Modulação do EGFR em complexos cumulus-oócitos bovinos cultivados in vitro e seus efeitos sobre o metabolismo, maturação e aquisição da competência oocitária e a transcrição gênica das células do cumulus e dos embriões
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Data
2018-08-27
Autores
Dall'Acqua, Priscila Chediek [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O receptor do fator de crescimento epidermal (EGFR) está relacionado com a retomada da meiose induzida por estímulo gonadotrófico. Nós utilizamos um inibidor do EGFR (AG1478) para inibir a retomada da meiose durante a pré-maturação (PMIV). No experimento I, foi avaliada a maturação oocitária, a expansão das células do cumulus, a funcionalidade das junções gap, a distribuição dos microfilamentos de actina e o conteúdo lipídico em oócitos e, a expressão gênica nas células do cumulus. No experimento II, foi avaliado o desenvolvimento embrionário, a taxa de eclosão, o número total de células, a taxa de apoptose, o conteúdo lipídico e expressão gênica nos embriões. No experimento I, os COC foram PMIV por 8h em meio com 1 μM de AG1478, inibidor do EGFR, (EGFR-_PreIVM), em seguida foram MIV por 22h (EGFR-_IVM); foram feitos 4 grupos controle: oócitos imaturos imediatamente após sua remoção do folículo (Control_Immat), oócitos MIV por 8h (Control_PreIVM), por 22h (Control_IVM_22h) e por 30h (Control_IVM_30h). No experimento II, os COC foram PMIV por 8h em meio com 1 μM do inibidor do EGFR, em seguida foram MIV por 22h (EGFR-); os COC do grupo controle foram MIV por 22h (Control). Em seguida, os oócitos foram FIV e cultivados por até 9 dias. No experimento I, o grupo EGFR-_PreIVM apresentou 53,1% dos oócitos em GV, com a maioria em GV1 (42,9%; P>0,05) ou em GV3 [28,6%, semelhante ao grupo Control_Immat (P>0,05) e menor que o grupo Control_PreIVM (P<0,05)]; após a MIV não foram encontradas diferenças (P>0,05) entre os grupos nas taxas de MII, mas o grupo Control_IMV_30h (9,1%) teve maiores (P<0,05) taxas de oócitos degenerados. A expansão das células do cumulus foi menor (P<0,05) no grupo EGFR-_PreIVM e as junções gap se mantiveram funcionais (P<0,05). Os microfilamentos de actina não diferiram entre os grupos (P>0,05) e estavam em sua maioria na categoria normal. O conteúdo lipídico foi menor (P<0,05) no grupo EGFR-_PreIVM comparado com Control_Immat, mas após a MIV foi maior (P<0,05) no grupo EGFR-_IVM do que no Control_IVM. Diferenças na expressão dos genes de maturação oocitária, expansão das células do cumulus, metabolismo, metabolismo lipídico, qualidade e desenvolvimento embrionário, regulação epigenética, estresse do retículo endoplasmático, defesa antioxidante e apoptose foram observadas após a PMIV e; nos genes de expansão das células do cumulus, metabolismo e qualidade embrionária após a MIV. No experimento II, as taxas de blastocistos, de eclosão, o número total de células e o conteúdo lipídico não diferiram (P>0,05) nos embriões avaliados. A apoptose foi menor (P<0,05) no grupo EGFR-. Os genes NANOG, RPLP0, H2AFZ, HMGCS1, ACSL1, GPAT3, FADS2, FASN, FDX1, BID, GPX4 e HIF1A foram mais expressos (P<0,05) no grupo EGFR-. Em conclusão o tratamento com inibidor do EGFR proporcionou bloqueio parcial da meiose com manutenção das junções gap funcionais, que foi relacionado com maior potencial de desenvolvimento dos oócitos, devido à redução da apoptose nos embriões e ao aumento na expressão de genes relacionados com desenvolvimento, sugerindo melhor qualidade embrionária.
