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Unraveling the Sperm Transcriptome by Next Generation Sequencing and the Global Epigenetic Landscape in Infertile Men Fadi Choucair

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Unraveling the Sperm Transcriptome by Next Generation Sequencing and the Global Epigenetic Landscape in Infertile Men Fadi Choucair Unraveling the sperm transcriptome by next generation sequencing and the global epigenetic landscape in infertile men Fadi Choucair To cite this version: Fadi Choucair. Unraveling the sperm transcriptome by next generation sequencing and the global epigenetic landscape in infertile men. Molecular biology. Université Côte d’Azur; Université libanaise, 2018. English. NNT : 2018AZUR4058. tel-01958881 HAL Id: tel-01958881 https://tel.archives-ouvertes.fr/tel-01958881 Submitted on 18 Dec 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE DE DOCTORAT Exploration du transcriptome spermatique par le séquençage nouvelle génération et le portrait épigénétique de l’infertilité masculine Unraveling the sperm transcriptome by next generation sequencing and the global epigenetic landscape in infertile men Fadi CHOUCAIR INSERM U1065, C3M Présentée en vue de l’obtention Devant le jury, composé de : du grade de docteur en interactions Mme RACHEL LEVY, PR, UMRS 938, UPMC M. FABIEN MONGELARD, MC, CRCL, ENS Lyon moléculaires et cellulaires Mme NINA SAADALLAH-ZEIDAN, PR, Université Libanaise de l’Université Côte d’Azur Mme Anne-Amandine CHASSOT, CR, iBV, Université de et de l’Université Libanaise Nice Mme VALÉRIE GRANDJEAN, CR, C3M, Université de Nice Dirigée par : Valérie Grandjean / Mira Mme MIRA HAZZOURI, PR, Université Libanaise Hazzouri Soutenue le : 6 Septembre 2018 ii Exploration du transcriptome spermatique par le séquençage nouvelle génération et le portrait épigénétique de l’infertilité masculine Jury Président du Jury Mme Anne-Amandine CHASSOT, CR, iBV, Université de Nice Rapporteurs Mme RACHEL LEVY, PR, UMRS 938, UPMC M. FABIEN MONGELARD, MC, CRCL, ENS Lyon Examinateurs Mme NINA SAADALLAH-ZEIDAN, PR, Université Libanaise Mme VALÉRIE GRANDJEAN, CR, C3M, Université de Nice Mme MIRA HAZZOURI, PR,Université Libanaise Invités M. IMAD ABOU JAOUDE, M.D., Hôpital Abou Jaoude, Liban M. JOHNNY AWWAD, M.D., PR, American University of Beirut Exploration du transcriptome spermatique par le séquençage nouvelle génération et le portrait épigénétique de l’infertilité masculine Résumé L’infertilité masculine est actuellement considérée comme un problème majeur qui pose une situation alarmante sur la santé publique. L’oligozoospermie, l’asthénozoospermie et la tératozoospermie sont les trois anomalies les plus connues des spermatozoïdes. Elles affectent, respectivement, la densité, la motilité et la morphologie des spermatozoïdes. Un spermatozoïde anormal est très souvent corrélé à des altérations génétiques et épigénétiques qui peuvent affecter considérablement le transcriptome. Dans ce sens, le sequençage aléatoire du transcriptome entier des spermatozoïdes ou RNA-seq constitue un outil puissant pour caractériser ces maladies. Jusqu’à présent, il n’existe aucune étude exploitant des données RNA-seq chez des hommes présentant de telles anomalies spermatiques. L’objectif principal de notre étude fût d’identifier des profils distincts des modifications du transcriptome de chaque phénotype d’infertilité pour ainsi révéler des gènes-signatures qui tamponnent une spermatogenèse pathologique Pour ce faire, les transcriptomes des spermatozoïdes de 60 sujets infertiles atteints soit d’oligozoospermie, d’asthénozoospermie ou de tératozoospermie ont été comparés à ceux de 20 patients fertiles. Ces analyses supervisées nous ont conduit à identifier: (i) les gènes clés spécifiques aux différentes anomalies des spermatozoïdes (ii) les voies de signalisation associées, (ii) les différents longs ARNs non codants dérégulés dans ces anomalies. Au niveau de l’oligozoospermie, les transcrits de spermatozoïdes dérégulés étaient associées à divers stades de la spermatogenèse, y compris le cycle cellulaire méiotique, l’assemblage du complexe synaptonémal, la cohésion des chromatides sœurs, les processus métaboliques de piRNA, le processus catabolique protéique dépendant de la voie de l’ubiquitine, à la réponse aux dommages de l'ADN et particulièrement le processus de fécondation. Quant à l’asthenozoospermia, la spermatogenèse, l’assemblage du cil, des voies métaboliques reliées à la spermatogenèse, la chimiotaxie et la physiologie des cellules immunitaires ont été significativement dérégulés. De plus, ce qui nous a intéressé au plus était l’analyse des transcrits sous-exprimés qui a permis l’identification de nombreux transcrits associées aux modifications