Efeito da rutina sobre a maturação in vitro de oócitos de ovelhas obtidos de folículos pré-antrais cultivados in vitro
DOI:
https://doi.org/10.5433/1679-0359.2024v45n1p207Palavras-chave:
Antioxidante, Flavonóide, Glutationa, Retomada de meiose, Apoptose.Resumo
Os objetivos deste estudo foram verificar o efeito da rutina sobre a maturação in vitro (MIV) de oócitos provenientes de folículos secundários de ovelhas cultivados in vitro e analisar o possível envolvimento da via mTOR na MIV, sob influência da rutina. Os folículos secundários foram cultivados por 18 dias em meio α-Mínimo Essencial (α-MEM) suplementado com albumina sérica bovina (BSA), insulina, glutamina, hipoxantina, transferrina, selênio, ácido ascórbico e leptina (meio controle: α-MEM+). Em seguida, os folículos foram avaliados quanto à morfologia, formação do antro e diâmetro folicular e taxa de oócitos totalmente crescidos (≥110 µm). Oócitos totalmente crescidos foram submetidos à MIV em meio de cultivo de tecidos 199 (TCM199) suplementado com soro fetal bovino (FBS), hormônio luteinizante (LH), hormônio folículo estimulante recombinante (rFSH) (meio controle MIV) ou neste meio com 0,1, 1 ou 10 µg.mL-1 de rutina. Ao final da MIV, os oócitos foram avaliados quanto à atividade mitocondrial, concentração de espécies reativas de oxigênio (ERO) e glutationa (GSH), porcentagem de retomada de meiose, fragmentação de DNA e envolvimento da via mTOR. Após 18 dias de cultivo in vitro, 77,5% dos folículos estavam normais e 77,7% tornaram-se folículos antrais, com 380,41 µm de diâmetro. Além disso, 70% dos oócitos que cresceram in vitro atingiram diâmetro ≥110 µm e foram submetidos à MIV. A concentração de 10 µg.mL-1 de rutina aumentou significativamente a porcentagem de oócitos que retomaram a meiose (47,27%) em comparação ao meio controle (30,43%). Houve um aumento significativo nas concentrações de ROS e GSH em oócitos maturados com 0.1 µg.mL-1 de rutina em comparação com os outros tratamentos com rutina (p < 0,05). Além disso, a maturação de oócitos em TCM199+ aumentou (p<0,05) o percentual de fragmentação de DNA (30%) comparado ao tratamento com 10 µg.mL-1 de rutina (0%). Após MIV, ambos os tratamentos maturados na presença ou ausência de rapamicina apresentaram porcentagem semelhante de retomada meiótica (61,76% para TCM199 + 10 µg.mL-1 de rutina e 70,73% para TCM199 + 10 µg.mL-1 de rutina + rapamicina) (p>0,05). Em conclusão, a concentração de 10 µg.mL-1 de rutina aumentou a retomada da meiose e reduziu os danos ao DNA.
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