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.

Melatonin protects against cisplatin-induced ovarian damage in mice via the MT1 receptor and antioxidant activity.

This study evaluated the receptor- and/or antioxidant stress-mediated mechanisms by which melatonin prevents the ovarian toxicity of cisplatin treatment. The expression of the MT1 receptor in mouse ovaries was investigated by immunohistochemistry. Pretreatment with melatonin (5, 10, or 20 mg/kg body weight, i.p.) before cisplatin (5 mg/kg body weight, i.p.) was administered to mice once daily for 3 days (phase I). The pharmacological modulation via melatonin type 1 and/or 2 receptors was analyzed by administration of receptor antagonists (luzindole: nonselective MT1/MT2 antagonist; 5 mg/kg body weight or 4-phenyl-2-propionamidotetralin: selective MT2 antagonist; 4 mg/kg body weight) once daily for 3 days, 15 min before the treatment with melatonin and cisplatin (phase II). Thereafter, the ovaries were harvested and used for histological (morphology and activation), immunohistochemical (PCNA, activated caspase-3 and bcl-2 expression), terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, and fluorescence (reactive oxygen species [ROS], glutathione [GSH], and active mitochondria levels) analyses. The expression of the MT1 protein in mouse ovaries was documented. Pretreatment with 20 mg/kg melatonin before cisplatin administration preserved the normal follicular morphology and cell proliferation rate, reduced apoptosis, ROS production, mitochondrial damage and increased GSH expression, as compared to the cisplatin treatment alone. Additionally, administration of the nonselective MT1/MT2 receptor antagonist inhibited the melatonin ovarian protection from the cytotoxic effects of cisplatin. However, administration of a selective MT2 antagonist did not modify the protective effects observed at 20 mg/kg melatonin. In conclusion, pretreatment with 20 mg/kg melatonin effectively protected the ovaries against cisplatin-induced damage. Moreover, the MT1 receptor and melatonin antioxidant effects mediated this cytoprotective activity.

Aortocaval fistula delays gastric emptying of liquid test meal in awake rats.

Arteriovenous anastomoses disrupt cardiovascular and renal homeostasis, eliciting hemodynamic adjustments, resetting the humoral pattern, and inducing cardiac hypertrophy. Because acute circulatory imbalance alters gut motor behavior, we studied the effects of arteriovenous fistula placement on the gastric emptying (GE) of a liquid meal in awake rats. After laparotomy, we created an aortocaval fistula (ACF) by aorta and cava wall puncture with a 21-, 23-, or 26-gauge needle. The ACF was not created in the control group, which underwent sham operation. After 12, 24, or 48 h, mean arterial pressure, heart rate, and central venous pressure were continuously recorded, and cardiac output was estimated by thermal dilution. The rats were then gavage fed a test meal (i.e., phenol red in glucose solution), and fractional dye retention was determined 10, 20, or 30 min later. The effect of prior bleeding on ACF-induced GE delay, the role of neuroautonomic pathways, and changes in plasma hormone levels (i.e., angiotensin II, arginine vasopressin, atrial natriuretic peptide, corticosterone, and oxytocin) were evaluated. When compared with the sham-operated group, ACF rats exhibited arterial hypotension, higher (P < 0.05) heart rate, central venous pressure, and cardiac output values and increased (P < 0.05) gastric dye retention, a phenomenon prevented by bilateral subdiaphragmatic vagotomy and hexamethonium treatment. Pirenzepine also impaired the occurrence of gastric delay in subjects with ACF. In addition to causing hyperkinetic circulation, ACF placement delayed the GE of liquid in awake rats, an effect that likely involves a parasympathetic pathway.

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.

Amburana cearensis leaf extract protects against cisplatin-induced ovarian damage through regulation of p-PTEN and p-Akt proteins in mice.

