Efeito do kaempferol sobre o desenvolvimento in vitro de folículos ovarianos ovinos

Abstract

The aim of this study was to investigate the effect of kaempferol on the in vitro culture of ovarian tissue and isolated secondary follicles in the ovine species. For this, two experimental phases were performed, divided in two chapters. For the culture of ovarian tissue (in situ culture – chapter 1), fragments of the cortex were cultured for 7 days in control medium (α-MEM+) or in α-MEM+ supplemented with different concentrations of kaempferol (0.1, 1, 10 or 100 μM). The control medium consisted of α-MEM supplemented with insulin, transferrin, selenium, glutamine, hypoxanthine, bovine serum albumin (BSA), penicillin and streptomycin, called α-MEM+. After culture, morphology, activation, growth and DNA fragmentation of the preantral follicles were analyzed. In addition, we performed a pharmacological inhibition of the phosphatidylinositol 3 kinase (PI3K) pathway and the immunohistochemistry for analysis of phosphorylated protein kinase B (pAKT) expression. In chapter 2, the secondary follicles were isolated and cultured for 12 days in α-MEM supplemented with BSA, insulin, glutamine and hypoxanthine (α-MEM: base medium without antioxidants) or in this medium also supplemented with 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), totaling 8 treatments. After culture of isolated follicles, the following parameters were analyzed: morphology, antrum formation, follicular diameter, percentage of oocytes ≥ 110 μm, glutathione (GSH) levels, mitochondrial activity and percentage of in vitro maturation. In Chapter 1, 10 μM kaempferol showed a higher percentage of follicular activation and cell proliferation than the other treatments (P <0.05) and a percentage of TUNEL positive cells similar to those of fresh and less control than other treatments (P <0.05). LY294002 significantly inhibited the activation of primordial follicle stimulated by α-MEM + and 10 μM kaempferol and reduced pAKT expression. Already in Chapter 2, the percentage of normal follicles was higher (P <0.05) in AO medium compared to other treatments and similar (P> 0.05) to α-MEM supplemented with 1 or 10 μM kaempferol. In addition, α-MEM plus 1 or 10 μM kaempferol and AO medium showed follicular diameters, fully grown oocytes and similar GSH levels (P> 0.05). The den formation was higher (P <0.05) in α-MEM + 1 μM kaempferol than in AO and similar (P> 0.05) α-MEM + 10 μM kaempferol. In addition, α-MEM supplemented with 1 μM kaempferol had higher (P <0.05) active mitochondria levels than α-MEM + 10 μM kaempferol and AO medium. There was no difference for meiotic recovery rates (P> 0.05). In conclusion, 10 μM kaempferol promotes activation, reduces DNA fragmentation, and increases pAKT expression of ovine preantral follicles cultured in situ. For secondary follicles, 1 μM of kaempferol can be used as the only antioxidant present in the base medium, replacing the addition of transferrin, selenium and ascorbic acid during in vitro culture and the meiotic recovery of oocytes from ovine secondary follicles.