RESUMO
Physical exercise differentially increases body temperature according to the time of day, which shows the importance of circadian rhythm in thermal regulation. Given its contribution in central pathways involved in thermoregulation, orexin A could play a role in the regulation of core body temperature during and after exercise. To test this hypothesis, we assessed the effect of exercise, performed at two times of day, on core temperature and on the amount of orexin A in the production zone, i.e., the dorsal hypothalamus. Forty-nine male Wistar rats underwent forced treadmill exercise during the HG phase and HL phase of core temperature. Basal core temperature was recorded continuously for 48 h by implanted telemetric sensors in 11 rats. Regulation of core temperature during exercise (20 min) and after each exercise (60 min) was modeled with a modified logistic-type function. During HG exercise, core temperature curve reached a significantly higher maximum (asymptote: +0.70 ± 0.10 °C) and took longer to attain the strongest inclination of the core temperature regulation curve (Xmid: 3.46 ± 0.72 min). After HG exercise, time of recovery was significantly longer than after HL exercise. In male rats, thermoregulatory response to acute physical exercise was influenced by the time of day. There was no effect of either physical activity or time of day on the level of orexin A in the dorsal hypothalamus. Our results suggest that orexin A in the dorsal hypothalamus is not involved in the effects of physical exercise on thermoregulation.
Assuntos
Regulação da Temperatura Corporal , Temperatura Corporal , Animais , Masculino , Ratos , Temperatura Corporal/fisiologia , Regulação da Temperatura Corporal/fisiologia , Hipotálamo/metabolismo , Orexinas/metabolismo , Ratos WistarRESUMO
BACKGROUND: Whether differentiated thyroid cancer (DTC) occurring concomitantly with Graves' disease (GD) is more aggressive and bound to a less favorable outcome is controversial. OBJECTIVE: Aim of this multicenter retrospective study was to compare baseline features and outcome of DTC patients with GD (DTC/GD+) or without GD (DTC/GD-). PATIENTS: Enrolled in this study were 579 patients referred to five endocrine units (Cagliari, Pavia, Pisa, Siena, and Varese) between 2005 and 2014: 193 patients had DTC/GD+ , 386 DTC/GD-. Patients were matched for age, gender and tumor size. They underwent surgery because of malignancy, large goiter size, or relapse of hyperthyroidism in GD. RESULTS: Baseline DTC features (histology, lymph node metastases, extrathyroidal extension) did not differ in the two groups, except for multifocality which was significantly more frequent in DTC/GD+ (27.5% vs. 7.5%, p < 0.0001). At the end of follow-up (median 7.5 years), 86% of DTC/GD+ and 89.6% DTC/GD- patients were free of disease. Patients with persistent or recurrent disease (PRD) had "biochemical disease" in the majority of cases. Microcarcinomas were more frequent in the DTC/GD+ group (60% vs. 37%, p < 0.0001) and had an excellent outcome, with no difference in PRD between groups. However, in carcinomas ≥ 1 cm, PRD was significantly more common in DTC/GD+ (24.4% vs. 11.5%; p = 0.005). In the whole group, univariate and multivariate analyses showed that GD+ , lymph node involvement, extrathyroidal invasion, multifocality and tall cell histotype were associated with a worse outcome. Female gender and microcarcinomas were favorable features. No association was found between baseline TSH-receptor antibody levels and outcome. Graves' orbitopathy (GO) seemed to be associated with a better outcome of DTC, possibly because patients with GO may early undergo surgery for hyperthyroidism. CONCLUSIONS: GD may be associated with a worse outcome of coexisting DTC only if cancer is ≥ 1 cm, whereas clinical outcome of microcarcinomas is not related to the presence/absence of GD.
Assuntos
Adenocarcinoma/mortalidade , Diferenciação Celular , Doença de Graves/complicações , Neoplasias da Glândula Tireoide/mortalidade , Tireoidectomia/mortalidade , Adenocarcinoma/etiologia , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Neoplasias da Glândula Tireoide/etiologia , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/cirurgiaRESUMO
BACKGROUND: Recidivism rates in substance-addicted patients placed in institutions according to §64 of the German legal code are approximately 50%, 3 years after discharge from inpatient treatment. The recidivism rates of patients with premature termination of inpatient treatment who had then been referred back to prison and were finally discharged into the community are unknown. RESEARCH QUESTION: Is premature termination of treatment a risk factor for recidivism? METHODS: Patients released from forensic treatment according to § 64 of the German legal code were followed up for violent and non-violent recidivism. Full data were acquired for Baden-Württemberg patients released in 2010 and 2011 with regular vs. premature termination of treatment. RESULTS: All measures revealed highly significant group differences: 48% of the patients discharged after subsequent prison sentences recidivated within the first year and 73% within 3 years after discharge. Among recidivists, the severity of offences was much higher (odds ratio > 3.8 each). Regularly discharged patients also re-offended to a remarkable extent (50%). DISCUSSION: Patients serving prison sentences after unsuccessful forensic treatment are a high-risk group for recidivism. Alternative concepts of clinical and legal treatment of this group should be developed.
