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Universidade De São Paulo Faculdade De Zootecnia E Engenharia De Alimentos Ana Clara Faquineli Cavalcante Mendes De Ávila

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Universidade De São Paulo Faculdade De Zootecnia E Engenharia De Alimentos Ana Clara Faquineli Cavalcante Mendes De Ávila UNIVERSIDADE DE SÃO PAULO FACULDADE DE ZOOTECNIA E ENGENHARIA DE ALIMENTOS ANA CLARA FAQUINELI CAVALCANTE MENDES DE ÁVILA Efeitos da comunicação entre células do sistema reprodutivo feminino e oócitos bovinos mediada por vesículas extracelulares de diferentes estágios do ciclo estral Pirassununga 2019 ANA CLARA FAQUINELI CAVALCANTE MENDES DE ÁVILA Efeitos da comunicação entre células do sistema reprodutivo feminino e oócitos bovinos mediada por vesículas extracelulares de diferentes estágios do ciclo estral Versão Corrigida Dissertação apresentada à Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo, como parte dos requisitos para a obtenção do título de Mestre em Ciências do programa de pós-graduação em Biociência Animal. Área de Concentração: Biociência Animal Orientador: Prof. Dr. Juliano Coelho da Silveira Pirassununga 2019 Ficha cataLográfica eLaborada peLo Serviço de BibLioteca e Informação, FZEA/USP, com os dados fornecidos pelo(a) autor(a) de Ávila, Ana Clara Faquineli Cavalcante Mendes d278e Efeitos da comunicação entre células do sistema reprodutivo feminino e oócitos bovinos mediada por vesículas extracelulares de diferentes estágios do ciclo estral / Ana Clara Faquineli Cavalcante Mendes de Ávila ; orientador Juliano Coelho da Silveira. -- Pirassununga, 2019. 194 f. Dissertação (Mestrado - Programa de Pós-Graduação em Biociência Animal) -- Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo. 1. Reprodução animal. 2. Bovinos. 3. Folículo ovariano. 4. Vesículas extracelulares. 5. miRNAs. I. da Silveira, Juliano Coelho, orient. II. Título. Permitida a cópia total ou parcial deste documento, desde que citada a fonte - o autor ANA CLARA FAQUINELI CAVALCANTE MENDES DE ÁVILA Efeitos da comunicação entre células do sistema reprodutivo feminino e oócitos bovinos mediada por vesículas extracelulares de diferentes estágios do ciclo estral Dissertação apresentada à Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo, como parte dos requisitos para a obtenção do título de Mestre em Ciências do programa de pós-graduação em Biociência Animal. Área de Concentração: Biociência Animal Data de aprovação: 04/09/2019 Banca Examinadora: Prof. Dr. Juliano Coelho da Silveira – Presidente da Banca Examinadora Faculdade de Zootecnia e Engenharia de Alimentos – FZEA- USP Profa. Dra. Anna Carolina Denicol University of California, Davis campus Profa. Dra. Gisele Zoccal Mingoti Faculdade de Medicina Veterinária de Araçatuba- FMVA- UNESP Profa. Dra. Paula Andrea de Albuquerque Salles Navarro Faculdade de Medicina de Ribeirão Preto – FMRP- USP Aos meus pais, Fábio e Heloisa, e a todos os profissionais que se dedicam à reprodução. AGRADECIMENTOS Agradeço imensamente ao meu orientador, Prof. Dr. Juliano Coelho da Silveira, pela dedicação a este trabalho, por todos os ensinamentos, pelas oportunidades, pela amizade e apoio durante estes três anos de mestrado. Ao Prof. Dr. Felipe Perecin por abrir as portas do Laboratório e por todos os ensinamentos. Agradeço também aos professores Dr. Flávio Vieira Meirelles, Dr. Luciano Andrade, Dra. Fabiana Bressan e Dra. Cláudia Leal pela colaboração. À Dra. Cibele Prado pela dedicação e disposição em nos ajudar. Ao Prof. Dr. Claude Robert, Alexandre Bastien, Dra. Isabelle Gilbert e Isabelle Laflamme pela oportunidade de intercâmbio e colaboração. Agradeço à Universidade de São Paulo, à Faculdade de Zootecnia e Engenharia de Alimentos e à Université Laval pela estrutura e apoio dos funcionários. Ao laboratório Multiusuário de Microscopia Eletrônica e de Microscopia Confocal, ao Laboratório de Genética Molecular e Bioinformática, situados na FMRP-USP, e ao Laboratório de Biotecnologia Animal, situado na ESALQ-USP, pela colaboração com as análises realizadas neste estudo. Agradeço também aos abatedouros Olhos d’água (Ipuã-MG) e Vale do Prata (São João da Boa vista-SP), pela doação dos ovários e à CRV Lagoa da Serra pela doação do sêmen. À CAPES e à Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; processos número: 2017/02037-0; 2018/14869-2; 2014/22887-0 e 2015/21829-9), pela concessão da bolsa de mestrado, pela bolsa de estágio de pesquisa no exterior e pelo auxílio financeiro para o desenvolvimento deste estudo. Agradeço aos meus pais, Fábio e Heloisa, pelo exemplo de profissionalismo, caráter, por todo apoio e amor incondicional. Ao meu irmão, João Victor e ao Lucas pelo carinho, paciência e companheirismo. Aos meus avós, tios, Rê e Malu por toda preocupação e amor. Aos meus amigos, Andréia, Paula, Dani, Lu e Pedro, que sempre estiveram ao meu lado. Aos meus amigos de laboratório, Gabriella, Alessandra, Maite, Monalisa, Marina, Juliano, Rafael, Tiago, Ricardo, Jorge, Julio, Rosane, Natália, Taismara, Ramon, Naira, Aline, Raquel, Lucas, KelLy, Maíra, Hugo, Letícia, Jéssica, Dewison, Victória Barillari, Victória Sanchez, João e Léo, agradeço pela convivência, todos os ensinamentos e momentos de aLegria. RESUMO DE ÁVILA, A.C.F.C.M. Efeitos da comunicação entre células do sistema reprodutivo feminino e oócitos bovinos mediada por vesículas extracelulares de diferentes estágios do ciclo estral. 