O papel do estradiol no oviduto bovino: evidências in vivo e in vitro
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Data
2020-10-15
Autores
Fontes, Patricia Kubo
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Universidade Estadual Paulista (Unesp)
Resumo
O oviduto desempenha diversas funções até a formação do embrião, como maturação e transporte final dos gametas, processo de fertilização, transporte do embrião e desenvolvimento inicial do embrião. No entanto, nenhuma dessas etapas precisa ocorrer obrigatoriamente no oviduto. A capacidade de desenvolver embriões até o estágio de blastocisto sem qualquer contato com o trato reprodutivo materno gerou a hipótese de que o oviduto seria secundário e não essencial no estágio inicial da vida. Hoje, no entanto, é amplamente conhecido que a interação embrião-oviduto, ou interação com produtos de células ovidutais, pode impactar o embrião de diferentes maneiras, especialmente em relação às características epigenéticas, metabolismo e vários marcadores de qualidade. Para reconectar o embrião em desenvolvimento às células ovidutais (OC), muitas abordagens artificiais foram delineadas. Nesta tese, investigamos as CO diante de duas dessas abordagens: os protocolos de superestimulação ovariana (SOV) e a cocultura in vitro de embrião-CO. No primeiro estudo, as vacas foram submetidas à: i) protocolo SOV apenas com hormônio folículo-estimulante (FSH), ii) protocolo SOV usando FSH com gonadotrofina coriônica equina (eCG), ou iii) ovulação única por sincronização hormonal. O uso de protocolos SOV permite o desenvolvimento de múltiplos embriões dentro do oviduto, possibilitando a interação dos embriões com as CO. Este primeiro estudo demonstrou que as CO de vacas submetidas à SOV com FSH + eCG apresentam níveis mais elevados de estradiol, que também modulou a expressão gênica de genes relacionados ao processo de fertilização. Em seguida, esse perfil hormonal foi aplicado em um sistema de cultivo in vitro de OC. As CO podem ser cultivadas para aplicação em sistema de cocultura com embriões, o que permite que o embrião produzido in vitro interaja com as CO. A cultura de OC ainda não possui protocolo padrão ouro. Portanto, para analisar o efeito dos diferentes perfis hormonais obtidos como resultado do primeiro estudo, foi necessário incluir outra variação no segundo estudo: diferentes perfis hormonais x CO coletadas em diferentes fases do ciclo estral. Ao analisar os dados obtidos na análise de RNAseq, após o cultivo in vitro, as OC coletadas durante a fase folicular apresentam um perfil de transcriptoma muito diferente do perfil das células coletadas na fase lútea, caso nenhum tratamento hormonal for aplicado. Porém, quando altos níveis de estradiol são aplicados na cultura in vitro dessas células, o perfil do transcriptoma das CO torna-se muito semelhante, independentemente do ciclo estral em que as CO foram coletadas. Em conclusão, essas duas abordagens artificiais para possibilitar a interação dos embriões com o oviduto modulam as CO em função dos perfis hormonais. Portanto, o perfil hormonal é um fator importante a ser considerado nas estratégias para estabelecer a interação de embriões e células ovidutais.