Epidermal growth factor receptor (EGFR) is related with meiosis resumption by gonadotropic stimulus. We used an EGFR inhibitor (AG1478) to block meiosis resumption during prematuration (PIVM). In experiment I, was evaluated oocyte maturation, cumulus cells expansion, gap junctions functionality, microfilaments distribution and lipid content in oocytes and, gene expression in cumulus cells. In experiment II, was assessed embryo development, hatching rates, total cell number, apoptosis, lipid content and gene expression in embryos. For experiment I, COC were PIVM for 8h in medium supplemented with 1μM of AG1478, an EGFR inhibitor, (EGFR-_PreIVM), followed by 22h IVM (EGFR-_IVM); four control groups were evaluated: immature oocytes immediately after follicle removal (Control_Immat), oocytes IVM for 8h (Control_PreIVM), oocytes IVM for 22h (Control_IVM_22h) and oocytes IVM for 30h (Control_IVM_30h). For experiment II, COC were PIVM for 8h in medium supplemented with 1μM of EGFR inhibitor, followed by 22h IVM (EGFR-); COC from control group were matured for 22h (Control). After that, oocytes were IVF and zygotes were cultured up to 9 days. In experiment I, EGFR-_PreIVM group had 53.1% of the oocytes blocked in GV, the majority in GV1 (42.9%; P>0.05) or in GV3 [28.6%, similar to Control_Immat (P>0.05) and lower than Control_PreIVM (P<0.05)]. After IVM, no differences (P>0.05) were found between groups in MII rates, but Control_IMV_30h (9.1%) had higher (P<0.05) rates of degenerated oocytes. Cumulus cells expansion was lower (P<0.05) in EGFR-_PreIVM and gap junctions maintained the functionality (P<0.05). Actin microfilaments were mostly distributed in normal category after PIVM and after IVM, and no differences were found (P>0.05). Lipid content was lower (P<0.05) in EGFR-_PreIVM in comparison to Control_Immat, but after IVM was higher (P<0,05) in EGFR-_IVM than Control_IVM. After PIVM differences (P>0,05) were found in genes for oocyte maturation, cumulus cells expansion, metabolism, lipid metabolism, embryo quality, embryo development, epigenetic regulation, endoplasmic reticulum stress, antioxidant defense and apoptosis; after IVM, the differences (P<0,05) were in genes for cumulus cells expansion, metabolism and embryo quality. In experiment II, blastocyst, hatching rates, lipid content and cell number did not differ between treatments (P>0.05). Apoptosis was lower (P<0.05) in EGFR-. The genes NANOG, RPLP0, H2AFZ, HMGCS1, ACSL1, GPAT3, FADS2, FASN, FDX1, BID, GPX4 and HIF1A were upregulated (P<0.05) in EGFR-. In conclusion, treatment with EGFR inhibitor lead to partial meiotic block with functional gap junctions, related to higher oocyte developmental potential, due to the lower apoptotic index and the increased expression of genes related do development, suggesting better embryo quality.
Epidermal growth factor receptor (EGFR) is related with meiosis resumption by gonadotropic stimulus. We used an EGFR inhibitor (AG1478) to block meiosis resumption during prematuration (PIVM). In experiment I, was evaluated oocyte maturation, cumulus cells expansion, gap junctions functionality, microfilaments distribution and lipid content in oocytes and, gene expression in cumulus cells. In experiment II, was assessed embryo development, hatching rates, total cell number, apoptosis, lipid content and gene expression in embryos. For experiment I, COC were PIVM for 8h in medium supplemented with 1μM of AG1478, an EGFR inhibitor, (EGFR-_PreIVM), followed by 22h IVM (EGFR-_IVM); four control groups were evaluated: immature oocytes immediately after follicle removal (Control_Immat), oocytes IVM for 8h (Control_PreIVM), oocytes IVM for 22h (Control_IVM_22h) and oocytes IVM for 30h (Control_IVM_30h). For experiment II, COC were PIVM for 8h in medium supplemented with 1μM of EGFR inhibitor, followed by 22h IVM (EGFR-); COC from control group were matured for 22h (Control). After that, oocytes were IVF and zygotes were cultured up to 9 days. In experiment I, EGFR-_PreIVM group had 53.1% of the oocytes blocked in GV, the majority in GV1 (42.9%; P>0.05) or in GV3 [28.6%, similar to Control_Immat (P>0.05) and lower than Control_PreIVM (P<0.05)]. After IVM, no differences (P>0.05) were found between groups in MII rates, but Control_IMV_30h (9.1%) had higher (P<0.05) rates of degenerated oocytes. Cumulus cells expansion was lower (P<0.05) in EGFR-_PreIVM and gap junctions maintained the functionality (P<0.05). Actin microfilaments were mostly distributed in normal category after PIVM and after IVM, and no differences were found (P>0.05). Lipid content was lower (P<0.05) in EGFR-_PreIVM in comparison to Control_Immat, but after IVM was higher (P<0,05) in EGFR-_IVM than Control_IVM. After PIVM differences (P>0,05) were found in genes for oocyte maturation, cumulus cells expansion, metabolism, lipid metabolism, embryo quality, embryo development, epigenetic regulation, endoplasmic reticulum stress, antioxidant defense and apoptosis; after IVM, the differences (P<0,05) were in genes for cumulus cells expansion, metabolism and embryo quality. In experiment II, blastocyst, hatching rates, lipid content and cell number did not differ between treatments (P>0.05). Apoptosis was lower (P<0.05) in EGFR-. The genes NANOG, RPLP0, H2AFZ, HMGCS1, ACSL1, GPAT3, FADS2, FASN, FDX1, BID, GPX4 and HIF1A were upregulated (P<0.05) in EGFR-. In conclusion, treatment with EGFR inhibitor lead to partial meiotic block with functional gap junctions, related to higher oocyte developmental potential, due to the lower apoptotic index and the increased expression of genes related do development, suggesting better embryo quality.
Descrição
Palavras-chave
Conteúdo lipídico, Desenvolvimento embrionário, Maturação oocitária in vitro, Sinalização do EGFR