The presence of oviductal fluid or oviductal cells during IVF help to improve embryo production in vitro but is not sufficient to reach high fertilization rates. Thus, our aim was to perform equine IVF either after sperm preincubation with oviductal fluid or in the presence of oviductal cells, and to evaluate the effect of cumulus removal from the oocyte or sperm preincubation with progesterone. In experiment 1 and 2, IVF was performed in the presence of porcine oviduct epithelial cells. The removal of cumulus cells from equine oocytes after in vitro maturation tended to increase the percentage of fertilization when fresh sperm was used (1/33 vs 4/31, p > 0.05) but had no effect when frozen sperm was used (1/32 vs 1/32). Equine sperm preincubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/14 vs 2/18 for fresh, 1/29 vs 1/25 for frozen). In experiment 3 and 4, IVF was performed after preincubation of sperm with porcine oviductal fluid. The removal of cumulus cells tented to increase the percentage of fertilization when fresh sperm was used (1/24 vs 3/26, p>0.05). Sperm preincubation with progesterone did not significantly influence the fertilization rate when fresh or frozen sperm was used (2/39 vs 2/36 for fresh, 2/37 vs 1/46 for frozen), but two 3-4 cells stage zygotes were obtained with fresh sperm preincubated with progesterone. This is an encouraging result for the setting up of an efficient IVF procedure in equine.
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
Relative transcript abundance in porcine cumulus cells collected from different sized follicles
(2020) Li, Y.; González-Brusi, L.; Romar, R.; Funahashi, H.; Izquierdo Rico, María José; Moros Nicolás, Carla; Biología Celular e Histología
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
Open Access
Comparative View on the Oviductal Environment during the Periconception Period.
(2020) González-Brusi, Leopoldo; Algarra, Blanca; Izquierdo Rico, María José; Avilés Sánchez, Manuel; Jíménez Movilla, María; Moros Nicolás, Carla; Biología Celular e Histología
The oviduct plays important roles in reproductive events: sperm reservoir formation, final gamete maturation, fertilization and early embryo development. It is well known that the oviductal environment affects gametes and embryos and, ultimately, the health of offspring, so that in vivo embryos are better in terms of morphology, cryotolerance, pregnancy rates or epigenetic profile than those obtained in vitro. The deciphering of embryo–maternal interaction in the oviduct may provide a better understanding of the embryo needs during the periconception period to improve reproductive efficiency. Here, we perform a comparative analysis among species of oviductal gene expression related to embryonic development during its journey through the oviduct, as described to date. Cross-talk communication between the oviduct environment and embryo will be studied by analyses of the secreted or exosomal proteins of the oviduct and the presence of receptors in the membrane of the embryo blastomeres. Finally, we review the data that are available to date on the expression and characterization of the most abundant protein in the oviduct, oviductin (OVGP1), highlighting its fundamental role in fertilization and embryonic development.
Open Access
Análisis molecular, proteómico y filogenético de lazona pelúcida de mamíferos
(2015-03-13) Moros Nicolás, Carla; Chevret, Pascale; Avilés Sánchez, Manuel; Izquierdo Rico, María José; Facultad de Veterinaria
INTRODUCCIÓN La zona pelúcida (ZP) es una matriz traslúcida, glicoproteica y acelular que rodea los ovocitos de los mamíferos implicada en importantes procesos durante la fecundación. Se ha considerado que la ZP estaba constituida por tres glicoproteínas (ZP1, ZP2 y ZP3) teniendo en cuenta estudios realizados en el ratón. Sin embargo, estudios posteriores demostraron que la ZP de cerda y de vaca también presentaba tres glicoproteínas, pero no estando presente en estos casos ZP1, siendo la composición ZP2, ZP3 y ZP4. Además, en estos últimos años se ha descrito que diferentes especies presentan cuatro glicoproteínas en su ZP, como es el caso de la mujer, la rata, el macaco coronado, el hámster, la coneja y la gata. OBJETIVOS: En la presente Tesis Doctoral ahondamos en la composición de la ZP en varios grupos de mamíferos: marsupiales, roedores y carnívoros. Estudiamos la evolución de los distintos genes y se analiza el comportamiento de la ZP de varias especies con diferente composición proteica mediante estudios funcionales como la fecundación in vitro (FIV) y la digestión de la ZP mediante proteasas. METODOLOGÍA: En este estudio realizamos un análisis in silico de las distintas especies de marsupiales y carnívoros disponibles en distintas bases de datos (Ensembl y Pubmed); así como un análisis molecular en un marsupial australiano (wallaby de Bennett y koala), un marsupial sudamericano (zarigüeya común), tres roedores (Mastomys coucha, Mus mattheyi y Mus pahari) y dos carnívoros (perro y zorro). Para ello, se aisló ARNm de ovarios de las distintas especies con el que se sintetizó ADNc y en otros casos se obtuvo ADNg a partir de tejidos. El ADN se empleó en las amplificaciones por PCR usando cebadores diseñados a partir de las secuencias de las distintas ZPs. A continuación, realizamos análisis filogenéticos a partir de alineamientos múltiples de las secuencias empleando SEAVIEW. El modelo de evolución se eligió empleando iMOLDETEST y el árbol se construyó usando el método de máxima