Efeito de um meio condicionado por células do Cumulus oophorus de bovinos na maturação e no potencial de desenvolvimento embrionário in vitro de oócitos desnudos

Detalhes bibliográficos
Ano de defesa: 2017
Autor(a) principal: Isabela Maria Alves de Avila
Orientador(a): Não Informado pela instituição
Banca de defesa: Não Informado pela instituição
Tipo de documento: Dissertação
Tipo de acesso: Acesso aberto
Idioma: por
Instituição de defesa: Universidade Federal de Minas Gerais
UFMG
Programa de Pós-Graduação: Não Informado pela instituição
Departamento: Não Informado pela instituição
País: Não Informado pela instituição
Palavras-chave em Português:
Link de acesso: http://hdl.handle.net/1843/SMOC-AVDPBF
Resumo: One of the challenges of reproductive biology and medicine is to understand the nature of the molecular and cellular processes that control oocyte developmental competence. This competence, acquired through maturation, a complex process that comprises nuclear and cytoplasmic maturation, involves a deep interaction between the oocyte and Cumulus oophorus cells. Cumulus oophorus cells are key for paracrine and endocrine signaling and for oocyte maturation; however, in many applications, removal of these cells, or oocyte denudation, is required before maturation begins or prior to in vitro fertilization. Because denudation reduces developmental competence, an efficient in vitro maturation system for denuded oocytes is needed, as no system has been well established in mammalians. Different systems use co-cultures of homologous and heterologous cells or media conditioned by these cells, to take advantage of factors secreted by them and optimize results in in vitro production. The present study evaluates the effect of a medium conditioned by a culture of bovine Cumulus-oophorus cells in the in vitro maturation of bovine denuded oocytes and their embryonic development potential. The experiment was carried out in the Laboratory of in vitro Production of Embryos at the School of Veterinary Medicine, Federal University of Minas Gerais, Brazil. The oocytes were aspirated from follicles between 2 and 8 mm in diameter from slaughterhouse ovaries. Grade I and II oocytes, half of which had been denuded by successive pipetting prior to maturation, were matured in control medium and in the conditioned media previously obtained from the culture of Cumulus oophorus cells. 2927 oocytes were matured, of which 894 were used for evaluation of maturation and 2033 for evaluation of embryonic development. The evaluation of in vitro oocyte maturation was performed based on nuclear maturation, cytoplasmic maturation (mitochondrial distribution), enzymatic dissolution of zona pellucida with chymotrypsin 1% and pronase 0.5% and ultrastructure by transmission electron microscopy. The evaluation of embryonic development potential was based on embryo cleavage and blastocyst rates. There were no significant differences in nuclear maturation of oocytes in control medium and conditioned medium, as well as no differences between nuclear maturation of CCOs and denuded oocytes. Cytoplasmic maturation in conditioned medium was not significantly different from that in control medium. Denuded oocytes presented a predominantly heterogeneous-peripheral mitochondrial distribution pattern, whereas CCOs had a predominant heterogeneous-perinuclear pattern. Denuded oocytes matured in the conditioned medium showed a significant reduction of the dissolution time of the zona pellucida in enzymatic dissolution assay with chymotrypsin 1%, but there were inconsistencies between the results obtained in the assays with the two enzymes. There were no significant differences between cleavage and blastocysts rates between oocytes matured in control medium and conditioned medium. In conclusion, under these experimental conditions, the conditioned medium from bovine Cumulus oophorus cells did not prove to be superior to the control medium in relation to the in vitro oocyte maturation of the denuded oocytes nor with respect to its embryonic development and, therefore, its incorporation into in vitro embryo production routines is not recommended. Further studies are still needed to better understand the events related to the acquisition of oocyte competence and to develop more efficient in vitro maturation media for denuded oocytes.