Objectives: To evaluate the effects of Amburana cearensis leaf extract against cisplatin-induced ovarian toxicity in mice and involvement of p-PTEN and p-Akt proteins. Materials and Methods: A. cearensis ethanolic leaf extract was analyzed by high-performance liquid chromatography (HPLC). Mice were pretreated once daily for 3 days as follows: (1) the control group was pretreated with oral administration (o.p.) of saline solution, followed by intraperitoneal (IP) injection of saline solution. The other groups were pretreated (o.p.) with (2) saline solution (cisplatin group), (3) N-acetylcysteine (positive control), with (4) 50, or (5) 200 mg/kg body weight of A. cearensis extract, followed by injection of 5 mg/kg body weight (IP) of cisplatin. The ovaries were harvested and destined for histological (follicular morphology), immunohistochemistry (apoptosis and cell proliferation), and fluorescence (reactive oxygen species [ROS], glutathione concentrations [GSH], and active mitochondria) analyses. Furthermore, immunoexpression of p-PTEN and p-Akt was evaluated to elucidate a potential mechanism by which A. cearensis extract could prevent cisplatin-induced ovarian damage. Results: After HPLC analysis, protocatechuic acid was detected in the extract. The pretreatment with N-acetylcysteine or A. cearensis extract maintained the percentage of normal follicles and cell proliferation, reduced apoptosis and ROS concentrations, and increased GSH concentrations and mitochondrial activity compared with cisplatin treatment. Furthermore, pretreatment with A. cearensis extract regulated p-PTEN and p-Akt immunoexpression after cisplatin exposure. Conclusion: Pretreatment with A. cearensis extract prevented cisplatin-induced ovarian damage through its anti-oxidant actions and by modulating the expression of phosphorylated PTEN and Akt proteins.

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.

Cyclooxygenase-2 contributes to functional changes seen on experimental hemorrhagic cystitis induced by ifosfamide in rat urinary bladder.

PURPOSE: Ifosfamide (IFS) is often involved in the occurrence of hemorrhagic cystitis due to direct contact of its metabolite acrolein with uroepithelium. It has been shown that COX-2 is involved in this pathogenesis. Thus, we aimed to study the functional changes on the urinary bladder in the putative modifications induced by IFS, as well as the COX-2 role in this process. MATERIALS AND METHODS: IFS-treated rats were evaluated by cystometrography in absence or presence of COX inhibitors indomethacin or etoricoxib or in the presence of mesna. Experiments with isolated strips of urinary bladder obtained from animals with IFS-induced cystitis, either treated or not treated with COX inhibitors or mesna, were performed. Histological analyses, immunohistochemistry for COX-2, and measurement of plasma PGE(2) were also performed. RESULTS: IFS treatment caused severe inflammation of the bladder tissue. Cystometrography recordings of IFS-treated rats revealed bladder with increased micturition frequency and enhanced filling intravesical pressure. Contractility of the isolated smooth muscle from the rat's bladder with IFS-induced cystitis showed decreased force development in response to KCl and CCh. Almost all effects induced by IFS were ameliorated by the use of COX inhibitors or mesna. Enzyme expression in the urinary bladder tissue was positive, and plasma concentration of PGE(2) was increased in IFS-treated animals and decreased significantly in etoricoxib-treated animals. CONCLUSIONS: IFS causes important changes in the micturition physiology in rats, and the inhibition of the isoenzyme COX-2 could be an important event that could prevent the detrimental effects elicited by IFS-induced hemorrhagic cystitis.

Sildenafil inhibits duodenal contractility via activation of the NO-K+ channel pathway.

Phosphodiesterase type-5 (PDE5) specifically cleaves cyclic guanosine monophosphate (cGMP), a key intracellular secondary messenger. The PDE5 inhibitor sildenafil is a well-known vasodilator that also has gastrointestinal myorelaxant properties. In the present study, we further investigated sildenafil-induced myorelaxation in rat isolated duodenum, assessing its interaction with nitric oxide (NO) synthase and K(+) channel opening. The spontaneous contractions of duodenal strips were reversibly inhibited by sildenafil (0.1-300 microM) in a concentration-dependent manner [mean (95% confidence interval); EC(50) = 6.8 (2.7-17.3) microM]. The sildenafil-induced myorelaxation was significantly decreased by the NO synthase inhibitor N-nitro-L-arginine methyl ester [increasing the EC(50) value to 41.9 (26.1-67.3) microM]. Sodium nitroprusside or forskolin pretreatments enhanced the sildenafil-induced myorelaxation. In isolated strips pretreated with BaCl(2) (0.2 mM), 4-aminopyridine (4-AP, 3 mM), or glybenclamide (1 microM), the sildenafil-induced EC(50) value was significantly increased to 32.8 (19.1-56.4), 27.1 (15.2-48.3) and 20.1 (16.4-24.7) microM, respectively. Minoxidil (50 microM) or diazoxide (100 microM) also significantly attenuated the sildenafil-induced potency. In conclusion, the NO synthase/cyclic nucleotide pathway activation is involved in sildenafil-induced inhibition of spontaneous duodenal contractions. Its pharmacological action seems to be influenced by K(+) channel opening, especially the voltage-sensitive ones, being inhibited by 4-AP and K(ATP) channels, sensitive to glybenclamide.