Kaempferol can be used as the single antioxidant in the in vitro culture medium, stimulating sheep secondary follicle development through the phosphatidylinositol 3-kinase signaling pathway

Abstract

This study evaluated the effect of addition of kaempferol alone or combined with other antioxidants (transferrin, selenium and ascorbic acid) on in vitro culture of sheep isolated secondary follicles and if PI3K pathway is involved in kaempferol action. Secondary follicles were isolated and cultured for 12 days in α-Minimal Essential Medium (α-MEM) supplemented with BSA, insulin, glutamine and hypoxanthine (α-MEM: antioxidant free-medium) or in this medium also added by transferrin, selenium and ascorbic acid (AO: base medium with antioxidants). Moreover, different concentrations of kaempferol (0.1; 1 or 10 μM) were added to the different base media (α-MEM or AO). After culture, glutathione (GSH) levels, mitochondrial activity and meiotic resumption were evaluated. In addition, inhibition of PI3K activity was performed through pretreatment in medium supplemented with LY294002. After 12 days, the percentage of normal follicles was higher (P < 0.05) in AO base medium than the other treatments and similar (P > 0.05) to α-MEM supplemented with 1 or 10 μM kaempferol Moreover, α-MEM plus 1 or 10 μM kaempferol and AO medium showed similar (P > 0.05) follicular diameter, fully-grown oocytes, and GSH levels. However, at the end of the culture, antrum formation was higher (P < 0.05) in α-MEM + 1 μM kaempferol than in AO, and similar (P > 0.05) to α-MEM + 10 μM kaempferol. In addition, oocytes cultured in α-MEM supplemented with 1 μM kaempferol showed greater (P < 0.05) levels of active mitochondria than α-MEM + 10 μM kaempferol and AO medium. The rates of meiotic resumption were similar (P > 0.05) among α-MEM + 1 μM kaempferol and AO medium. LY294002 significantly inhibited antrum formation, follicular diameter and the percentage of fully grown oocytes stimulated by 1 μM kaempferol. In conclusion, 1 μM kaempferol can be used as the single antioxidant present in the base medium, replacing the addition of transferrin, selenium and ascorbic acid during in vitro culture of ovine secondary follicles, maintaining follicular survival, increasing active mitochondria levels, and promoting the oocyte meiotic resumption. Moreover, the development of the ovine secondary follicle stimulated by kaempferol is mediated by PI3K pathway.