Publication : Traduction des ARNM maternels d'ovocytes bovins impliqués dans le développement embryonnaire
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2006
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Richard, François
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Durant sa croissance, l’ovule entrepose des organelles, des protéines et des ARNm dits maternels qui permettront de supporter les premières étapes du développement embryonnaire dans le but primordial d’activer le génome embryonnaire. Un ovocyte capable de supporter cette étape est dit compétent au développement embryonnaire. Le premier objectif avait pour but de déterminer des populations d’embryons ayant différents niveaux de compétence au développement. Le développement d’une nouvelle approche, la triple sélection consiste à utiliser en même temps deux caractères reliés à l’ovule tels que la taille d’origine de son follicule et la morphologie de son complexe cumulus-ovocyte, et un caractère relié à l’embryon, soit le temps du premier clivage. Les différentes populations générées par la sélection permettront ainsi la recherche des facteurs de compétence au développement. Le deuxième objectif avait pour but de connaître le protéome traduit de la traduction des ARNm maternels lors du développement embryonnaire, ainsi que de connaître les besoins constants en protéines de l’embryon, mais également de l’ovocyte, soit les protéines "housekeeping" maternels. Cette expérience a démontré clairement que suite à la reprise de la méiose (Coenen et al., 2004) et ensuite de la fécondation, la traduction des ARNm maternels présente un continuum d’évènements qui mène à l’activation du génome embryonnaire. Malgré un réservoir commun d’ARNm, les besoins lors de la maturation de l’ovocyte et lors du développement de l’embryon sont loin d’être les mêmes. Seulement une cinquantaine de protéines sont communément traduites lors de ces deux évènements cellulaires. Onze de ces protéines ont été identifiées : les protéines de choc thermique 71 et 70, la cyclophiline A (2 fois), la glutathione-S-transférase mu 5, la protéine épithéliale liant les acides gras, la 2,3-biphosglycérate mutase, la sous-unité epsilon TCP-1 de la chaperonine, l’ubiquitine carboxyl-terminal hydrolase isozyme L1, l’enzyme conjuguant l’ubiquitine E2D3 et l’isoforme alpha et gamma de l’actine. Le troisième objectif avait pour but de déterminer des facteurs de compétence au niveau de l’embryon à 2-cellules basé sur les populations observées lors de la triple sélection. Nous ii avons compris la traduction des ARNm maternels dans les embryons incompétents au développement. Ces derniers présentent une traduction basale des ARNm maternels, tandis que les plus compétents présentent bon nombre de protéines exclusives qui représentent des facteurs de compétence potentiels. Afin d’identifier ces facteurs de compétence, nous avons pu observer que les protéines traduites lors du développement embryonnaire ne sont que transitoires, observation supportée également par la seconde expérience. Les protéines ne s’accumulent pas dans le cytoplasme rendant ainsi leur identification très difficile. Malgré tout, six protéines ont été identifiées: la thiolase cytosolique, l’isocitrate déshydrogénase, la peroxiredoxine 6, la sous-unité alpha du proteasome-6 et la thiamine triphosphatase. Deux protéines « housekeeping » d’origine maternelle découvertes durant l’expérience précédente sont également moins présentes dans les embryons non compétents. De ces expériences de protéomique a découlé une nouvelle méthode de confirmation de l’identification des protéines, la confirmation in silico. Le génome et le protéome de Bos taurus étant limité, l’identification de nos protéines a souvent été faite soit chez d’autres espèces ou uniquement à partir de fragments d’ADN des banques de EST de Bos taurus. Cette méthode permet donc de reconstruire le messager bovin, d’en traduire la protéine bovine, d’identifier le patron peptidique théorique et de le comparer à nos résultats de séquençage, augmentant ainsi le recouvrement de notre protéine. En conclusion, la protéomique de la compétence au développement de l’ovocyte bovin a permis l’identification de candidats qui comparativement à la génomique sont plus restreints. Une nouvelle méthode a découlé de ces expériences. La confirmation in silico permet maintenant la confirmation des protéines identifiées sans utilisation de matériel supplémentaire.
