Immunolocalization of melatonin receptor type 1 in the sheep ovary and involvement of the PI3K/Akt/FOXO3a signaling pathway in the effects of melatonin on survival and in vitro activation of primordial follicles
Ricássio de Sousa Barberino, T.J.S. Macedo, T.L.B.G. Lins, Vanúzia Gonçalves Menezes, Regina Lúcia dos Santos Silva, Alane Pains Oliveira do Monte, Raimundo C. Palheta, Johan Smitz, M.H.T. Matos
Abstract
Abstract This study characterized the expression of melatonin receptor type 1 (MT 1 ) protein in sheep ovaries, evaluated melatonin effects on primordial follicle survival and development after in vitro culture of ovarian tissue and verified the possible involvement of the phosphatidylinositol‐3‐kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway in the melatonin actions. Ovine ovarian fragments were cultured in α‐modified minimum essential medium alone (α‐MEM + ) or supplemented with 100, 500, or 1000 pg/ml melatonin for 7 days. PI3K inhibition was performed through pretreatment of ovarian fragments with LY294002. Thereafter, immunohistochemistry was performed to evaluate the expression of cleaved caspase‐3, Akt, phosphorylated‐Akt, and phosphorylated‐FOXO3a (p‐FOXO3a). The immunohistochemical localization of the MT 1 receptor protein was documented in sheep preantral and antral follicles. After in vitro culture, 100 pg/ml melatonin showed higher follicular survival and activation than α‐MEM + and other melatonin concentrations. After PI3K inhibition, there was an increase in cleaved caspase‐3‐positive follicles, and a decrease in the primordial follicle activation, Akt phosphorylation, and nuclear exclusion of p‐FOXO3a. In conclusion, MT 1 receptor protein is present in the sheep ovary. Furthermore, 100 pg/ml melatonin maintains survival and stimulates activation of primordial follicles through the PI3K/Akt/FOXO3a signaling pathway after in vitro culture of sheep ovarian tissue.