Assuntos
Crime/legislação & jurisprudência , Crime/psicologia , Pacientes Desistentes do Tratamento/psicologia , Prisioneiros/legislação & jurisprudência , Prisioneiros/psicologia , Psicoterapia/legislação & jurisprudência , Transtornos Relacionados ao Uso de Substâncias/reabilitação , Adulto , Feminino , Seguimentos , Psiquiatria Legal/legislação & jurisprudência , Alemanha , Humanos , Masculino , Estudos Prospectivos , Psicotrópicos , Recidiva , Fatores de Risco , Transtornos Relacionados ao Uso de Substâncias/psicologiaRESUMO
Primary thyroid leiomyosarcoma (LMS) is an extremely rare tumor. We report a case of a 47-year-old male with a rapidly growing neck mass and disfagia. Preoperative investigations were diagnostic of anaplastic carcinoma. Total thyroidectomy with partial esophagectomy and dissection of right infrahyoid muscles was performed. Through histolological and immunohistochemical evaluations a primary thyroid high-grade LMS was diagnosed. At 2 months of follow-up a local recurrence was detected and consequently the patient was submitted to chemotherapy with partial response. He is still alive 9 months after surgery. Diagnosis of primary thyroid LMS is difficult due to its similarity to other more common thyroid tumors. To date, there is no standard therapy and prognosis is poor.
Assuntos
Leiomiossarcoma/cirurgia , Neoplasias da Glândula Tireoide/cirurgia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Terapia Combinada , Doxorrubicina/administração & dosagem , Esofagectomia/métodos , Esôfago/patologia , Esôfago/cirurgia , Humanos , Ifosfamida/administração & dosagem , Leiomiossarcoma/diagnóstico , Leiomiossarcoma/tratamento farmacológico , Leiomiossarcoma/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia/tratamento farmacológico , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia , TireoidectomiaRESUMO
Gait is one of the most basic movements, and walking activity accomplished in dual task conditions realistically represents daily life mobility. Much is known about diurnal variations of gait components such as muscle power, postural control, and attention. However, paradoxically only little is known about gait itself. The aim of this study was to analyze whether gait parameters show time-of-day fluctuation in simple and dual task conditions. Sixteen young subjects performed sessions at five specific hours (06:00, 10:00, 14:00, 18:00 and 22:00 h), performing a single (walking or counting) and a dual (walking and counting) task. When performing gait in dual task conditions, an additional cognitive task had to be carried out. More precisely, the participants had to count backwards from a two-digit random number by increments of three while walking. Spatio-temporal gait parameters and counting performance data were recorded for analysis. Walking speed significantly decreased, while stride length variability increased when the task condition switched from single to dual. In the single-task condition, diurnal variations were observed in both walking speed and counting speed. Walking speed was higher in the afternoon and in the evening (14:00 and 22:00 h) and lower in the morning (10:00 h). Counting speed was maximum at 10:00 and 14:00 h and minimum at 18:00 h. Nevertheless, no significant diurnal fluctuation was substanytiated in the dual task condition. These results confirm the existing literature about changes in gait between single and dual task conditions. A diurnal pattern of single-task gait could also be highlighted. Moreover, this study suggests that diurnal variations faded in complex dual task gait, when the cognitive load nearly reached its maximum. These findings might be used to reduce the risk for falls, especially of the elderly.
Assuntos
Ritmo Circadiano , Marcha , Acidentes por Quedas , Idoso , Cognição , Humanos , Equilíbrio Postural , CaminhadaRESUMO
Earth's gravity acts both as a mechanical stimulus on the body and as a sensory stimulus to the vestibular organ, which is transmitted into the brain. The vestibular system has been recently highlighted as the cornerstone of the multisensory cortex and of the dorsal hippocampus related to spatial cognition. Consequently, we have hypothesized that the vestibular sensory perception of gravity by the otoliths might also play a crucial role during the first stages of development in both sensorimotor and cognitive functions and the construction and perception of the 'self' and related functions of orientation and navigation. We have investigated an original mouse model (Head Tilted mice, B6Ei.GL-Nox3het/J) suffering from a selective congenital absence of vestibular otolithic gravisensors. We report that mouse pups suffered from a delay in the acquisition of sensorimotor reflexes, spatial olfactory guidance, path integration, and ultrasonic communication, while maternal care remained normal. We demonstrate that development has a critical period dependent on the vestibular otolithic sensory perception of gravity, probably temporally between the somesthetic and visual critical periods. The symptoms expressed by the congenital otolithic-deficient mice are similar to validated mouse models of autism and highlight the significance of vestibular graviception in the pathophysiology of development.