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spelling Efeito de um meio condicionado por células do Cumulus oophorus de bovinos na maturação e no potencial de desenvolvimento embrionário in vitro de oócitos desnudosmaturação in vitroCumulus oophorusmeio condicionadooócitos desnudosReprodução animalBovinoCumulus oophorusEmbriãoOocitoOne of the challenges of reproductive biology and medicine is to understand the nature of the molecular and cellular processes that control oocyte developmental competence. This competence, acquired through maturation, a complex process that comprises nuclear and cytoplasmic maturation, involves a deep interaction between the oocyte and Cumulus oophorus cells. Cumulus oophorus cells are key for paracrine and endocrine signaling and for oocyte maturation; however, in many applications, removal of these cells, or oocyte denudation, is required before maturation begins or prior to in vitro fertilization. Because denudation reduces developmental competence, an efficient in vitro maturation system for denuded oocytes is needed, as no system has been well established in mammalians. Different systems use co-cultures of homologous and heterologous cells or media conditioned by these cells, to take advantage of factors secreted by them and optimize results in in vitro production. The present study evaluates the effect of a medium conditioned by a culture of bovine Cumulus-oophorus cells in the in vitro maturation of bovine denuded oocytes and their embryonic development potential. The experiment was carried out in the Laboratory of in vitro Production of Embryos at the School of Veterinary Medicine, Federal University of Minas Gerais, Brazil. The oocytes were aspirated from follicles between 2 and 8 mm in diameter from slaughterhouse ovaries. Grade I and II oocytes, half of which had been denuded by successive pipetting prior to maturation, were matured in control medium and in the conditioned media previously obtained from the culture of Cumulus oophorus cells. 2927 oocytes were matured, of which 894 were used for evaluation of maturation and 2033 for evaluation of embryonic development. The evaluation of in vitro oocyte maturation was performed based on nuclear maturation, cytoplasmic maturation (mitochondrial distribution), enzymatic dissolution of zona pellucida with chymotrypsin 1% and pronase 0.5% and ultrastructure by transmission electron microscopy. The evaluation of embryonic development potential was based on embryo cleavage and blastocyst rates. There were no significant differences in nuclear maturation of oocytes in control medium and conditioned medium, as well as no differences between nuclear maturation of CCOs and denuded oocytes. Cytoplasmic maturation in conditioned medium was not significantly different from that in control medium. Denuded oocytes presented a predominantly heterogeneous-peripheral mitochondrial distribution pattern, whereas CCOs had a predominant heterogeneous-perinuclear pattern. Denuded oocytes matured in the conditioned medium showed a significant reduction of the dissolution time of the zona pellucida in enzymatic dissolution assay with chymotrypsin 1%, but there were inconsistencies between the results obtained in the assays with the two enzymes. There were no significant differences between cleavage and blastocysts rates between oocytes matured in control medium and conditioned medium. In conclusion, under these experimental conditions, the conditioned medium from bovine Cumulus oophorus cells did not prove to be superior to the control medium in relation to the in vitro oocyte maturation of the denuded oocytes nor with respect to its embryonic development and, therefore, its incorporation into in vitro embryo production routines is not recommended. Further studies are still needed to better understand the events related to the acquisition of oocyte competence and to develop more efficient in vitro maturation media for denuded oocytes.Um dos desafios da medicina e biologia reprodutiva é entender a natureza dos processos moleculares e celulares que controlam a competência oocitária para o desenvolvimento. Esta competência, adquirida por meio da maturação, processo complexo que compreende a maturação nuclear e citoplasmática, envolve uma interação profunda entre oócito e células do Cumulus oophorus. As células do Cumulus oophorus são chave para a sinalização parácrina e endócrina e para a maturação oocitária; no entanto, em muitas aplicações é necessária sua remoção, ou desnudamento dos oócitos, antes do início da maturação in vitro. Como o desnudamento reduz a competência de desenvolvimento, um sistema eficiente de maturação in vitro para oócitos desnudos é necessário, e até o presente nenhum sistema está bem estabelecido para mamíferos. Diferentes sistemas usam co-cultivos de células homólogas e heterólogas ou meios