Caracterização do Grb10 em complexos cumulus-oócito oriundos de folículos bovino de diferentes tamanhos

Abstract

The objective of this study was to investigate the mRNA expression and protein localization of Grb10 gene in bovine cumulus-oocyte complexes (COCs) from different follicle sizes. Firstly, it was investigated the mRNA expression to correlate with maturation rates. COCs from follicles at 1-3, 4-6, 6-8 and >8mm were used to evaluate Grb10 gene expression by qRT-PCR assay and nuclear maturation rates. It was observed that more competent oocytes (from follicles at 6-8 and >8mm; P<0.05), had lower Grb10 mRNA expression levels when compared to the oocytes from follicles at 1-3 and 4-6mm (P<0.05). After it was performed an immunofuorescence analysis in COCs from different follicle sizes (1-3, 4-6, 6-8 and >8mm) to investigate Grb10 protein localization. Samples were incubated with primary antibody: Polyclonal rabbit anti-Grb10 (1:100). Primary antibody was detected using goat anti-rabbit IgG antibody conjugated with Alexa Fluor 488 (1:500). Positive fuorescence signal was detected in all analyzed samples but less evident in COCs from largest follicles. These results characterized Grb10 gene in bovine COC and provide evidences for its involvement during oocyte molecular maturation.