Assuntos
Orientação/fisiologia , Percepção Espacial/fisiologia , Vestíbulo do Labirinto/fisiologia , Animais , Encéfalo , Córtex Cerebral , Cognição/fisiologia , Feminino , Gravitação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Orientação Espacial/fisiologia , Membrana dos Otólitos/fisiologia , Sensação/fisiologia , Lobo Temporal , Vestíbulo do Labirinto/crescimento & desenvolvimentoRESUMO
The aim of the study was to test the effect of total sleep deprivation on performance and time-of-day pattern of subjective visual vertical (SVV) and postural control. Nineteen healthy, young participants (4 women and 15 men 21.9 ± 1.2 yr) were engaged in two counterbalanced experimental sessions with or without total sleep deprivation. Oral temperature, Karolinska Sleepiness Scale, and visual analogic scale for fatigue, postural control, and SVV were randomly measured every 4 h, from 0600 to 2200. A linear mixed model was used to capture the effect of time of day and sleep condition as factors. A classical adjusted COSINOR function was then used to modelize this daily variation. After the control night of sleep, SVV as well as oral temperature, sleepiness, and fatigue showed significant time-of-day variation, contrasting with measures of postural control which remained stable across the day. After sleep deprivation, SVV showed no diurnal variation, but its mean deviation value increased by 29%. Postural control capability also decreased after sleep deprivation, with a higher center of pressure surface (+70.4%) and total length (+7.37%) but remained stable throughout the day. These results further confirm the negative effect of sleep loss on postural control capability. Even if a direct relationship cannot be confirmed, the disruption of SVV capacity after sleep deprivation could strongly play a role in postural control capacity changes. Sleep deprivation should be considered as a potent factor involved in balance loss and subsequent fall. NEW & NOTEWORTHY The topic of sleep deprivation and postural control is not understood, with discrepancy among results. This study described that postural control displays a stable level throughout the day and that sleep deprivation, even if it increases postural sway, does not affect this stable diurnal pattern. The modification of the perception of the vertical level after sleep deprivation could strongly play a role in the observed changes in postural control capacity.
Assuntos
Equilíbrio Postural/fisiologia , Postura/fisiologia , Privação do Sono/fisiopatologia , Sono/fisiologia , Adulto , Ritmo Circadiano/fisiologia , Fadiga/fisiopatologia , Feminino , Humanos , Masculino , Vigília/fisiologia , Adulto JovemRESUMO
LH-RH effects on LH/hCG receptors content, on basal and LH-stimulated cAMP and cGMP release by cultured porcine granulosa cells, were investigated by radioreceptor analysis and radioimmunoassay. It was found that LH-RH additions (10, 100, 1000 or 10,000 ng/ml medium) increased the number of LH/hCG binding sites in granulosa cells. LH or hCG (0.1, 1, 10 or 100 ng/ml) increased both cAMP and cGMP secretion by the cell culture. LH-RH alone (0.1, 1, 10, 100 or 1000 ng/ml) had the same stimulating effect on both cyclic nucleotides. The addition of LH-RH (100 ng/ml) to the medium supplemented with LH (10, 100, 1000, 10,000 or 100,000 ng/ml) enhanced LH-stimulated cAMP and cGMP output from the culture. The present observations suggest that LH-RH can act as synergist of LH and of hCG increasing a number of LH/hCG receptors and stimulating basal and LH-induced cyclic nucleotide release by ovarian cells.
