Kaempferol promotes primordial follicle activation through the phosphatidylinositol 3‐kinase/protein kinase B signaling pathway and reduces DNA fragmentation of sheep preantral follicles cultured in vitro

Abstract

The aim of this study was to evaluate the effects of kaempferol on the morphology, follicular activation, growth, and DNA fragmentation of ovine preantral follicles cultured in situ, and the effects of a phosphatidylinositol 3 kinase (PI3K) inhibitor and the expression of phosphorylated protein kinase B (pAKT) after culture. Ovine ovarian fragments were fixed for histological and terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) analyses (fresh control) or cultured in α‐MEM+ alone (control) or with different concentrations of kaempferol (0.1, 1, 10, or 100\u2009μM) for 7 days. Follicles were classified as normal or atretic, primordial or growing, and the oocyte and follicle diameters were measured. Proliferating cells were analyzed and DNA fragmentation was evaluated by the TUNEL assay. Inhibition of PI3K activity was performed through pretreatment in media added with 50\u2009µM LY294002 for 1\u2009hr and pAKT immunohistochemistry was performed after culture in the absence or presence of LY294002. After culture, the percentage of normal follicles was similar among the treatments (p\u2009>\u20090.05), except for 100\u2009µM kaempferol, which had less normal follicles (p\u2009<\u20090.05). Moreover, kaempferol at 10\u2009μM showed a higher percentage of follicular activation and cell proliferation than the other treatments (p\u2009<\u20090.05) and a percentage of TUNEL‐positive cells similar to that in the fresh control and lower than other treatments (p\u2009<\u20090.05). LY294002 significantly inhibited primordial follicle activation stimulated by α‐MEM+ and 10\u2009μM kaempferol and reduced pAKT expression in those follicles. In conclusion, 10\u2009μM kaempferol promotes primordial follicle activation and cell proliferation through the PI3K/AKT pathway and reduces DNA fragmentation of ovine preantral follicles cultured in vitro.