condicionados por células para tirar proveito de fatores por elas secretados e otimizar os resultados na produção in vitro. Avaliou-se o efeito de um meio condicionado por células do Cumulus oophorus de bovinos na maturação in vitro de oócitos bovinos desnudos e no seu potencial de desenvolvimento embrionário. O experimento foi realizado no Laboratório de Produção in vitro de Embriões da Escola de Veterinária da Universidade Federal de Minas Gerais, Brasil. Os oócitos foram aspirados de folículos entre 2 e 8 mm de diâmetro, de ovários obtidos em abatedouro. Oócitos de graus I e II, metade dos quais desnudada por pipetagens sucessivas previamente à maturação, foram maturados em meio controle e no meio condicionado desenvolvido previamente a partir do cultivo de células do Cumulus oophorus de bovinos. Foram maturados 2927 oócitos (1525 complexos Cumulus-oócito (CCOs) e 1402 oócitos desnudados), dos quais 894 foram utilizados para avaliação da maturação e 2033 para avaliação do desenvolvimento embrionário. A avaliação da maturação oocitária in vitro foi feita com base na maturação nuclear, maturação citoplasmática (distribuição mitocondrial), tempo de digestão enzimática da zona pelúcida com quimotripsina 1% e pronase 0,5% e ultraestrutura por microscopia eletrônica de transmissão. Adicionalmente, foi feita a avaliação da expansão das células do Cumulus dos CCOs maturados. A avaliação do potencial de desenvolvimento embrionário foi feita com base nas taxas de clivagem e blastocisto dos embriões. Não houve diferenças significativas na maturação nuclear dos oócitos maturados em meio controle e em meio condicionado, bem como não houve diferenças entre a maturação nuclear de CCOs e oócitos desnudos. A maturação citoplasmática em meio condicionado não foi significativamente diferente da em meio controle. Oócitos desnudos apresentaram padrão de distribuição mitocondrial predominantemente heterogêneo-periférico, enquanto CCOs apresentaram padrão heterogêneo-perinuclear predominante. Oócitos desnudos maturados no meio condicionado apresentaram redução significativa do tempo de digestão da zona pelúcida em ensaio de digestão enzimática com quimotripsina 1%, porém houve inconsistências entre os resultados obtidos nos ensaios com as duas enzimas. Não houve diferenças significativas entre taxas de clivagem e blastocistos entre os oócitos maturados em meio controle e meio condicionado. Conclui-se que, nas condições experimentais, o meio condicionado obtido a partir de células do Cumulus oophorus de bovinos não se mostrou superior ao meio controle com relação à maturação oocitária in vitro dos oócitos bovinos desnudos e nem com relação ao seu desenvolvimento embrionário. Portanto, não se recomenda a sua incorporação às rotinas de produção in vitro de embriões. Continuam necessários estudos para melhor compreensão dos eventos relacionados à aquisição da competência oocitária e para que se desenvolvam meios de maturação mais eficientes para oócitos desnudos.Universidade Federal de Minas GeraisUFMGAntonio de Pinho Marques JuniorJoao Henrique Moreira VianaRivia Mara LamaitaIsabela Maria Alves de Avila2019-08-12T21:54:06Z2019-08-12T21:54:06Z2017-08-31info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/1843/SMOC-AVDPBFinfo:eu-repo/semantics/openAccessporreponame:Repositório Institucional da UFMGinstname:Universidade Federal de Minas Gerais (UFMG)instacron:UFMG2019-11-14T23:03:38Zoai:repositorio.ufmg.br:1843/SMOC-AVDPBFRepositório InstitucionalPUBhttps://repositorio.ufmg.br/oairepositorio@ufmg.bropendoar:2019-11-14T23:03:38Repositório Institucional da UFMG - Universidade Federal de Minas Gerais (UFMG)false
dc.title.none.fl_str_mv Efeito de um meio condicionado por células do Cumulus oophorus de bovinos na maturação e no potencial de desenvolvimento embrionário in vitro de oócitos desnudos
title Efeito de um meio condicionado por células do Cumulus oophorus de bovinos na maturação e no potencial de desenvolvimento embrionário in vitro de oócitos desnudos
spellingShingle Efeito de um meio condicionado por células do Cumulus oophorus de bovinos na maturação e no potencial de desenvolvimento embrionário in vitro de oócitos desnudos
Isabela Maria Alves de Avila
maturação in vitro
Cumulus oophorus
meio condicionado
oócitos desnudos
Reprodução animal
Bovino
Cumulus oophorus
Embrião
Oocito
title_short Efeito de um meio condicionado por células do Cumulus oophorus de bovinos na maturação e no potencial de desenvolvimento embrionário in vitro de oócitos desnudos
title_full Efeito de um meio condicionado por células do Cumulus oophorus de bovinos na maturação e no potencial de desenvolvimento embrionário in vitro de oócitos desnudos
title_fullStr Efeito de um meio condicionado por células do Cumulus oophorus de bovinos na maturação e no potencial de desenvolvimento embrionário in vitro de oócitos desnudos
