Proteínas ligantes dos receptores de fatores de crescimento em complexo cumulus-oócito bovino

Abstract

The aim of this study was to characterize the Grb10 and Grb14 mRNA and protein expression in COCs derived from follicles of different stages of development (1-3, 4-6, 6-8 and >8mm in diameter) and determinate the involvement of the estradiol in the Grb10 and/or Grb14 mRNA expression. Firstly, a pool of the 80 COCs from follicles at 3 to 8mm was used to demonstrate Grb14 and Grb10 mRNA expression in denuded oocytes and respective cumulus cells. The expression was detected in oocyte and cumulus cells. To characterize the experimental model, oocytes from different follicle size were submitted to nuclear maturation assessment and evaluated for competence to reach metaphase II. Maturation competence began in oocytes from follicles larger than 2mm in diameter (P<0,01) and increased throughout follicular development, since, oocytes from follicles of 1-3mm and 4-6mm had lower maturation rate than those from follicles of 6-8 and >8mm (P<0,05). In the first experiment, Grb10 and Grb14 mRNA expression was assessed by qRT-PCR in COCs from different follicle size. The expression of Grb10 mRNA in oocytes from 1-3 and 4-6mm follicles was higher (P<0,05) than those from 6-8 and >8mm follicles. However the Grb14 mRNA relative expression was higher in oocytes of group 1-3mm decreasing (P<0,05) as the follicle size increase. In addition, we performed the localization of the Grb10 protein by immunofluorescence analysis. Positive fluorescence signal for Grb10 was detected in all analyzed samples. To evaluate an hormonal influence on the Grb10 and Grb14 mRNA expression, oocytes from follicles at 3-8mm were cultured in the presence of follicular cells in medium supplemented with 17β-estradiol and/or fulvestrant (an estradiol antagonist). The mRNA expression did not show statistical difference between treatments groups (P>0,05). Based on this results, we conclude that Grb10 and Grb14 mRNA and Grb10 protein are expressed COCs in cattle. In addition, the differential expression observed in COCs from different follicle size suggest an involvement of the Grb10 and Grb14 genes in the bovine oocyte competence.