Assuntos
AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Hormônio Liberador de Gonadotropina/fisiologia , Células da Granulosa/metabolismo , Hormônio Luteinizante/fisiologia , Receptores do LH/metabolismo , Animais , Células Cultivadas , Feminino , Masculino , Ovário/metabolismo , Radioimunoensaio , Ensaio Radioligante , SuínosRESUMO
The aim of our in vitro experiments was to discover the effects of steroid hormones (progesterone, androstenedione and estradiol-17 beta, 10, 100, 1000 or 10,000 pg/ml medium) on the output of cyclic nucleotides adenosine 2',3'-cyclic monophosphate (cAMP) and guanosine 2',3'-cyclic monophosphate (cGMP) by granulosa cells isolated from human ovaries. The dramatic increase of cAMP release after progesterone treatment was observed. Low doses of androstenedione (10 or 100 pg/ml) significantly decreased while higher doses (1000 or 10,000 pg/ml) tended to increase cAMP output. All doses of estradiol significantly inhibited cAMP release. Low doses (10 or 100 pg/ml) of all investigated steroid inhibited while higher doses (1000 or 10,000 pg/ml) stimulated cGMP output. cGMP-stimulating effects of progesterone and androstenedione were expressed significantly more than that of estradiol. The influence of progestagen, androgen and estrogen on cyclic nucleotide release by granulosa cells may suggest the involvement of cAMP- and cGMP-dependent intracellular mechanisms in the realization of steroid hormone effects within human ovaries.
Assuntos
Androstenodiona/farmacologia , AMP Cíclico/biossíntese , GMP Cíclico/biossíntese , Estradiol/farmacologia , Células da Granulosa/metabolismo , Progesterona/farmacologia , Adulto , Células Cultivadas , Feminino , Células da Granulosa/efeitos dos fármacos , Humanos , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/metabolismo , RadioimunoensaioRESUMO
OBJECTIVES: The aim of the present study was to determine the activity of superoxide dismutase (SOD), total antioxidant status (TAS) of the hen granulosa cells, and selected biochemical parameters, including calcium, phosphorus, sodium, potassium, glucose, cholesterol, proteins, in the culture medium of granulosa cells after exposing them to ascorbic acid in vitro conditions. METHODS: Ovarian granulosa cells of hens were incubated with various doses of ascorbic acid (E1 0.09 mg/ml, E2 0.13 mg/ml, E3 0.17 mg/ml, E4 0.33 mg/ml, E5 0.5 mg/ml). RESULTS: Ascorbic acid did not manifest antioxidant potential and higher doses of ascorbic acid (0.17; 0.33 and 0.5 mg/ml) decreased the activity of SOD in granulosa cells. Vitamin application resulted in a significantly (p<0.05) higher accumulation of Na+ and K+ in culture media of granulosa cells and decreased the concentration of glucose and proteins. CONCLUSION: These results indicate that ascorbic acid might be involved in the regulation of selected biochemical and physiological processes in ovarian granulosa cells.
Assuntos
Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Meios de Cultura/farmacologia , Células da Granulosa/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Células Cultivadas , Galinhas , Meios de Cultura/metabolismo , Relação Dose-Resposta a Droga , Feminino , Glucose/metabolismo , Células da Granulosa/metabolismo , Potássio/metabolismo , Proteínas/metabolismo , Sódio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
Hypergravity disrupts the circadian regulation of temperature (Temp) and locomotor activity (Act) mediated through the vestibular otolithic system in mice. In contrast, we do not know whether the anatomical structures associated with vestibular input are crucial for circadian rhythm regulation at 1 G on Earth. In the present study we observed the effects of bilateral vestibular loss (BVL) on the daily rhythms of Temp and Act in semipigmented rats. Our model of vestibular lesion allowed for selective peripheral hair cell degeneration without any other damage. Rats with BVL exhibited a disruption in their daily rhythms (Temp and Act), which were replaced by a main ultradian period (τ <20 h) for 115.8 ± 68.6 h after vestibular lesion compared with rats in the control group. Daily rhythms of Temp and Act in rats with BVL recovered within 1 wk, probably counterbalanced by photic and other nonphotic time cues. No correlation was found between Temp and Act daily rhythms after vestibular lesion in rats with BVL, suggesting a direct influence of vestibular input on the suprachiasmatic nucleus. Our findings support the hypothesis that the vestibular system has an influence on daily rhythm homeostasis in semipigmented rats on Earth, and raise the question of whether daily rhythms might be altered due to vestibular pathology in humans.
Assuntos
Ritmo Circadiano/fisiologia , Vestíbulo do Labirinto/fisiologia , Animais , Temperatura Corporal/fisiologia , Hipergravidade , Masculino , Atividade Motora/fisiologia , Ratos , Ratos Long-EvansRESUMO
Insomniacs report decreased performance in daily routines, which may have detrimental consequences for car driving. We compared changes over time in driving performance (measured as Standard Deviation of Lateral Position - SDLP) and background EEG between 20 untreated insomnia patients (52-70 years old) and 21 normal sleepers (54-73 years old) during a 1h on-the-road driving test after a normal night of sleep, in the morning. SDLP did not differ between groups and increased slightly over time to similar degrees in both groups. EEG alpha and beta power were lower in insomniacs as compared to normal sleepers. Alpha and beta power slightly reduced during driving in normal sleepers but remained at a constant low level in insomniacs. Changes in EEG power and SDLP were not related. It is concluded that on-the-road driving performance does not differ between older insomniacs and older normal sleepers and that changes in spectral EEG measures of cortical arousal and in driving performance are not related.
