RESUMO
In this study, we investigated the effect of leptin on the ovarian metalloproteinase system in the rat during the ovulatory process. Ovulation was induced in immature rats primed with gonadotropins. In both in vitro and in vivo experiments, we measured i) the protein expression of the ovarian metalloproteinases (matrix metalloproteinases, MMPs) and their tissue inhibitors (TIMPs) by western blot; ii) the gelatinase activity of the ovarian MMPs by zymography; and iii) the inhibitory action of TIMPs by reverse zymography. Using cultures of ovarian explants, leptin increased the activity but not the protein expression of MMP-2 and MMP-9 in both culture medium and ovarian tissue, and the protein expression of TIMPs, without a higher inhibitory action of the gelatinase activity. These results suggest either that the increase in TIMP proteins was not sufficient or that the inhibitory actions of TIMPs were impaired to suppress the MMP activity when the ovaries were directly exposed to leptin. To study the in vivo effect, rats received an acute treatment with high doses of leptin to inhibit ovulation. This treatment increased the expression of both the latent and the active forms of MMP-2 but did not result in a greater activity of MMP-2. In addition, the inhibitory action of TIMP-2 was also increased by this treatment. These results suggest that the administration of high doses of leptin could be regulating the follicle wall degradation, at least in part, by increasing the action of the ovarian TIMP-2 as a result of an extraovarian mechanism or signaling pathway.
Assuntos
Leptina/farmacologia , Metaloproteinases da Matriz/metabolismo , Ovário/efeitos dos fármacos , Inibidores Teciduais de Metaloproteinases/metabolismo , Análise de Variância , Animais , Western Blotting , Relação Dose-Resposta a Droga , Feminino , Leptina/metabolismo , Ovário/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
To investigate the expression of leptin receptors (Ob-R) in the rat hypothalamus-pituitary-ovarian axis, immature rats were treated with eCG/hCG and Ob-R expression was evaluated by western blot analysis. The Ob-R expression increased 24 h after eCG administration in all the tissues assayed. In the hypothalamus, these levels immediately decreased to those obtained without treatment. In the pituitary, the Ob-R expression continued to be elevated 48 h after eCG administration, whereas the hCG injection did not modify these levels. Similar results were obtained with the ovarian long isoform. To assess the effect of leptin on its receptors, Ob-R was assessed in hypothalamus, pituitary and ovarian explants cultured in the presence or absence of leptin (0.3-500 ng/ml). In the hypothalamus, we found a biphasic effect: the Ob-R expression was either reduced or increased at low or high concentrations of leptin respectively. LH-releasing hormone secretion increased at 1 ng/ml. In the pituitary, Ob-R increased at 10 or 30 ng/ml of leptin for the long and short isoforms respectively. Leptin also induced an increase in LH release at 30 ng/ml. In the ovarian culture, the presence of leptin produced an increase in Ob-R expression at different ranges of concentrations and a dose-dependent biphasic effect on the progesterone production. In conclusion, all these results clearly suggest that leptin is able to modulate the expression of its own receptors in the reproductive axis in a differential way. Moreover, the positive or negative effect that leptin exerts on the ovulatory process may be dependent on this regulation.
Assuntos
Expressão Gênica/efeitos dos fármacos , Hipotálamo/metabolismo , Leptina/farmacologia , Ovário/metabolismo , Hipófise/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Animais , Western Blotting , Gonadotropina Coriônica/farmacologia , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/efeitos dos fármacos , Imuno-Histoquímica , Técnicas In Vitro , Ovário/efeitos dos fármacos , Hipófise/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
The aims of this study were to investigate the negative action of leptin on some intraovarian ovulatory mediators during the ovulatory process and to assess whether leptin is able to alter the expression of its ovarian receptors. Immature rats primed with gonadotrophins were used to induce ovulation. Serum leptin concentration was diminished 4 h after human chorionic gonadotrophin (hCG) administration, whereas the ovarian expression of leptin receptors, measured by western blot, was increased by the gonadotrophin treatment. Serum progesterone level, ovulation rate and ovarian prostaglandin E (PGE) content were reduced in rats primed with equine chorionic gonadotrophin (eCG)/hCG and treated with acute doses of leptin (five doses of 5 mug each). These inhibitory effects were confirmed by in vitro studies, where the presence of leptin reduced the concentrations of progesterone, PGE and nitrites in the media of both ovarian explants and preovulatory follicle cultures. We also investigated whether these negative effects were mediated by changes in the expression of the ovarian leptin receptors. Since leptin treatment did not alter the expression of ovarian leptin receptor, the inhibitory effect of leptin on the ovulatory process may not be mediated by changes in the expression of its receptors at ovarian level, at least at the concentrations assayed. In summary, the ovulatory process was significantly inhibited in response to an acute treatment with leptin, and this effect may be due, at least in part, to the direct or indirect impairment of some ovarian factors, such as prostaglandins and nitric oxide.