The oviduct plays many roles in the embryo formation, as gametes final maturation and transport, fertilization process, embryo transport, and embryo early development. However, none of these steps need to occur in the oviduct mandatorily. The ability to develop embryos up to the blastocyst stage without any contact with the maternal reproductive tract hypothesized that the oviduct would be minor and inessential at the early stage of life. Today, however, it is widely known that the embryo-oviduct interaction, or interaction with oviductal cells’ products, can impact the embryo in different ways, especially regarding epigenetic characteristics, metabolism, and several quality markers. To reconnect the embryo in development to the oviductal cells (OC), many artificial approaches have been designed. In this thesis, we investigated the OC from two of these approaches: the ovarian superstimulation protocols (OVS) and the embryo-OC in vitro coculture. In the first study, cows were submitted to: i) OVS protocol only with follicle-stimulating hormone (FSH), ii) OVS protocol using FSH with equine chorionic gonadotrophin (eCG), or iii) single ovulation by hormone synchronization. The use of OVS protocols allows the development of multiples embryos inside the oviduct, making possible the interaction of the embryos with the OC. This first study demonstrated that OC from cows submitted to OVS using FSH+eCG has higher levels of estradiol, which also modulated gene expression of genes related to the fertilization process. Next, this hormone profile was applied in an in vitro culture system of OC. The OC can be cultured for application in a coculture system with embryos, which enable the in vitro produced embryo to interact with the OC. The culture of OC has still no gold standard protocol. Therefore, to analyze the effect of different hormone profiles obtained as a result of the first study, it was necessary to include another variation in the second study: different hormone profiles x OC collected in a different phase of the estrous cycle. When analyzing the data obtained from the RNAseq analysis, after in vitro culture, OC collected during the follicular phase presents a very different transcriptome profile than cells collected during the luteal phase, if no hormone treatment is applied. However, when high levels of estradiol are applied in the in vitro culture, the transcriptome profile of the OC become very similar to each other, independently of the estrous cycle when OC was collected. In conclusion, these two artificial approaches to enable the interaction of embryos with the oviduct modulate the OC depending on the hormone levels presented. Therefore, the hormone profile is an important factor to be considered in strategies to establish the interaction of embryos and oviductal cells.
The oviduct plays many roles in the embryo formation, as gametes final maturation and transport, fertilization process, embryo transport, and embryo early development. However, none of these steps need to occur in the oviduct mandatorily. The ability to develop embryos up to the blastocyst stage without any contact with the maternal reproductive tract hypothesized that the oviduct would be minor and inessential at the early stage of life. Today, however, it is widely known that the embryo-oviduct interaction, or interaction with oviductal cells’ products, can impact the embryo in different ways, especially regarding epigenetic characteristics, metabolism, and several quality markers. To reconnect the embryo in development to the oviductal cells (OC), many artificial approaches have been designed. In this thesis, we investigated the OC from two of these approaches: the ovarian superstimulation protocols (OVS) and the embryo-OC in vitro coculture. In the first study, cows were submitted to: i) OVS protocol only with follicle-stimulating hormone (FSH), ii) OVS protocol using FSH with equine chorionic gonadotrophin (eCG), or iii) single ovulation by hormone synchronization. The use of OVS protocols allows the development of multiples embryos inside the oviduct, making possible the interaction of the embryos with the OC. This first study demonstrated that OC from cows submitted to OVS using FSH+eCG has higher levels of estradiol, which also modulated gene expression of genes related to the fertilization process. Next, this hormone profile was applied in an in vitro culture system of OC. The OC can be cultured for application in a coculture system with embryos, which enable the in vitro produced embryo to interact with the OC. The culture of OC has still no gold standard protocol. Therefore, to analyze the effect of different hormone profiles obtained as a result of the first study, it was necessary to include another variation in the second study: different hormone profiles x OC collected in a different phase of the estrous cycle. When analyzing the data obtained from the RNAseq analysis, after in vitro culture, OC collected during the follicular phase presents a very different transcriptome profile than cells collected during the luteal phase, if no hormone treatment is applied. However, when high levels of estradiol are applied in the in vitro culture, the transcriptome profile of the OC become very similar to each other, independently of the estrous cycle when OC was collected. In conclusion, these two artificial approaches to enable the interaction of embryos with the oviduct modulate the OC depending on the hormone levels presented. Therefore, the hormone profile is an important factor to be considered in strategies to establish the interaction of embryos and oviductal cells.
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Palavras-chave
Oviduto, Células epiteliais do oviduto bovino, Cultivo 3D in vitro, Estradiol, Superestimulação ovariana, Oviduct, Bovine oviduct epithelial cell, 3D in vitro culture, Ovarian superstimulation