2019. 194 f. Dissertação (Mestrado) – Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, 2019. As técnicas de reprodução assistida (TRA) são alternativas para infertiLidade e aumento da produção de animais de interesse. Apesar de amplamente utiLizadas, as TRA podem causar consequências negativas aos indivíduos gerados desde o estabelecimento da gestação até a vida adulta. A tentativa de mimetizar o ambiente in vivo é uma opção para aprimorar as TRA. Vesículas extracelulares (VEs) secretadas por células são alvos de estudos recentes devido suas funções de comunicação intercelular no ambiente in vivo. As VEs são encontradas em fluidos corporais e possuem material bioativo, como miRNAs, que regulam o funcionamento de células a partir da modulação pós-transcricional. A hipótese deste estudo é que VEs pequenas secretadas por células foliculares e células do oviduto favoreçam a maturação e a fertilização in vitro de complexos cumulus-oócitos (CCOs). O objetivo do estudo foi caracterizar e avaliar os efeitos de VEs do fluido folicular (FF) e do oviduto obtidos em diferentes momentos do ciclo estral de bovinos durante a produção in vitro. Para isso, VEs foram isoladas de FF e do Lavado de oviduto bovino, caracterizadas e utiLizadas como suplementação durante a maturação e a fertilização in vitro, respectivamente. Os resuLtados demonstraram que as VEs do fluido de folículos em diferentes momentos do ciclo estral possuem diferentes perfis de miRNAs. A suplementação com VEs pequenas de FF durante a maturação in vitro (MIV) causou a internalização dessas VEs e do seu RNA pelos CCOs, mas não afetou as taxas de blastocistos. Porém, foi demonstrado que as VEs de diferentes origens foliculares moduLam vias como PI3K-Akt e de transporte de RNA e processos biológicos relacionados à maturação oocitária, à ovulação e à resposta imune em células do cumulus. As VEs obtidas do oviduto em diferentes momentos do ciclo estral não afetaram as taxas de produção e a qualidade de blastocistos no modelo utiLizado. Em conclusão, este estudo demonstrou que as VEs pequenas são modificadas de acordo com sua origem folicular e que impactam processos biológicos em células do cumulus de bovinos. Palavras-chave: Bovinos. MicroRNAs. Fluido folicular. Oviduto. Vesículas extracelulares. ABSTRACT DE ÁVILA, A.C.F.C.M. The effects of communication between female reproductive system cells and bovine oocytes mediated by extracellular vesicles of different estrous cycle stages. 2019. 194f. M.Sc. Dissertation – Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, 2019. Assisted reproductive techniques (ART) are alternatives to infertility and to increase animals’ production. Although these techniques are widely used, they can cause negative consequences since early pregnancy until adult life. Attempts to mimic in vivo environment are applied to improve ARTs. Extracellular vesicles (EVs) secreted by cells are targets of recent studies due to their function in intercellular communication. These vesicles are in body fluids and have bioactive material, as miRNAs, that regulate cell functions by post-transcriptional modulation. The hypothesis of this study is that small EVs secreted by follicular and oviductal cells improve the in vitro maturation and fertilization of cumulus-oocyte complexes (COCs). Our goal was to characterize and to evaluate the effects of small EVs from follicular and oviductal fluids obtained from different bovine estrous cycle stages during in vitro embryo production. For this, EVs were isolated from bovine follicular fluid (FF) and oviductal flushing, characterized and used as supplementation during in vitro maturation and fertilization, respectively. The results demonstrated that small EVs from follicles at different estrous cycle stage have different miRNA profile. The small EVs from FF and their RNA were taken up by COCs during in vitro maturation but did not change blastocyst production rates. However, we demonstrated that small EVs from different follicle origin modulate pathways such as PI3K-Akt and RNA transport and biological processes related to oocyte maturation, ovulation and immune responses in cumulus cells. Small EVs from oviduct at different estrous cycle stage did not improve embryo production rates and embryo quality in our model. Therefore, this study demonstrated
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