Efeito de fatores de crescimento no cultivo in situ sobre o desenvolvimento e apoptose de folículos pré-antrais ovinos

Abstract

The manipulation and culture of oocytes included in ovarian preantral follicles is one of the main tools currently used for studies of the initial folliculogenesis and rescue of primary oocytes, to cultivate them in vitro until maturation. The objective of this research was to study an efficient culture system for this stage of follicles, using IGF-1, EGF and FSH. Ovarian cortex fragments from sheep, were cultured in situ for eight days in αMEM+ added with IGF-1, FSH and EGF, isolated and/or associated. Samples were analyzed by classical histology and immunohistochemistry (TUNEL), in order to measare follicular diameters and classify follicles as primordial and developing (primary and secondary), normal / abnormal and with or without apoptosis. The relative mRNA expressions of the apoptosis-related genes, Bcl2, Bax and Caspases 3 and 9 in the ovarian fragments were evaluated by the real-time PCR technique. The culture with different concentrations of IGF-1, demonstrated that 50 ng/mL provided greater activation of follicle growth, compared to the other concentrations (P <0.05). FSH-associated IGF-1 improved follicular activation, normality, and reduced apoptosis (P <0.05). The relative expression of Caspase 9 was reduced (P <0.05) in cultures containing IGF-1, associated or not to FSH, but no difference was observed for the other genes (P> 0.05). Likewise, the association of IGF-1 to EGF with or without FSH addition was efficient for improving the in vitro culture of ovarian sheep fragments for 8 days, either in morphological quality or in the reduction of apoptosis (P <0.05 ). It is concluded that 50 ng/ml of IGF-1 is the most effective concentration for the development of cultured preantral follicles included in ovary ovarian tissue, for 8 days. The association of IGF-1 to EGF or FSH is efficient to improve the development of follicles and reduce the levels of apoptosis in these culture conditions, favoring the balance between the studied apoptotic factors.