Assuntos
Condução de Veículo , Eletroencefalografia , Distúrbios do Início e da Manutenção do Sono/fisiopatologia , Fatores Etários , Idoso , Nível de Alerta/fisiologia , Estudos de Casos e Controles , Potenciais Evocados/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
The aim of our studies was to examine whether IGF-binding protein (IGFBP)-4 is involved in the control of the secretion of various ovarian substances and also the mediation of the effects of several hormones and growth factors on this secretion. For this purpose, we carried out the transfection of porcine granulosa cells with a cDNA sense construct, increasing IGFBP-4 synthesis. We then compared the release of IGFBP-3, progesterone, oxytocin and IGF-I by control and transfected cells cultured with and without porcine LH (100 ng/ml), porcine GH (100 ng/ml), IGF-I (10 ng/ml), oxytocin (10 ng/ml) and estradiol-17beta (100 ng/ml). The concentration of IGFBP-4 produced was assessed using ligand blotting, and the release of progesterone, oxytocin, IGF-I and IGFBP-3 was evaluated using RIA/IRMA techniques. It was observed that GH, IGF-I, estradiol, LH and oxytocin alter the progesterone, oxytocin, IGF-I and IGFBP-3 release by porcine ovarian granulosa cells. Transfection of these cells with an IBFBP-4 cDNA expression construct significantly increased the IGFBP-4 accumulation in cell-conditioned medium. Furthermore, this transfection significantly reduced progesterone, oxytocin and IGFBP-3 release, and increased IGF-I output in cells cultured in the absence or presence of GH, IGF-I, estradiol and LH. The addition of oxytocin, but not of other tested substances, fully or partially prevented the effects of IGFBP-4 overexpression on IGFBP-3, IGF-I, but not on progesterone release. The present results suggested that IGFBP-4, as well as GH, IGF-I, estradiol, LH and oxytocin, is a potent regulator of porcine ovarian steroid (progesterone), nonapeptide hormone (oxytocin), growth factor (IGF-I) and growth factor-binding protein (IGFBP-3) release. IGFBP-4 is an inhibitor of basal progesterone, oxytocin and IGFBP-3 release and a stimulator of IGF-I output by porcine ovarian cells. The action of IGFBP-4 on the ovary can be mediated by (1) inhibition of oxytocin release, (2) suppression of receptor/postreceptor events induced by other hormones and IGF-I and (3) stimulation of IGF-I release.
Assuntos
Células da Granulosa/metabolismo , Proteína 5 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Fator de Crescimento Insulin-Like I/farmacologia , Transfecção , Animais , Estradiol/farmacologia , Feminino , Células da Granulosa/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Hormônio Luteinizante/farmacologia , Ocitocina/metabolismo , Ocitocina/farmacologia , Progesterona/metabolismo , Radioimunoensaio , Proteínas Recombinantes/farmacologia , SuínosRESUMO
The aim of our in vitro experiments was to examine if IGF binding protein (IGFBP)-3 is involved in control of bovine ovarian secretory activity. For this purpose we performed the transfection of bovine granulosa cells with cDNA sense and antisense constructs increasing or inhibiting IGFBP-3 synthesis. The release of IGFBP-3, progesterone, oxytocin, IGF-I and prostaglandins F (PGF) and E (PGE) by control and transfected cells was compared. The transfected ovarian cells were cultured with and without bLH (100 ng/ml), bGH (100 ng/ml), IGF-I (10 ng/ml), oxytocin (10 ng/ml) and oestradiol-17beta (100 ng/ml). The concentration of IGFBP-3 produced was assessed using ligand and western blotting and secretion of progesterone, oxytocin, IGF-I, PGF and PGE was evaluated using RIA/IRMA techniques. Transfection of cells with the sense IGFBP-3 cDNA construct resulted in the expected increase in IGFBP-3 release, whereas the antisense IGFBP-3 construct induced the expected reduction in IGFBP-3 output. The granulosa cells transfected to overexpress IGFBP-3 had an increase in IGF-I, PGF and PGE release, and a decrease in basal and hormone- or growth factor-induced accumulation of progesterone and oxytocin. The granulosa cells transfected to have reduced IGFBP-3 expression gave primarily significant opposite findings. The present results suggest the involvement of IGFBP-3 in control of bovine ovarian steroid, peptide hormone, growth factor and prostaglandin release. IGFBP-3 is a physiological stimulator of IGF-I and prostaglandin release and an inhibitor of steroid and peptide hormone output.
