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Oocyte Intercommunication

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Oocyte Intercommunication Identification of large molecule transfer in cumulus cell - oocyte intercommunication Thèse Angus Macaulay Doctorat en sciences animales Philosophiae doctor (Ph.D.) Québec, Canada © Angus Macaulay, 2015 Résumé Mes travaux explorent, chez la vache, le transfert entre les cellules du cumulus et l’ovocyte des transcrits d’ARN et d’autres larges molécules qui, selon notre hypothèse, pourraient être exportées des cellules somatiques à l’ovocyte, ceci afin de soutenir sa maturation. L’étude des projections transzonales (PTZ) reliant les cellules du cumulus à l’ovocyte a révélé qu’elles sont de morphologie irrégulière, qu’elles peuvent contenir des organelles (mitochondries) et des structures cellulaires (ribosomes) et qu’elles se retractent au cours de la maturation ovocytaire. Des microvésicules pouvant transférer de larges molécules ont été identifiées à la jonction entre les cellules. Pour déterminer si l’ARN est empaqueté préalablement à son transfert entre les cellules, nous avons analysé les transcrits présents dans les PTZ et avons constaté qu’ils se déplacent vers l’ovocyte en maturation. Parmi les transcrits retrouvés dans les PTZ, certains étaient commun à ceux dont l’abondance augmente dans l’ovocyte en cours de maturation de même qu’à ceux retrouvés dans les polyribosomes de l’ovocyte en maturation, suggérant ainsi le transfert et l’utilisation des transcrits provenant des cellules du cumulus. Un transcrit synthétique ajouté aux cellules du cumulus avant qu’elles ne s’attachent à l’ovocyte a été transféré à ce dernier, confirmant ainsi le potentiel des cellules à transférer des ARNm et possiblement leurs protéins associées à l’ovocyte. Nouse avons observé, sur une échelle de temps, que les transcrits sont accumulés dans les PTZ au cours des heures suivant l’abattage de l’animal, mais préalablement à l’aspiration de l’ovocyte. Le retrait des cellules du cumulus et de cette période d’accumulation des transcrits se traduit par un faible taux de maturation des ovocytes. La nécessité d’avoir des vésicules d’ARN transférées à l’ovocyte a été testée grâce à des inhibiteurs de la synthèse d’ARN, de son transport et de la formation des vésicules. L’inhibition de chacune de ces étapes a compromis la maturation des ovocytes. III En se penchant sur les mécanismes impliqués dans le transfert des transcrits, nous avons découvert un candidat potentiel jouant un rôle dans l’insuffisance ovarienne précoce. Cette protéine liant les ARNm, Fragile X mental retardation (FMRP), a été retrouvée dans les cellules folliculaires et dans l’ovocyte tout au long de la folliculogenèse et de l’ovogenèse. Nous avons constaté que FMRP s’associe à la machinerie traductionnelle et aux protéines des granules d’entreposage dans l’ovocyte. L’inhibition de la protéine a significativement réduit le taux de blastocyste. En démontrant que des transcrits exogènes contribuent au développement de l’ovocyte et influencent sa maturation, nos recherches ajoutent un niveau de compréhension additionnel aux mécanismes de communication intercellulaire menant à la production de gamètes de bonne qualité. De futures expériences axées la caractérisation du transfert des transcrits et des mécanismes spécifiques en jeu contribuera à accroître notre compréhension de la compétence ovocytaire. IV Abstract The reports in this thesis explored the potential for large molecule communication between the cumulus cell and the oocyte hypothesizing that large molecules, including RNA, could be transferred to the oocyte for support during maturation. Exploration of the transzonal projections (TZPs) connecting the cumulus cells to the oocyte revealed that they are irregular in shape, can contain large organelles (mitochondria) and small cellular structures (ribosomes), and that these connections retract during oocyte maturation. Microvesicles were identified at the intercellular articulations capable of sharing large molecules between the cells. To determine if RNA is transferring as cargo between cells, nascent as well as total transcripts were evaluated in the TZPs and found moving towards the oocyte during maturation. Of the transcripts found in the TZPs during maturation, some were common to those increasing in abundance in the oocyte during maturation, and on the polyribosomes of the maturing oocyte, thus suggesting transfer and use of cumulus cell transcripts. A synthetic transcript provided to some cumulus cells for reconstruction with, and transfer to the oocyte, confirmed the potential to transfer mRNA and possibly proteins. Temporally, RNA transcripts were found to accumulate in TZPs during the hours post slaughter but prior to oocyte aspiration. Removal of the cumulus cells and this period of accumulation resulted in poor oocyte maturation. The requirement of vesicle mediated RNA transfer to the oocyte was tested. Inhibitors against RNA synthesis, transport, and vesicle formation were explored and found to reduced oocyte maturation. Focusing on mechanisms that could mediate transference, we assessed an RNA binding protein candidate with implications in premature ovarian insufficiency. Fragile X mental retardation protein (FMRP) was found in follicular cells and the oocyte throughout folliculogenesis and oogenesis. Based on known roles in translation control, FMRP was V shown associated with translational machinery and storage granule proteins in the oocyte. Knockdown of this protein resulted in compromised rates of blastocyst formation. Knowing that exogenous transcripts contribute to oocyte development, and influence the molecular aspects of oocyte maturation adds another layer to our understanding of intercellular communication in the production of a healthy gamete. Future characterization of the transferred transcripts and the mechanisms in control of this process will improve our understanding of oocyte health and competence. VI Table of contents Résumé .................................................................................................................................. III Abstract .................................................................................................................................. V Table of contents ................................................................................................................. VII List of figures ....................................................................................................................... XI List of tables ....................................................................................................................... XIII List of abbreviations and symbols ...................................................................................... XV Acknowledgments ............................................................................................................. XXI Foreword .......................................................................................................................... XXV 1 Introduction ....................................................................................................................... 1 1.1 The oocyte; a cornerstone in reproductive biology ...................................................... 1 1.1.1 Ovarian follicular development and oocyte growth ................................................. 2 1.1.1.1 Primordial germ cells establish the follicle population ....................................... 2 1.1.1.2 The primordial follicle ......................................................................................... 3 1.1.1.3 The primary follicle; morphology and activation ................................................ 3 1.1.1.4 The secondary follicle ......................................................................................... 5 1.1.1.5 Antral follicle development ................................................................................. 5 1.1.1.6 Follicular Atresia ................................................................................................. 8 1.2 Intercellular signalling in the cumulus oocyte complex governs growth and maturation of the oocyte ...................................................................................................... 10 1.2.1 Paracrine communication ....................................................................................... 10 1.2.2 Communication by small molecule transfer .......................................................... 12 1.3 Meiosis Resumption and Oocyte Maturation ............................................................. 14 1.3.1 In vitro maturation and embryo production ........................................................... 16 1.3.2 Maturation of Denuded Oocytes ............................................................................ 19 1.3.3 Challenges for In vitro embryo production ............................................................ 21 1.4 RNA is stored by the oocyte ....................................................................................... 22 1.4.1 Establishing the stores of RNA in the oocyte ........................................................ 23 1.4.2 Oocytes undergo transcriptional arrest .................................................................. 25 1.4.3 The duration of transcriptional silencing is species specific .................................. 28 1.5 How the oocyte packages, stores, and later uses RNA .............................................. 30 1.5.1 Shortening of the polyA tail ..................................................................................
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