verosimilitud mediante el programa PHYLM. Por otro lado, se realizó un análisis por proteómica en los roedores analizados para determinar la composición proteica de su ZP. Por último, se realizaron análisis funcionales determinando el tiempo de digestión completa de la ZP usando tripsina en roedores y se realizan ensayos de FIV homólogos y heterólogos empleando roedores de diferentes especies. RESULTADOS Y CONCLUSIONES: Marsupiales: La composición de la ZP es diferente en los marsupiales australianos y sudamericanos, pudiendo estar formada por 4 a 6 glicoproteínas. En el orden Didelphimorphia de los marsupiales sudamericanos, la ZP podría estar formada por ZP1, ZP2, ZP3-b y ZP3-c y en los marsupiales australianos podría estar formada por ZP1, ZP2, ZP3-a, ZP3-b, ZP3-c y ZP4. Detectándose pseudogenización del gen ZP4 en los marsupiales del orden Didelphimorphia además de en dos xenartros, el armadillo y el perezoso. Roedores: En el ovario de Mastomys coucha, Mus mattheyi y Mus pahari se expresa el ARNm de ZP1, ZP2, ZP3 y ZP4 demostrándose la presencia de estas proteínas mediante análisis proteómico. La pseudogenización de ZP4 en la subfamilia Murinae se estima que ocurrió hace 5-7 millones de años, afectando únicamente al subgénero Mus. La ZP de ratonas con 4 glicoproteínas se digiere antes que la ZP de ratonas con 3 glicoproteínas y los espermatozoides de ratones con 3 proteínas en su ZP pueden fecundar el ovocito de ratones con 4 proteínas en su ZP. Carnívoros: En el ovario de la hurona se expresa el ARNm de ZP1, ZP2, ZP3 y ZP4, mientras que en el ovario de la zorra se expresa el ARNm de ZP2, ZP3 y ZP4, estando ZP1 pseudogenizado. La pseudogenización de ZP1 parece afectar únicamente a la familia Canidae. INTRODUCTION: The zona pellucida (ZP), a translucent, glycoproteic and acellular matrix that surrounds the mammalian oocytes, is involved in important steps during fertilization. Based on studies in mouse, it was long considered that the ZP is formed of three glycoproteins (ZP1, ZP2 and ZP3). However, later studies demonstrated that pig and cow ZP is also formed of three glycoproteins, although ZP1 is not present in these cases, the composition being ZP2, ZP3 and ZP4. Furthermore, in more recent years other species have been found to contain four glycoproteins in their ZP, e.g. human, bonnet macaque, hamster, rabbit and cat. OBJECTIVES: In the present thesis we take a deep look at the ZP composition in placental mammals (carnivores and rodents) and in metatherian mammals (marsupials). We also study the evolution of the different ZP genes and analyze the ZP behaviour in several species of different protein composition by means of functional studies, such as in vitro fertilization (IVF) and ZP digestion with proteases. METHODS: An in silico analysis was made of the marsupial and carnivore species for which information is available in the databases Ensembl and Pubmed. In addition we made a molecular analysis of two Australasian marsupials (Bennett’s wallaby and koala), one South American marsupial (Common opossum), three mice (Mastomys coucha, Mus mattheyi and Mus pahari) and two carnivores (dog and fox). For this, mRNA was isolated from the ovaries and cDNA was synthesized; in other cases, gDNA was obtained from tissues. The DNA was used in PCR amplifications and primers were designed according to the different ZP sequences. Next, a phylogenetic analysis was made using SEAVIEW from multiple alignments of the obtained sequences. The evolution model was chosen using iMOLDETEST and the phylogenetic tree was constructed using the maximum likelihood method with the PHYLM program. In addition, a proteomic analysis was conducted in the studied mice to determine the protein composition of their ZP. Finally, functional analyses were made, calculating ZP digestion time using tripsine and doing homologous and heterologous IVF analysis using different mice species. RESULTS AND CONCLUSIONS: Marsupials: ZP composition differs between Australasian and South American marsupials; since it may be formed of four to six glycoproteins. In the South American order Didelphimorphia, the ZP is probably formed of four glycoproteins (ZP1, ZP2, ZP3-b and ZP3-c), whilst in Australasian marsupials the ZP is probably formed of six proteins (ZP1, ZP2, ZP3-a, ZP3-b, ZP3-c and ZP4). A ZP4 pseudogenization was detected in the Didelphimorphia order of marsupials and also in two xenarthrans, the armadillo and the tree sloth. Rodents: In the ovaries of Mastomys coucha, Mus mattheyi and Mus pahari, the mRNA of ZP1, ZP2, ZP3 and ZP4 is expressed and the presence of these proteins was demonstrated by proteomic analysis. The pseudogenization of ZP4 in the Murinae subfamily is estimated to date from around 5-7 million years, affecting only the subgenus Mus. The digestion of ZP from mice with 4 glycoproteins occurs before than the digestion from mice with 3 glycoproteins. Furthermore, mice with 3 ZP proteins can fertilize mice with 4 ZP proteins. Carnivores: The mRNA of ZP1, ZP2, ZP3 and ZP4 is expressed in ferret ovaries, whilst in fox the mRNA of ZP2, ZP3 and ZP4 is expressed, ZP1 being a pseudogene. Such ZP1 pseudogenization seems to only affect the Canidae family.
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Papel de la zona pelúcida en la fecundación
(Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, 2017) Algarra, B.; Ballesta, J.; Izquierdo Rico, María José; Avilés Sánchez, Manuel; Jíménez Movilla, María; Moros Nicolás, Carla; Biología Celular e Histología