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.

This study characterized the expression of melatonin receptor type 1 (MT1 ) 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 MT1 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, MT1 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.

Epigallocatechin-3-gallate attenuates cyclophosphamide-induced damage in mouse ovarian tissue via suppressing inflammation, apoptosis, and expression of phosphorylated Akt, FOXO3a and rpS6.

This study was conducted to evaluate the protective effects of epigallocatechin-3-gallate (EGCG) against ovarian toxicity in cyclophosphamide-treated mice and to verify the possible involvement of phosphorylated Akt, FOXO3a and rpS6 in the EGCG actions. Mice received saline solution (i.p.; control) or a single dose of cyclophosphamide (200 mg/kg body weight, i.p.) or mice were pretreated with N-acetylcysteine (150 mg/kg body weight, i.p.; positive control) or with EGCG (5, 25 or 50 mg/kg body weight, i.p.) once daily for three days followed by injection with single dose of cyclophosphamide (200 mg/kg body weight, i.p.). Thereafter, the mice were euthanized, and the ovaries were harvested and destined to histological (follicular morphology and activation), immunohistochemistry (cleaved caspase-3 and TNF-α) and fluorescence (mitochondrial activity and GSH concentrations) analyses. Furthermore, we examined the participation of p-Akt, p-FOXO3a and p-rpS6 in the protective effects of EGCG in cyclophosphamide-induced ovarian damage by immunohistochemical staining. The results showed that pretreatment with N-acetylcysteine or EGCG at 25 and 50 mg/kg before cyclophosphamide administration preserved the normal follicular morphology, prevented primordial follicle loss, reduced atresia, inflammation, and mitochondrial damage, and increased GSH concentrations compared to the only cyclophosphamide treatment. Additionally, pretreatment with 25 mg/kg EGCG regulated phosphorylated Akt, FOXO3a and rpS6 after cyclophosphamide treatment. In conclusion, short-time pretreatment with 25 mg/kg EGCG can prevent follicle loss in cyclophosphamide-treated mice by reducing oxidative damage, inflammation, and apoptosis, and regulating of p-Akt, p-FOXO3a and p-rpS6.

Melatonin Attenuates Cyclophosphamide-Induced Primordial Follicle Loss by Interaction with MT1 Receptor and Modulation of PTEN/Akt/FOXO3a Proteins in the Mouse Ovary.

This study evaluated the protective effect of melatonin before cyclophosphamide administration on ovarian function and its potential mechanism in a mouse model. Two studies were performed. In the first, mice were pretreated with melatonin (10, 20, or 30 mg/kg body weight, i.p.) once daily for 3 days, followed by injection with a single dose of cyclophosphamide (200 mg/kg body weight, i.p.) 30 min after the last melatonin injection. The second study analyzed whether melatonin type 1 and/or 2 receptors mediate the effects of melatonin on the ovary through administration of non-selective MT1/MT2 antagonist (luzindole) or selective MT2 antagonist (4-PPDOT) before the treatment with melatonin plus cyclophosphamide. After treatment groups, the ovaries were harvested and destined to histology, immunohistochemistry, and fluorescence analyses. Lastly, we examined the p-PTEN, p-Akt, and p-FOXO3a participation in the protective effect of melatonin in cyclophosphamide-induced ovarian damage. Results demonstrated that pretreatment with 20 mg/kg melatonin before cyclophosphamide administration showed more morphologically normal follicles, attenuated primordial follicle loss, decreased growing follicle atresia and mitochondrial damage, and increased GSH concentrations. Furthermore, treatment with luzindole blocked the protective effects of melatonin against the damage caused by cyclophosphamide. Additionally, pretreatment with 20 mg/kg melatonin regulated the PTEN/Akt/FOXO3a signaling pathway components after cyclophosphamide treatment. In conclusion, pretreatment with 20 mg/kg melatonin prevented primordial follicle loss and reduced apoptosis and oxidative damage in the mouse ovary during experimental chemotherapy with cyclophosphamide. Furthermore, the MT1 receptor and PTEN/Akt/FOXO3a proteins mediated these cytoprotective effects.

Gallic acid promotes the in vitro development of sheep secondary isolated follicles involving the phosphatidylinositol 3-kinase pathway.

This study was conducted to evaluate the effect of addition of gallic acid as the single antioxidant to the base medium for in vitro culture of sheep secondary follicles and if the phosphatidylinositol 3-kinase (PI3K) pathway is involved in the action of gallic acid. Secondary follicles were isolated and cultured for 12 days in α-MEM supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium, and ascorbic acid (control medium: α-MEM+) or in α-MEM supplemented with BSA, insulin, glutamine, hypoxanthine and different concentrations of gallic acid (25, 50 or 100 µM), thus replacing transferrin, selenium and ascorbic acid in the medium. Follicle morphology, glutathione (GSH), and mitochondrial activity, and meiotic resumption were evaluated. Furthermore, inhibition of PI3K pathway was performed by pretreatment with LY294002. After 12 days of culture, the follicle survival in a medium containing 100 µM gallic acid was similar (P > 0.05) to α-MEM+ and greater (P < 0.05) compared with other gallic acid concentrations. Antrum formation, follicle diameter, GSH, and mitochondrial activity, and meiotic resumption, however, were greater (P < 0.05) when 100 µM gallic acid was included in the α-MEM+ culture medium compared with the control medium. Furthermore, LY294002 inhibited (P < 0.05) follicle survival, development, and meiotic resumption stimulated by 100 µM gallic acid. In conclusion, concentration of 100 µM of gallic acid can be a substitute for transferrin, selenium, and ascorbic acid in the base medium during in vitro culture of sheep secondary follicles, inducing follicle development likely through the PI3K pathway.

Rutin prevents cisplatin-induced ovarian damage via antioxidant activity and regulation of PTEN and FOXO3a phosphorylation in mouse model.

The aims of the present study were to evaluate the protective effects of rutin during cisplatin-induced ovarian toxicity in mice and to verify the possible involvement of the phosphatase and tension homolog (PTEN)/Forkhead box O3a (FOXO3a) pathway in the rutin actions. Mice received saline solution (control, 0.15 M, i.p.) or cisplatin (5 mg/Kg body weight, i.p.) or they were pretreated with N-acetylcysteine (positive control; 150 mg/Kg of body weight [p.o.]) or with rutin (10, 30 or 50 mg/Kg body weight, p.o.) before cisplatin (5 mg/Kg body weight, i.p.) once daily for 3 days. Next, the ovaries were harvested and destined to histological (follicular morphology and activation), immunohistochemical (cell proliferation and apoptosis) and fluorescence (reactive oxygen species [ROS], glutathione [GSH] and mitochondrial activity) analyses. Moreover, the expression of phosphorylated PTEN (p-PTEN) and FOXO3a (p-FOXO3a) were evaluated to investigate a molecular mechanism by which rutin would prevent the cisplatin-induced ovarian damage. The results showed that pretreatment with N-acetylcysteine or 10 mg/Kg rutin before cisplatin preserved the percentage of normal follicles and cell proliferation, reduced apoptosis and ROS levels and increased active mitochondria and GSH levels compared to the cisplatin treatment (P < 0.05). Cisplatin treatment increased p-PTEN and decreased p-FOXO3a expression in follicles, which was prevented by 10 mg/kg rutin. In conclusion, treatment with 10 mg/Kg rutin has the potential to protect the ovarian follicles against cisplatin-induced toxicity through its antioxidant effects and PTEN/FOXO3a pathway.