des histones. Nous avons aussi mis en évidence une sous expression des gènes différentiellement exprimés qui définit la tératozoospermie. Cette sous expression est associée au système ubiquitine-protéasome, à l’organisation du cytosquelette, au cycle cellulaire, à la SUMOylation en réponse aux dommages de l'ADN et aux protéines de réparation ainsi qu’à de nombreux modulateurs épigénétiques. Les gènes signature de l'oligozoospermie ont été liés au processus de fécondation et les composants de la matrice extracellulaire, tandis que ceux de la tératozoospermie sont liés à la spermatogenèse et la morphogenèse cellulaire, alors que les gènes signature de l'asthénozoospermie sont impliqués dans l'assemblage du ribosome et du flagelle. En complément de cette étude, nous avons réalisé une étude très globale du paysage épigénétique du sperme des hommes infertiles. Nous avons, ainsi comparé les niveaux des espèces réactives de l’oxygène (ERO), de méthylation de l’ADN, ainsi que l’intégrité de la chromatine dans les spermatozoïdes de 30 individus infertiles avec ceux de 33 individus fertiles. Nos analyses montrent des niveaux élevés d’espèces réactives de l’oxygène chez les individus infertiles. Ces niveaux sont d’une part négativement corrélés avec les niveaux de méthylation globale de l’ADN et d’autre part négativement corrélés avec ceux de la 5-hydroxyméthylcytosine et de la 5-formylcytosine (intermédiaire dans le processus de déméthylation active). Ces derniers suggèrent qu’une infertilité associée au stress oxydatif conditionne l’épigénome du sperme. En conclusion, l’ensemble de notre travail apporte des ressources précieuses et originales dans la compréhension des pathologies de sperme. Mots clés : spermatozoïdes ; infertilité masculine ; transcriptomique ; RNA-seq ; épigénome. ii Unraveling the sperm transcriptome by next generation sequencing and the global epigenetic landscape in infertile men Abstract Male infertility is actually considered as a public alarming health problem. The sperm pathologies spectrum ranges between different phenotypes including oligozoospermia, asthenozoospermia and teratozoospermia depending on the sperm conventional parameters abnormalities. Abnormal sperm is characterized by genetic alterations and epigenetic alterations which can affect the transcriptome extensively. These alterations in RNA profiles are retrospectively indicative of aberrant spermatogenic events. RNA-seq is a powerful tool for comprehensive characterization of whole transcriptome. To date, RNA-seq analysis of sperm from infertile men has not been reported. Our objectives are: (i) recognize key clusters, key pathways and specific gene transcripts for different sperm abnormalities; (ii) catalog the spermatozoal lncRNAs in different sperm pathologies; (iii) identify signature genes which are mechanistically important in the cascade of events driving a pathological spermatogenesis; (iii) portray the global epigenetic landscape in sperm from infertile men. Expression data from 60 sperm samples from 3 groups of infertile men (oligozoospermia, asthenozoospermia, and teratozoospermia) were generated on Illumina HiSeq platform, compared to 20 fertiles, and the resulting gene expression patterns were analyzed for functional enrichment. Our supervised analyses identified numerous differentially expressed genes between fertile and infertile men. In oligozoospermia, the deregulated spermatozoal transcripts were associated with various stages of spermatogenesis including meiotic cell cycle, synaptonemal complex assembly, sister chromatid cohesion, piRNA metabolic process, ubiquitin-dependent protein catabolic process, cellular response to DNA damage stimulus and interestingly fertilization. As for asthenozoospermia, spermatogenesis, cilium assembly, metabolic-related pathways, chemotaxis and immune cell physiology were most significantly differentially expressed. Interestingly, numerous transcripts associated with histone modifications were highly down-regulated. With regards to teratozoospermia, we evidenced sperm-specific differentially expressed genes which are involved in the ubiquitin-proteasome, cytoskeleton organization, the cell cycle pathway, SUMOylation of DNA damage response and repair proteins, as well as many putative epigenetic modulators of gene expression.. We also attempted to identify distinct patterns of gene expression changes that were definite to the different abnormal sperm phenotypes in infertile men relative to controls. Signature genes of oligozoospermia were over-enriched by genes involved in fertilization and extracellular matrix components, while signature genes of teratozoospermia were enriched by genes involved
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