Immunolocalization of leptin and its receptor in the sheep ovary and in vitro effect of leptin on follicular development and oocyte maturation.

The aims of the study were to characterize leptin and it is receptor (LEPR) proteins immunoexpression in ovine ovaries and to evaluate the effects of leptin on development of secondary follicles cultured in vitro. The ovaries were collected and fixed for immunohistochemical analysis. Additional pairs of ovaries were collected and secondary follicles were isolated and cultured, for 18 days, in α-MEM+ alone or supplemented with 10 or 25 ng/mL of leptin. The antrum formation and fully grown oocytes rates were higher in 25 ng/mL leptin than all treatments. GSH levels and mitochondrial activity were higher in 10 or 25 ng/mL leptin than α-MEM+. 25 ng/mL leptin showed a higher percentage of MII than the α-MEM+. In conclusion, leptin and its receptor are expressed in ovine ovaries and 25 ng/mL leptin promoted higher in vitro maturation rates by improving follicular development, GSH levels and mitochondrial activity of ovine oocytes compared to control medium.

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

We aimed to study the effects of resveratrol on the morphology, DNA fragmentation, follicular activation and cell proliferation after in vitro culture of ovine ovarian tissue, and to verify if PI3K pathway is involved in resveratrol action in the sheep ovary. Ovaries were collected and divided into fragments. One fragment was fixed for histology (fresh control). The remaining fragments were cultured for 7 days in control medium (α-MEM+ ) alone or with resveratrol (2, 10 or 30 µM). After culture, ovarian tissue was destined to morphological analysis. TUNEL and proliferating cell nuclear antigen (PCNA) analyses were performed in the fresh control, α-MEM+ and 2 µM resveratrol. Inhibition of PI3K activity was performed through pre-treatment with LY294002. The percentage of normal follicles was similar between α-MEM+ and 2 µM resveratrol, and higher than those in other resveratrol treatments. An increase in follicular activation was observed in all treatments compared to fresh control. DNA fragmentation decreased in tissues cultured in 2 µM resveratrol compared to α-MEM+ . Moreover, PCNA-positive cells were higher in 2 µM resveratrol than in α-MEM+ . LY294002 inhibited follicular activation stimulated by α-MEM+ and 2 µM resveratrol. In conclusion, 2 µM resveratrol promotes primordial follicle activation compared to the fresh control by reducing DNA fragmentation and stimulating granulosa cell proliferation through activation of the PI3K pathway.