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

Abstract

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) α-MEM + -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.

Resveratrol promotes in vitro activation of ovine primordial follicles by reducing DNA damage and enhancing granulosa cell proliferation via phosphatidylinositol 3‐kinase pathway

“…These data indicate that the PI3K pathway is potentially involved in ovine primordial follicle growth in vitro, which is in agreement with our previous study (Santos et al, 2017) and those reported for other species (rat: Keating et al, 2009;Abramovich et al, 2010; mice: Zhao et al, 2014;Gorre et al, 2014;cat: Thuwanut, Comizzoli, Wildt, Keefer, & Songsasen, 2017). A possible explanation for the involvement of the PI3K pathway in the activation observed in the α-MEM + is the presence of insulin in the medium, which acts primarily by activating this pathway (Khorami, Movahedi, Huzwah, & Sokhini, 2015).…”

“…The culture was performed according to Santos et al (2017). In the laboratory, ovarian cortex samples from each ovarian pair were cut into slices approximately 3 mm × 3 mm (1-mm thick) in size using a needle and scalpel.…”

“…However, until now, there has been minimal attention to what signalling pathways regulate the activation of primordial follicles in domestic species. To our knowledge, only one study has reported that PI3K is involved in follicular activation in sheep (Santos et al, ).…”

“…Primordial follicle activation is characterized by the transformation of the surrounding pregranulosa cells from a flattened to a cuboidal shape and by their proliferation, followed by the growth of the oocytes (Hirshfield, 1991). Some factors have been identified as being positive regulators of follicular activation in ovine species such as kit ligand (Cavalcante et al, 2016) and epidermal growth factor (Santos et al, 2017). In addition, many studies have demonstrated that phosphatidylinositol 3 kinase (PI3K) is one of the major signalling pathways that are involved in the control of follicular activation in mice (Gorre et al, 2014;Zhao et al, 2014) and rat (Abramovich, Irusta, Parborell, & Tesone, 2010;Keating, Mark, Sen, Sipes, & Hoyer, 2009) ovaries.…”

“…apoptosis, growth, oocyte, PI3K, polyphenol reported that PI3K is involved in follicular activation in sheep (Santos et al, 2017).…”