Efeito do IGF-1 e da PAPP-A sobre aspectos do metabolismo lipídico e estresse oxidativo de oócitos e embriões bovinos produzidos in vitro
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Data
2020-06-15
Autores
Franchi, Fernanda Fagali [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A proteína plasmática A associada à gravidez (PAPP-A) é uma metaloprotease, que tem como função aumentar a biodisponibilidade do fator de crescimento semelhante a insulina tipo 1 (IGF-1), um peptídeo com diversas funções em prol do bom desenvolvimento oocitário e embrionário. Assim, a temática principal dessa tese foi utilizar a PAPP-A como um potencializador dos efeitos atribuídos ao IGF-1 durante a PIVE de bovinos. No primeiro artigo, investigamos o efeito da adição de PAPP-A durante a MIV suplementada com soro fetal bovino (SFB). Os complexos cumulus-oócitos (CCOs) foram maturados in vitro por 24 horas na presença ou ausência de SFB e, posteriormente, foram fertilizados e cultivados in vitro. Analisamos o conteúdo lipídico dos oócitos e blastocistos usando o corante Sudan-Black B, a taxa de blastocistos produzidos e a expressão gênica de 96 genes por RTqPCR. Não houve diferença no conteúdo lipídico dos oócitos e blastocistos avaliados, nem na taxa de blastocistos produzidos. No entanto, a presença de PAPP-A durante a MIV diminuiu a expressão de 22 genes relacionados à síntese de lipídeos, apoptose e estresse celular e qualidade embrionária nos blastocistos produzidos. No segundo artigo, os CCOs foram maturados (tratamento na MIV) ou os embriões cultivados (tratamento no CIV) por 24h com adição de IGF-1, IGF-1/PAPP-A, ou na ausência de ambos, em um ambiente estresse-induzido pela presença de menadiona. Avaliamos nos oócitos a progressão da meiose, concentração de espécies reativas de oxigênio (EROs) e o potencial de membrana mitocondrial por técnicas de coloração utilizando sondas específicas. Também analisamos as concentrações de glutationa total (GSH) nos CCOs, blastocistos e meios de MIV e CIV, além da taxa de blastocistos produzidos e a expressão gênica dos embriões por RTqPCR. A combinação IGF-1/PAPP-A resultou nas menores concentrações de EROs em oócitos e blastocistos e também modulou positivamente nos embriões genes envolvidos na defesa antioxidativa, metabolismo lipídico e qualidade embrionária, além de aumentar a taxa de blastocistos produzidos quando utilizadas na CIV. Assim, concluímos que o uso da PAPP-A, durante a PIVE de bovinos, foi efetivo como uma estratégia para maximizar as ações mediadas por IGF-1. Especificamente, reiteramos que a combinação IGF-1/PAPP-A teve efeitos mais marcantes, potencializando a proteção antioxidativa e a qualidade embrionária, além de aumentar quantitativamente o número de blastocistos produzidos em um ambiente estresse-induzido.
Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease which role is to increase insulin-like growth factor type 1 (IGF-1), a peptide with several beneficial functions for oocyte and embryo development. Thus, the thesis main subject was using the PAPP-A as an enhancer of IGF-1 attributed-effects during bovine in vitro production. In the first article, we investigated the effects of PAPP-A addition during IVM supplemented with fetal bovine serum (FBS). The cumulus-oocyte complexes (COCs) were in vitro matured for 24 hours in the presence or absence of FBS, and after, in vitro fertilized and cultured. We analyzed the oocytes and blastocysts lipid content by Sudan-Black B staining, the blastocysts yield, and the expression of 96 genes by RTqPCR. There was no difference in oocytes and blastocysts lipid content or in the blastocysts yield. However, the PAPP-A addition during IVM downregulated the expression of 22 genes related to lipid synthesis, apoptosis and cell stress, and embryonic quality in the blastocysts. In the second article, the COCs were matured (IVM treatment) or the embryos were cultured (IVC treatment) for 24 hours with IGF-1, IGF-1/PAPP-A addition, or in absence of both at a stress environment-induced by the menadione addition. We assessed the meiosis progression, reactive oxygen species (ROS) concentration and mitochondrial membrane potential levels in the oocytes using staining techniques. We also analyzed the concentrations of total glutathione (GSH) in COCs, blastocysts, and IVM and IVC media, besides the blastocysts yield and the expression of 96 genes by RTqPCR. The IGF-1/PAPP-A combination showed the lower ROS concentrations in oocytes and blastocysts and also positively modulated genes involved in antioxidative defense, lipid metabolism, and embryonic quality, besides increasing the blastocysts yield when used in IVC. Thus, we conclude the PAPP-A uses during bovine IVP was effective to improve the IGF-1 mediated-actions. Specifically, the IGF-1/PAPP-A shows the highlight effects, increasing antioxidative protection and embryonic quality, besides the number of blastocysts in a stress-induced environment.
Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease which role is to increase insulin-like growth factor type 1 (IGF-1), a peptide with several beneficial functions for oocyte and embryo development. Thus, the thesis main subject was using the PAPP-A as an enhancer of IGF-1 attributed-effects during bovine in vitro production. In the first article, we investigated the effects of PAPP-A addition during IVM supplemented with fetal bovine serum (FBS). The cumulus-oocyte complexes (COCs) were in vitro matured for 24 hours in the presence or absence of FBS, and after, in vitro fertilized and cultured. We analyzed the oocytes and blastocysts lipid content by Sudan-Black B staining, the blastocysts yield, and the expression of 96 genes by RTqPCR. There was no difference in oocytes and blastocysts lipid content or in the blastocysts yield. However, the PAPP-A addition during IVM downregulated the expression of 22 genes related to lipid synthesis, apoptosis and cell stress, and embryonic quality in the blastocysts. In the second article, the COCs were matured (IVM treatment) or the embryos were cultured (IVC treatment) for 24 hours with IGF-1, IGF-1/PAPP-A addition, or in absence of both at a stress environment-induced by the menadione addition. We assessed the meiosis progression, reactive oxygen species (ROS) concentration and mitochondrial membrane potential levels in the oocytes using staining techniques. We also analyzed the concentrations of total glutathione (GSH) in COCs, blastocysts, and IVM and IVC media, besides the blastocysts yield and the expression of 96 genes by RTqPCR. The IGF-1/PAPP-A combination showed the lower ROS concentrations in oocytes and blastocysts and also positively modulated genes involved in antioxidative defense, lipid metabolism, and embryonic quality, besides increasing the blastocysts yield when used in IVC. Thus, we conclude the PAPP-A uses during bovine IVP was effective to improve the IGF-1 mediated-actions. Specifically, the IGF-1/PAPP-A shows the highlight effects, increasing antioxidative protection and embryonic quality, besides the number of blastocysts in a stress-induced environment.
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Palavras-chave
Pappalisina, Fator de crescimento, EROs, IGFPB, IGF-1 livre, Biodisponibilidade, PAPP-A, Pappalysin, Growth factor, Bioavailability