title_full_unstemmed Efeito de um meio condicionado por células do Cumulus oophorus de bovinos na maturação e no potencial de desenvolvimento embrionário in vitro de oócitos desnudos
title_sort Efeito de um meio condicionado por células do Cumulus oophorus de bovinos na maturação e no potencial de desenvolvimento embrionário in vitro de oócitos desnudos
author Isabela Maria Alves de Avila
author_facet Isabela Maria Alves de Avila
author_role author
dc.contributor.none.fl_str_mv Antonio de Pinho Marques Junior
Joao Henrique Moreira Viana
Rivia Mara Lamaita
dc.contributor.author.fl_str_mv Isabela Maria Alves de Avila
dc.subject.por.fl_str_mv maturação in vitro
Cumulus oophorus
meio condicionado
oócitos desnudos
Reprodução animal
Bovino
Cumulus oophorus
Embrião
Oocito
topic maturação in vitro
Cumulus oophorus
meio condicionado
oócitos desnudos
Reprodução animal
Bovino
Cumulus oophorus
Embrião
Oocito
description One of the challenges of reproductive biology and medicine is to understand the nature of the molecular and cellular processes that control oocyte developmental competence. This competence, acquired through maturation, a complex process that comprises nuclear and cytoplasmic maturation, involves a deep interaction between the oocyte and Cumulus oophorus cells. Cumulus oophorus cells are key for paracrine and endocrine signaling and for oocyte maturation; however, in many applications, removal of these cells, or oocyte denudation, is required before maturation begins or prior to in vitro fertilization. Because denudation reduces developmental competence, an efficient in vitro maturation system for denuded oocytes is needed, as no system has been well established in mammalians. Different systems use co-cultures of homologous and heterologous cells or media conditioned by these cells, to take advantage of factors secreted by them and optimize results in in vitro production. The present study evaluates the effect of a medium conditioned by a culture of bovine Cumulus-oophorus cells in the in vitro maturation of bovine denuded oocytes and their embryonic development potential. The experiment was carried out in the Laboratory of in vitro Production of Embryos at the School of Veterinary Medicine, Federal University of Minas Gerais, Brazil. The oocytes were aspirated from follicles between 2 and 8 mm in diameter from slaughterhouse ovaries. Grade I and II oocytes, half of which had been denuded by successive pipetting prior to maturation, were matured in control medium and in the conditioned media previously obtained from the culture of Cumulus oophorus cells. 2927 oocytes were matured, of which 894 were used for evaluation of maturation and 2033 for evaluation of embryonic development. The evaluation of in vitro oocyte maturation was performed based on nuclear maturation, cytoplasmic maturation (mitochondrial distribution), enzymatic dissolution of zona pellucida with chymotrypsin 1% and pronase 0.5% and ultrastructure by transmission electron microscopy. The evaluation of embryonic development potential was based on embryo cleavage and blastocyst rates. There were no significant differences in nuclear maturation of oocytes in control medium and conditioned medium, as well as no differences between nuclear maturation of CCOs and denuded oocytes. Cytoplasmic maturation in conditioned medium was not significantly different from that in control medium. Denuded oocytes presented a predominantly heterogeneous-peripheral mitochondrial distribution pattern, whereas CCOs had a predominant heterogeneous-perinuclear pattern. Denuded oocytes matured in the conditioned medium showed a significant reduction of the dissolution time of the zona pellucida in enzymatic dissolution assay with chymotrypsin 1%, but there were inconsistencies between the results obtained in the assays with the two enzymes. There were no significant differences between cleavage and blastocysts rates between oocytes matured in control medium and conditioned medium. In conclusion, under these experimental conditions, the conditioned medium from bovine Cumulus oophorus cells did not prove to be superior to the control medium in relation to the in vitro oocyte maturation of the denuded oocytes nor with respect to its embryonic development and, therefore, its incorporation into in vitro embryo production routines is not recommended. Further studies are still needed to better understand the events related to the acquisition of oocyte competence and to develop more efficient in vitro maturation media for denuded oocytes.
publishDate 2017
dc.date.none.fl_str_mv 2017-08-31
2019-08-12T21:54:06Z
2019-08-12T21:54:06Z
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dc.publisher.none.fl_str_mv Universidade Federal de Minas Gerais
UFMG
publisher.none.fl_str_mv Universidade Federal de Minas Gerais
UFMG
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