Assuntos
Leptina/fisiologia , Ovário/fisiologia , Ovulação/efeitos dos fármacos , Animais , Western Blotting/métodos , Depressão Química , Feminino , Gonadotropinas Equinas/farmacologia , Leptina/sangue , Ovário/química , Ovário/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/análise , Receptores para Leptina , Maturidade Sexual , Técnicas de Cultura de TecidosRESUMO
The aims of this study were to investigate the effects of chronic feed deprivation on the ovulatory process, and to assess whether leptin administration is able to alter these effects. Prepuberal rats subjected to food restriction and primed with gonadotrophins were used. Body and ovarian weights were significantly decreased in proportion to the severity of the food restriction. Only the most severe feed deprivation was able to inhibit the ovulation rate. Either buffer or leptin was daily administrated to prepuberal rats fed either ad libitum or with a severe food restriction. Serum progesterone, ovulation rate and ovarian prostaglandin E2 were reduced in rats subjected to food restriction and stimulated by daily administration of leptin in rats fed ad libitum. Negative effects produced by a severe food restriction were partially reversed by chronic administration of leptin. The ovarian endothelium nitric oxide synthase expression was strongly inhibited in rats with food restriction and once again, leptin administration reversed this effect. In summary, the ovulatory process was significantly inhibited in response to a severe decrease in food intake, at least in part, to the direct or indirect impairment of some ovarian factors production as prostaglandins and nitric oxide. Chronic treatment with leptin enhanced the ovulatory process in comparison with control animals, and partially prevented these negative effects produced by a severe malnutrition.
Assuntos
Privação de Alimentos/fisiologia , Leptina/metabolismo , Ovulação/fisiologia , Animais , Dinoprostona/biossíntese , Dinoprostona/metabolismo , Feminino , Leptina/administração & dosagem , Leptina/sangue , Leptina/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Ovulação/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The aim of this study was to investigate the relationship between beta-endorphin and nitric oxide (NO) during the ovulatory process in rats. Immature rats were treated with equine chorionic gonadotrophin-hCG to induce ovulation. An intrabursal injection of beta-endorphin stimulated nitric oxide synthase (NOS) activity. This effect was completely reversed when naltrexone was co-injected with beta-endorphin. The stimulatory action of beta-endorphin on NOS activity was studied to determine whether it was exerted via prostaglandins. Treatment with prostaglandin E(2) (PGE(2)) completely reversed the beta-endorphin-induced stimulation of NOS activity. Moreover, intrabursal injection of meloxicam, an inhibitor of cyclooxygenase 2, increased NOS activity, but this effect was not altered by co-injection with beta-endorphin. The presence of both endothelial NOS (eNOS) and inducible NOS (iNOS) in the ovary at 10 h after hCG treatment was studied by western blot analysis. Local administration of beta-endorphin inhibited the expression of eNOS protein, whereas expression of iNOS protein was not detectable. Ovarian beta-endorphin content was diminished at 10 h after hCG injection. Treatment with prostaglandin synthesis inhibitors in vivo augmented the ovarian beta-endorphin content. In conclusion, these results indicate that beta-endorphin stimulates the activity of ovarian NOS indirectly by inhibiting prostaglandin production.