Assuntos
Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Hormônios/farmacologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Animais , Bovinos , DNA Antissenso/genética , DNA Complementar/genética , Estradiol/farmacologia , Feminino , Hormônio do Crescimento/farmacologia , Técnicas In Vitro , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Hormônio Luteinizante/farmacologia , Ocitocina/metabolismo , Ocitocina/farmacologia , Progesterona/metabolismo , Prostaglandinas E/metabolismo , Prostaglandinas F/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
The aims of this study on porcine ovarian granulosa cells were to examine the effect of GH on oxytocin (OT), IGF-I and IGF-I receptors, IGF-binding protein-3 (IGFBP-3), progesterone and prostaglandin E (PGE), as well as to determine whether IGF-I and/or OT may be mediators of GH action. The cells were cultured either with porcine GH (pGH) (1 ng/ml to 10 microg/ml or 100 ng/ml only), antiserum against IGF-I (0.1%), antiserum against OT (0.1%) or a combination of GH (10 ng/ml) with antiserum against IGF-I or antiserum against OT (0.1%). The secretion of IGF-I, OT, IGFBP-3, progesterone and PGE was determined using RIA/IRMA, whilst the IGF-I binding sites were measured using a radioreceptor assay. It was observed that pGH increased the secretion of IGF-I and the abundance of IGF-I binding sites in granulosa cells. Furthermore, GH inhibited OT release, stimulated progesterone and PGE output, but had no significant effect on IGFBP-3 secretion. Immunoneutralization of IGF-I by antiserum against IGF-I inhibited PGE secretion, but it did not influence progesterone or IGFBP-3 secretion. Binding of OT by antiserum suppressed IGFBP-3, PGE, but not progesterone secretion. Neither immunoneutralization of IGF-I nor OT substantially prevented the effects of GH on progesterone, IGFBP and PGE. These observations demonstrate the involvement of GH, IGF-I and OT in the control of porcine ovarian secretory activity and the ability of GH to regulate IGF-I and OT production and IGF-I reception. Nevertheless, lack of correlation between the effects of GH, antiserum against IGF-I and antiserum against OT, as well as the inability of blockade of IGF-I or OT to prevent the effects of GH, suggests that IGF-I and OT, despite their dependence on GH, do not mediate GH action on ovarian cells.
Assuntos
Células da Granulosa/efeitos dos fármacos , Hormônio do Crescimento/farmacologia , Suínos/metabolismo , Animais , Técnicas de Cultura de Células , Meios de Cultivo Condicionados , Feminino , Células da Granulosa/metabolismo , Hormônio do Crescimento/fisiologia , Soros Imunes , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/biossíntese , Fator de Crescimento Insulin-Like I/fisiologia , Ocitocina/antagonistas & inibidores , Ocitocina/biossíntese , Ocitocina/fisiologia , Progesterona/biossíntese , Prostaglandinas E/biossínteseRESUMO
Thrombopoietin (TPO) is known to be involved in megakariocytopoesis, but its role in the control of ovarian function is unknown. The aims of this study were to determine whether TPO can regulate the proliferation, apoptosis and secretory activity of ovarian cells, to identify possible intracellular mediators of TPO action, especially protein kinase A (PKA), and to define their interrelationships within ovarian cells. We investigated the effect of TPO treatment (0, 1, 10 or 100 ng/ml) on the following characteristics of cultured porcine ovarian follicles, determined using SDS-PAGE and Western blotting, immunocytochemistry, RIA and ELISA: the expression of intracellular peptides associated with proliferation (PCNA), apoptosis (Bax), tyrosine kinase (TK, phosphotyrosine), Cdc2/p34 kinase, PKA and the transcription factor CREB-1, and the secretion of progesterone, androstenedione, estradiol-17beta, oxytocin, inhibin A, inhibin B, IGF-I, transforming growth factor-2beta (TGF-2beta) and IGF-binding protein 3 (IGFBP-3). The involvement of PKA-dependent pathways was examined by evaluating the effect of a PKA blocker (KT5720, 1 microg/ml), either alone or in combination with TPO, on the parameters listed above. A TPO-induced increase in expression of PCNA, Bax, PKA, TK, Cdc2/p34 and CREB was observed. Furthermore, TPO was able to inhibit androstenedione, estradiol, TGF-2beta and IGFBP-3 secretion, and to stimulate oxytocin, inhibin A, inhibin B and IGF-I secretion. Progesterone secretion was not stimulated. The PKA blocker KT5720, when given alone, reduced the