During the growth and the maturation of the oocyte, organelles, proteins and mRNA known as maternal are stockpiled in order to prepare and supply material to the developing embryo in the main goal to activate the embryonic genome. An oocyte that is able to supply to this step is called developmental competent. The objective of the first experiment was to determine an embryos population with different level of developmental competence. The triple selection lies in two characters related to the oocyte, which are the follicular size and the morphology of the COC and one is related to the embryo, which is the time of first cleavage post insemination. The different population generated by this selection has provided a population highly competent to development and a population lacking developmental competence that will be useful for the study of developmental competence. The objective of the second experiment was the study of the proteomic of the translated maternal mRNA during the early embryo development. We wanted to know which proteins play the role of maternal housekeeping proteins during oocyte maturation and early embryo development. This experiment has clearly demonstrated that when the oocyte resume meiosis (Coenen et al., 2004) and after fertilization, the translated protein pattern follows a continuum events which conducts embryo to his activation. Although they are supply by the same reservoir of mRNA, the needs of the oocyte and the embryo are different. Only about fifty proteins are commonly translated during oocyte maturation and early development. Eleven of them have been identified: HSC71; HSP70; CypA (2 times); UCHL1; GSTM5; Cct5; E-FABP; 2,3-BPGM, ubiquitin-conjugating enzyme E2D3; and β- actin/γ-actin. The third experiment consists of the discovery of factors related to developmental competence. Three populations from the triple selection with high, low and developmental competence was compared by a proteomic study. This study has shown that developmental incompetent embryos translate a minimum of maternal mRNA, which the majority is the embryo housekeeping found in the previous study. Whereas high and low developmental iv competent embryos present shared and exclusive translated proteins, these proteins represent potential factors related to developmental competence. By trying to identify these proteins, we have found that most translated proteins are transient and are not accumulated in the cytoplasm. In spite of this observation, we have identified six proteins: cytosolic thiolase, isocitrate dehydrogenase 1, peroxiredoxin 6, proteasome-α6, hypothetical protein My027 and thiamine triphosphatase. Two maternal housekeeping proteins were also missing for the incompetent 2-cell embryos: UCH-L1 and CypA. From these experiments, a new method of protein identification confirmation, called in silico confirmation, was developed. As the genome and the proteome of Bos taurus is limited, the protein identification was made in foreign species or from Bos taurus EST databases. This method as made possible to rebuilt the bos taurus messager, to translate the bovine protein, find his theoretical peptide mass fingerprint and compare it to the experimental spectrum and thus increase the number of matched peptides. In conclusion, the proteomic study on the developmental competence of the bovine oocyte has identified of a reduce number of candidates compared to genomic studies. A new method has been developed during this work. The in silico confirmation allowed the confirmation of protein identification without additional material.
During the growth and the maturation of the oocyte, organelles, proteins and mRNA known as maternal are stockpiled in order to prepare and supply material to the developing embryo in the main goal to activate the embryonic genome. An oocyte that is able to supply to this step is called developmental competent. The objective of the first experiment was to determine an embryos population with different level of developmental competence. The triple selection lies in two characters related to the oocyte, which are the follicular size and the morphology of the COC and one is related to the embryo, which is the time of first cleavage post insemination. The different population generated by this selection has provided a population highly competent to development and a population lacking developmental competence that will be useful for the study of developmental competence. The objective of the second experiment was the study of the proteomic of the translated maternal mRNA during the early embryo development. We wanted to know which proteins play the role of maternal housekeeping proteins during oocyte maturation and early embryo development. This experiment has clearly demonstrated that when the oocyte resume meiosis (Coenen et al., 2004) and after fertilization, the translated protein pattern follows a continuum events which conducts embryo to his activation. Although they are supply by the same reservoir of mRNA, the needs of the oocyte and the embryo are different. Only about fifty proteins are commonly translated during oocyte maturation and early development. Eleven of them have been identified: HSC71; HSP70; CypA (2 times); UCHL1; GSTM5; Cct5; E-FABP; 2,3-BPGM, ubiquitin-conjugating enzyme E2D3; and β- actin/γ-actin. The third experiment consists of the discovery of factors related to developmental competence. Three populations from the triple selection with high, low and developmental competence was compared by a proteomic study. This study has shown that developmental incompetent embryos translate a minimum of maternal mRNA, which the majority is the embryo housekeeping found in the previous study. Whereas high and low developmental iv competent embryos present shared and exclusive translated proteins, these proteins represent potential factors related to developmental competence. By trying to identify these proteins, we have found that most translated proteins are transient and are not accumulated in the cytoplasm. In spite of this observation, we have identified six proteins: cytosolic thiolase, isocitrate dehydrogenase 1, peroxiredoxin 6, proteasome-α6, hypothetical protein My027 and thiamine triphosphatase. Two maternal housekeeping proteins were also missing for the incompetent 2-cell embryos: UCH-L1 and CypA. From these experiments, a new method of protein identification confirmation, called in silico confirmation, was developed. As the genome and the proteome of Bos taurus is limited, the protein identification was made in foreign species or from Bos taurus EST databases. This method as made possible to rebuilt the bos taurus messager, to translate the bovine protein, find his theoretical peptide mass fingerprint and compare it to the experimental spectrum and thus increase the number of matched peptides. In conclusion, the proteomic study on the developmental competence of the bovine oocyte has identified of a reduce number of candidates compared to genomic studies. A new method has been developed during this work. The in silico confirmation allowed the confirmation of protein identification without additional material.
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thèse de doctorat