Assuntos
Dinoprostona/farmacologia , Óxido Nítrico/metabolismo , Ovário/metabolismo , Ovulação/fisiologia , beta-Endorfina/farmacologia , Animais , Western Blotting/métodos , Gonadotropina Coriônica/farmacologia , Dinoprostona/metabolismo , Feminino , Indometacina/farmacologia , Meloxicam , Modelos Animais , Naltrexona/farmacologia , Óxido Nítrico Sintase/análise , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Ovário/química , Antagonistas de Prostaglandina/farmacologia , Ratos , Ratos Sprague-Dawley , Tiazinas/farmacologia , Tiazóis/farmacologia , beta-Endorfina/análise , beta-Endorfina/metabolismoRESUMO
In this research we examined the mechanisms by which ethanol (EtOH) inhibits luteinizing hormone-releasing hormone (LHRH) release from incubated medial basal hypothalamic explants. EtOH (100 mM) stimulated the release of two inhibitory neurotransmitters: gamma-aminobutyric acid (GABA) and beta-endorphin. EtOH also inhibited NO production, indicative of a suppression of nitric oxide synthase (NOS) activity. This inhibition was reversed by naltroxone (10(-8) M), a micro-opioid receptor blocker, indicating that the inhibition of NOS by EtOH is mediated by beta-endorphin. EtOH also blocked N-methyl-d-aspartic acid-induced LHRH release, but the blockade could not be reversed by either the GABA receptor blocker, bicuculline (10(-5) M), naltroxone (10(-8) M), or both inhibitors added together. However, increasing the concentration of naltrexone (10(-6) M) but not bicuculline (10(-4) M) reversed the inhibition. When we lowered the concentration of EtOH (50 mM), the EtOH-induced blockade of LHRH release could be reversed by either bicuculline (10(-5) M), naltroxone (10(-8) M), or the combination of the two blockers. Therefore, GABA is partially responsible for the blockade of N-methyl-d-aspartic acid-induced LHRH release. The block by GABA was exerted by inhibiting the activation of cyclooxygenase by NO, because it was reversed by prostaglandin E(2), the product of activation of cyclooxygenase. Because the inhibition caused by the higher concentration of EtOH could not be reduced by bicuculline (10(-4) M) but was blocked by naltroxone (10(-6) M), the action of alcohol can be accounted for by stimulation of beta-endorphin neurons that inhibit LHRH release by inhibition of activation of NOS and stimulation of GABA release.
Assuntos
Etanol/farmacologia , Hormônio Liberador de Gonadotropina/metabolismo , Hipotálamo/efeitos dos fármacos , Animais , Ácido Araquidônico/farmacologia , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Dinoprostona/farmacologia , Etanol/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Hipotálamo/metabolismo , Técnicas In Vitro , Masculino , N-Metilaspartato/farmacologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I , Nitroprussiato/farmacologia , Ratos , Ratos Wistar , beta-Endorfina/metabolismo , beta-Endorfina/farmacologia , Ácido gama-Aminobutírico/metabolismoRESUMO
beta-Endorphin blocks release of luteinizing hormone (LH)-releasing hormone (LHRH) into the hypophyseal portal vessels by stimulating mu-opiate receptors, thereby inhibiting secretion of LH. LHRH release is controlled by release of nitric oxide from nitricoxidergic (NOergic) neurons in the basal tuberal hypothalamus. To determine whether beta-endorphin exerts its inhibitory action on this NOergic pathway, medial basal hypothalami (MBH) from male rats were incubated with beta-endorphin (10(-8) M). beta-Endorphin decreased basal secretion of LHRH, and significantly inhibited the release of prostaglandin E2 (PGE2), a known stimulant of LHRH release. Incubation of MBH with beta-endorphin at various concentrations (10(-9)-10(-6) M) in vitro decreased the activity of NO synthase (NOS) (measured by the conversion of [14C]arginine to labeled citrulline). Conversely, the activity of NOS was increased by the mu-receptor antagonist, naltrexone (10(-8) M). Not only was the inhibitory action of beta-endorphin on LHRH and PGE2 release blocked by naltrexone (10(-8) M), but it increased NOS activity and LHRH and PGE2 release. beta-Endorphin also stimulated gamma-aminobutyric acid (GABA) release. Because GABA inhibits both nitroprusside (NP-induced PGE2 and LHRH release by blocking the activation of cyclooxygenase by NO, this is another mechanism by which beta-endorphin inhibits NP-induced PGE2 and LHRH release. The results indicate that beta-endorphin stimulates mu-opioid receptors on NOergic neurons to inhibit the activation and consequent synthesis of NOS in the MBH. beta-Endorphin also blocks the action of NO on PGE2 release and, consequently, on LHRH release, by stimulating GABAergic inhibitory input to LHRH terminals that blocks NO-induced activation of cyclooxygenase and consequent PGE2 secretion.