expression of Bax and TGF-2beta, augmented the expression of PKA, CREB and oxytocin, but did not influence the secretion of progesterone, androstenedione, estradiol, IGFBP-3, inhibins A and B or IGF-I. When given together with TPO, the PKA blocker prevented or reversed the action of TPO on PKA, CREB, androstenedione, estradiol, IGFBP-3, oxytocin, but not its effect on Bax, TGF-2beta or inhibin B. On the other hand, treatment with KT5720 augmented the effect of TPO on progesterone, inhibin A and IGF-I. These results provide the first evidence that TPO may be a potent regulator of ovarian function (e.g. proliferation, apoptosis and the secretion of peptide hormones, steroids, growth factors and growth factor-binding protein, as well as of the expression of some intracellular messengers). Furthermore, they demonstrated the importance of PKA in controlling these functions and in mediating the effects of TPO on ovarian cells. It remains possible that other (TK- and Cdc2/p34-dependent) intracellular mechanisms are also involved in mediating TPO action on the ovary.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Folículo Ovariano/efeitos dos fármacos , Trombopoetina/farmacologia , Androstenodiona/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteína Quinase CDC2/metabolismo , Carbazóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Estradiol/metabolismo , Feminino , Immunoblotting/métodos , Imuno-Histoquímica/métodos , Indóis/farmacologia , Inibinas/metabolismo , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Ocitocina/metabolismo , Fosfotirosina/metabolismo , Progesterona/metabolismo , Pirróis/farmacologia , Suínos , Fator de Crescimento Transformador beta/metabolismoRESUMO
The aim of this study was to investigate the actions of insulin-like growth factor I (IGF-I) on the secretory and proliferative functions of rabbit ovarian cells and on early embryogenesis. It was found that addition of IGF-I at a lower concentration (1 ng/ml) stimulated progesterone secretion by cultured rabbit granulosa cells, whilst higher concentrations of IGF-I (10, 100 ng/ml) were inhibitory. IGF-I had no effect on estradiol secretion. Cyclic AMP secretion was slightly increased after addition of IGF-I at 10 ng/ml, but not by higher concentrations. Cyclic GMP secretion was stimulated by IGF-I at 100 ng/ml only. A blocker of protein kinase A, Rp-cAMPS, did not alter progesterone and estradiol secretion but did prevent the action of IGF-I on progesterone secretion. An immunocytochemical study demonstrated that IGF-I significantly increased the proportion of proliferating cell nuclear antigen-positive (PCNA-positive) cells. Rp-cAMP did not change cell proliferation but partially prevented the proliferation-stimulating effect of IGF-I. IGF-I (100 ng/ml) significantly increased the proportion of divided zygotes and the number of embryos reaching the morula/blastocyst stage. Blockers of PKA, Rp-cAMPS and KT5720, reversed the effects of IGF-I on zygote cleavage and embryo development. Addition of IGF-I (100 ng/ml) significantly increased MAPK within the cells (proportion showing immunoreactivity to ERK-1 and ERK-3 antibodies and intensity of a 42 kDa band related to ERK-2). Rp-cAMPS suppressed the basal ERK-2 immunoreactivity but not that of ERK-1 or ERK-3. It completely inhibited the IGF-I-induced activation of ERK-3 but not that of ERK-1 or ERK-2. This in vitro study demonstrates that IGF-I is a potent stimulator of ovarian secretion, proliferation and embryogenesis in rabbit. Its effects are mediated by cAMP/PKA- and, probably by, MAPK-dependent intracellular mechanisms.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , AMP Cíclico/biossíntese , GMP Cíclico/biossíntese , Desenvolvimento Embrionário e Fetal/fisiologia , Células da Granulosa/citologia , Fator de Crescimento Insulin-Like I/fisiologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Animais , Divisão Celular/fisiologia , Células Cultivadas , Desenvolvimento Embrionário , Feminino , Gravidez , Antígeno Nuclear de Célula em Proliferação/metabolismo , CoelhosRESUMO
