Protein Localization of Epidermal Growth Factor in Sheep Ovaries and Improvement of Follicle Survival and Antrum Formation In Vitro

Abstract

The aims of this study were to characterize EGF protein expression in ovine ovaries and to verify the effect of EGF on the in vitro development of isolated pre-antral follicles. After collection, ovarian tissue was fixed for immunohistochemical analysis. Additional pairs of ovaries were collected, and secondary follicles were cultured for 18 days in α-MEM(+) (control) alone or supplemented with EGF (1, 10 or 50 ng/ml). The immunostaining for EGF was observed in oocytes from pre-antral and antral follicles, in granulosa cells of primary and secondary follicles, as well as in cumulus and mural cells of antral follicles. After 18 days, the results showed that treatment with 50 ng/ml EGF significantly increased the percentage of morphologically normal follicles compared with the control group (α-MEM(+) ) and significantly reduced the precocious extrusion of oocytes and increased the percentage of antral follicles compared with the control and 1 ng/ml EGF. All the treatments induced a progressive and significant increase of the follicular diameter throughout the period of culture. However, there were no significant differences in follicular diameter or in the daily growth rate among treatments. In conclusion, this study demonstrated the presence of EGF in ovine ovaries. Moreover, 50 ng/ml EGF increased the percentage of normal follicles and improved antrum formation in isolated ovine follicles after 18 days of in vitro culture.

Use of protocatechuic acid as the sole antioxidant in the base medium for in vitro culture of ovine isolated secondary follicles

“…The present study was the first to evaluate the effect of different concentrations of PCA on the in vitro culture of ovarian follicles. The three antioxidants selenium, transferrin and ascorbic acid have been generally added to the in vitro culture of ovine isolated follicles at the same concentrations used in the current study, maintaining between 60 and 80% of follicular survival at day 12 of culture (Barberino et al., ; Santos et al., ). After culture of ovarian follicles, selenium or its active form (sodium selenite) reduced ROS concentration and increased the total antioxidant capacity and the levels of glutathione peroxidase (GSH‐Px) (Abedelahi et al., ), which is a selenium‐dependent protein (Vega, Fernández‐Sánchez, Fernández, Menéndez, & Sanz‐Medel, ).…”

“…Therefore, the addition of antioxidant substances to the in vitro culture medium improves cellular development through the balance between ROS and the antioxidant system (Abedelahi, Salehnia, Allameh, & Davoodi, ; Talebi, Zavareh, Kashani, Lashgarbluki, & Karimi, ). Some antioxidants such as selenium, transferrin and ascorbic acid have been commonly added to the base culture medium of ovine ovarian follicles (Barberino et al., ; Luz et al., , ; Santos et al., ). Recently, a substance that has demonstrated important antioxidant action is protocatechuic acid (Guan, Bao, Jiang, & An,; Li, Wang, Chen, & Chen1, ).…”

Estratégias para a melhoria da eficiência do cultivo folicular in vitro: Importância da suplementação do meio e estudo das alterações epigenéticas

“…A proteína albumina sérica bovina (BSA) é muito utilizada no meio de CIV por favorecer o desenvolvimento folicular e a produção de oócitos meioticamente competentes (Rodrigues et al, 2010), bem como manter a estrutura tridimensional folicular (Itoh, Kacchi, Abe, Sendai, & Hoshi, 2002). Há ainda outras substâncias, como hormônios: insulina, FSH, hormônio do crescimento (GH) e tiroxina (Ferreira et al, 2016;Cadenas et al, 2017;Ferreira et al, 2020;Paes et al, 2020), fatores de crescimento: fator semelhante à insulina tipo 1 (IGF-1), fator de crescimento epidérmico (EGF), fator fibroblástico básico (FGF-2) e fator endotelial vascular (VEGF; Costa et al, 2014;Santos et al, 2014a;Santos et al, 2014b;Cadenas et al, 2017), MC produzido a partir de CTMs (Bezerra et al, 2019) e antioxidantes, como anetol (Sá et al, 2020) que também ao serem adicionados ao meio estimulam e favorecem o desenvolvimento dos folículos cultivados in vitro.…”

Growth differentiation factor‐9 improves development, mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles

“…However, in sheep, the current culture system still has low efficiency with a small number of mature oocytes and embryos produced. In an attempt to improve these results, the addition of growth factors in the culture medium has been used to enhance the growth of secondary follicles (Cunha et al, ; Santos et al, ). Among these factors, the importance of growth differentiation factor‐9 (GDF‐9) in the follicular development is remarkable.…”