Análisis molecular, proteómico y filogenético de lazona pelúcida de mamíferos

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

Collecte in vivo d’ovocytes d’ânesses et chronologie de la maturation in vitro

peer reviewedLa plupart des espèces d’ânes sauvages et domestiques est en danger d’extinction. Leur préservation nécessite la cryoconservation du sperme, des ovocytes et des embryons. L’objectif de cette étude était de mettre au point une technique de collecte in vivo et de maturation in vitro des ovocytes d’ânesses. Les ovocytes ont été collectés par ponction folliculaire transvaginale sous guide échographique, placés dans un milieu de maturation in vitro pendant 24, 30, 34 ou 38 heures puis analysés. Quatre-vingt-douze ovocytes ont été collectés dans 193 follicules (48%) avec une moyenne de 4,2 ovocytes par ânesse. Après 34 heures de maturation in vitro, 45% des ovocytes ont été matures. Pour la première fois une technique de collecte in vivo et de maturation in vitro d’ovocytes d’ânesses a été développée. Cette technique permet d’envisager la cryoconservation et la fécondation in vitro de ces ovocytes

New Insights into the Mammalian Egg Zona Pellucida

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Analysis of ZP1 gene reveals differences in zona pellucida composition in carnivores

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ZP4 Is Present in Murine Zona Pellucida and Is Not Responsible for the Specific Gamete Interaction

International audienceMammalian eggs are surrounded by an extracellular matrix called the zona pellucida (ZP). This envelope participates in processes such as acrosome reaction induction, sperm binding, protection of the oviductal embryo, and may be involved in speciation. In eutherian mammals, this coat is formed of three or four glycoproteins (ZP1–ZP4). While Mus musculus has been used as a model to study the ZP for more than 35 years, surprisingly, it is the only eutherian species in which the ZP is formed of three glycoproteins Zp1, Zp2, and Zp3, Zp4 being a pseudogene. Zp4 was lost in the Mus lineage after it diverged from Rattus , although it is not known when precisely this loss occurred. In this work, the status of Zp4 in several murine rodents was tested by phylogenetic, molecular, and proteomic analyses. Additionally, assays of cross in vitro fertilization between three and four ZP rodents were performed to test the effect of the presence of Zp4 in murine ZP and its possible involvement in reproductive isolation. Our results showed that Zp4 pseudogenization is restricted to the subgenus Mus , which diverged around 6 MYA. Heterologous in vitro fertilization assays demonstrate that a ZP formed of four glycoproteins is not a barrier for the spermatozoa of species with a ZP formed of three glycoproteins. This study identifies the existence of several mouse species with four ZPs that can be considered suitable for use as an experimental animal model to understand the structural and functional roles of the four ZP proteins in other species, including human

Composition of marsupial zona pellucida: a molecular and phylogenetic approach

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Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species

Remerciements : Plate-formes CIRE et PIC, INRA, UMR PRC Physiologie de la Reproduction et des Comportements, 37380 NouzillyOviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization

Evidence of haptoglobin in the porcine female genital tract during oestrous cycle and its effect on in vitro embryo production

Abstract Recent evidence supports involvement of the acute phase protein haptoglobin in numerous events during mammalian reproduction. The present study represents an in-depth investigation of haptoglobin expression and secretion in the porcine oviduct and uterus, and assesses its effect on porcine in vitro embryo production. A systematic study was made of sows in different oestrous stages: late follicular, early luteal and late luteal stages. Relative haptoglobin mRNA abundance was quantified by RT-qPCR. In addition, expression of the protein was analysed by immunohistochemistry and the results were complemented by Western-blot and proteomic analyses of the oviductal and uterine fluids. In vitro porcine fertilization and embryo culture were carried out in the presence of haptoglobin. The results indicate that haptoglobin mRNA expression in the porcine oviduct and uterus is most abundant during the late luteal stage of the oestrous cycle. By means of Western blot and proteomic analyses haptoglobin presence was demonstrated in the oviduct epithelium and in the oviductal and uterine fluids in different stages of the oestrous cycle. The addition of haptoglobin during gamete co-incubation had no effect on sperm penetration, monospermy or efficiency rates; however, compared with the control group, blastocyst development was significantly improved when haptoglobin was present (haptoglobin: 64.50% vs. control: 37.83%; p < 0.05). In conclusion, the presence of haptoglobin in the oviduct and uterus of sows at different stages of the oestrous cycle suggests that it plays an important role in the reproduction process. The addition of haptoglobin during in vitro embryo production improved the blastocyst rates