Animal Reproduction (AR)
Animal Reproduction (AR)
Original Article

Blocking the PI3K pathway or the presence of high concentrations of EGF inhibits the spontaneous activation of ovine primordial follicles in vitro

Luciana da Paz dos Santos, Jamile Maiara da Silva Santos, Vanúzia Gonçalves Menezes, Ricássio de Sousa Barberino, Bruna Bortoloni Gouveia, Agnes Yasmin Pitombeira Cavalcante, Thae Lanne Barbosa Gama Lins, Rodrigo José de Sousa Gonçalves, Juliana Jales de Hollanda Celestino, Maria Helena Tavares de Matos

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The aims of this study were to verify the effects of Epidermal Growth Factor (EGF) on the morphology, primordial follicle activation, growth and proliferation of granulosa cells of ovine follicles cultured in situ, as well as the effect of a PI3K inhibitor on the follicular activation. Ten ovine ovaries were divided into fragments, being one fixed for histological analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+ ) alone or supplemented with EGF (1, 10, 50, 100 or 200 ng/mL). Follicles were classified as normal or atretic, as primordial or growing, and the oocyte and follicle diameters were measured. PCNA immunohistochemistry was performed in the fresh control and in treatment that showed the best results for follicular activation. Pharmacologic inhibition of PI3K activity was performed through pretreatment in media added with 50 µM LY294002 for 1 h. The percentage of normal follicles decreased (P < 0.05) after 7 days of culture in all treatments compared to the fresh control. A significant reduction in the percentage of primordial follicles and an increase (P < 0.05) in the growing ones were observed in all treatments compared to fresh control. Furthermore, both the control medium and 1 ng/mL EGF promoted an increase (P < 0.05) in follicular activation compared to other EGF treatments. The PCNA-positive cells in the EGF treatment were higher (P < 0.05) than in fresh control and α-MEM+ . Pretreatment of ovarian tissue with PI3K inhibitor significantly inhibited (P < 0.05) αMEM+ -stimulated primordial follicle activation, but had no effect on EGF-stimulated activation (P > 0.05). In conclusion, PI3K pathway mediates the in vitro spontaneous activation of sheep primordial follicles. Moreover, EGF may act indirectly on follicular activation by promoting granulosa cell proliferation at 1 ng/mL, and EGF inhibited follicle activation in concentrations similar or higher than 10 ng/mL.


culture, folliculogenesis, preantral follicle, sheep.


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