Efeito do fator de crscimento semelhante à insulina-I (IGF-I) e do fator de crescimento e diferenciação-9 (GDF-9) sobre o desenvolvimento in vitro de folículos pré-antrais ovinos

Abstract

The objective of this work was to study the effect of the insulin like growth factor-1(IGF-1) and growth differentiation factor-9 (GDF-9) on the in vitro development of sheep preantral follicles. For this, three experimental phases were developed. The first phase evaluated the effect of IGF-I on the in vitro culture of sheep isolated secondary follicles. Sixteen ovaries were allocated for the immunolocalization of IGF-I protein. Isolated secondary follicles of other ovaries were cultured for 18 days in control medium (MEM+) or MEM+ supplemented with different concentrations of IGF-I (10, 50 and 100 ng/mL) or the same treatments with the addition of 750 ng/ml of FSH. At the end of the culture, fluorescence, in vitro maturation (IVM) or LH receptor (LHR) expression analyzes were performed. Phase 2 evaluated the influence of GDF-9 on the development of preantral follicles cultured in situ. For this, ovarian fragments were cultured for 7 days in MEM or in MEM supplemented with of GDF-9 (1, 50, 100, 200 or 400 ng/mL). In addition, a second culture in MEM or MEM + 50 ng/mL of GDF-9 was performed in the presence or absence of the PI3K pathway inhibitor. At the end of culture, follicular survival, activation and diameter, immunolocalization of PCNA, activated caspase-3 and pAKT proteins were evaluated. In the phase three, isolated secondary follicles were cultured for 12 days in MEM or MEM + GDF-9 (1, 10, 50 or 100 ng/mL). At the end of culture, oocytes were submitted to IVM, follow by mitochondrial activity and ROS analysis. The results of phase 1 showed that 50 ng/mL of IGF-I + FSH showed more fully-grown oocytes (P <0.05). ROS levels were higher in 100 ng/mL IGF-I + FSH compared to the other groups, with a concomitant decrease of GSH level and mitochondrial activity (P <0.05). In the second phase, follicular survival was higher with the concentration of 50 ng/ml of GFD-9 (P <0.05) compared to other treatments (except 1 ng/mL) concomitant with a greater proliferation and lower apoptosis compared to the control, through of PI3K/AKT pathway. In the last phase, the follicles cultured in 100 ng/ml GDF-9 showed a higher percentage of fully-grown oocytes, MI and higher mitochondrial activity compared to other groups (P <0.05). In conclusion, this study demonstrated the presence of IGF-I protein in sheep ovary. In addition, 50 ng/mL of IGF-I + FSH promotes follicular development and meiotically competent oocytes. The concentration of 50 ng/ml of GDF-9 maintains the survival of primordial follicles cultured in situ, increases cell proliferation and decreases apoptosis via the PI3K/AKT pathway. In isolated secondary follicles, GDF-9 increases the percentage of fully-grown oocytes able to resume meiosis, and enhances mitochondrial activity after IVM.