The aim of the present study was to examine the role of cGMP-dependent intracellular mechanisms in control of ovarian functions. In the first series of experiments we studied the effects of the cGMP analogues 8-pCPT-cGMP (0.001-100 nM), Rp-8-pCPT-cGMPS (0. 01-100 nM), Rp-8-Br-cGMPS (0.01-100 nM), and Rp-8-Br-PET-cGMPS (0.01-100 nM) on the release of progesterone, insulin-like growth factor I (IGF-I) and oxytocin by cultured porcine granulosa cells. In a second series of experiments, the effects of Rp-8-Br-PET-cGMPS (50 nM) and KT5822 (100 ng/ml), specific inhibitor of cGMP-dependent protein kinase (PKG), on cAMP, PKA, oxytocin and the occurrence of apoptosis in cultured cells were compared. The release of hormones and IGF-I into the culture medium was evaluated using a RIA, while the percentage of cells containing visible oxytocin, cAMP, as well as the regulatory and catalytic subunits of PKA was assessed using immunocytochemistry. Occurrence of apoptosis in these cells was detected using the TUNEL method. The stimulatory (8-pCPT-cGMP and Rp-8-pCPT-cGMPS), inhibitory (Rp-8-Br-cGMPS) and biphasic (Rp-8-Br-PET-cGMPS) effect of cGMP analogues on progesterone release was observed. All cGMP analogues used suppressed IGF-I release. All cGMP analogues decreased oxytocin release, but 8-pCPT-cGMP and Rp-8-Br-cGMPS, when given at low doses (0.01-0.1 and 1-10 nM, respectively) stimulated oxytocin output. Both, Rp-8-Br-PET-cGMPS and KT5822 increased the rate of incidence of apoptosis and percentage of cells containing immunoreactive cAMP. Both Rp-8-Br-PET-cGMPS and KT5822 decreased the proportion of cells containing immunoreactive oxytocin and regulatory subunit of PAK KT5822, but not Rp-8-Br-PET-cGMPS, increased the number of cells containing catalytic subunit of PKA. The present observations suggest the involvement of cGMP and PKG in control of the production of steroid, nonapeptide hormone, growth factor, cAMP and cAMP-dependent PKA, as well as the induction of apoptosis in porcine ovarian cells.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Células da Granulosa/efeitos dos fármacos , Suínos , Animais , Domínio Catalítico , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Feminino , Células da Granulosa/citologia , Células da Granulosa/enzimologia , Células da Granulosa/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Ocitocina/metabolismo , Progesterona/metabolismo , Subunidades Proteicas , RadioimunoensaioRESUMO
The aim of our in vitro experiments was to study the effects of EGF on rabbit ovarian cells, as well as the possible mechanisms of these effects. The influence of EGF on steroidogenesis, proliferation, cyclic nucleotides and MAP-kinase in rabbit granulosa cells were studied. Results of RIA showed, that EGF stimulated the release of progesterone (1-100 ng/ml), cAMP (at 100 ng/ml), cGMP (1-100 ng/ml). EGF effect on estradiol output was biphasic: at dose 1 ng/ml it inhibited, whilst at 100 ng/ml it strongly increased estradiol secretion. Immunocytochemical study demonstrated an EGF-induced (10 ng/ml) increase in the proportion of cells revealing proliferating cell nuclear antigen (41% vs 24.7% in control, p < 0.01). EGF (10 ng/ml) increased the proportion of cells with immunoreactivity to ERK-1 (more than two-fold) and ERK-3 (three-fold) members of the MAP-kinase family. Moreover, EGF induced the translocation of ERK-1 to the nucleus, whilst preferentially cytoplasmic localization of ERK-3 was not changed after EGF addition. This can indicate regulation of ERK-1 and -3 by EGF, as well as differential patterns of ERK-1 and ERK-3 expression in response to EGF in cultured granulosa cells. - These results indicate that EGF can be a stimulator of proliferation, steroidogenesis and cyclic nucleotide release by rabbit granulosa cells. Stimulation of cAMP and cGMP release, and activation of ERK-related MAP kinase in granulosa cells after EGF addition indicates the involvement of these intracellular messengers in mediating the EGF action on the ovary.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Células da Granulosa/citologia , Células da Granulosa/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Núcleo Celular/ultraestrutura , Células Cultivadas , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Feminino , Células da Granulosa/efeitos dos fármacos , Cinética , Antígeno Nuclear de Célula em Proliferação/análise , Coelhos , Esteroides/metabolismoRESUMO
The aim of our in vitro experiments was to study the role of oxytocin (OT), cAMP/protein kinase A (PKA), and mitogen-activated protein kinase (ERKs MAP-kinase) in the control of ovarian cell functions as well as the role of PKA and MAPK in mediating OT effects on these processes. The whole porcine ovarian follicles were cultured in the presence or absence of OT (1, 10, 100 ng/ml), PKA inhibitor Rp-cAMPS (10 nM), MAP-kinase inhibitor PD98059 (1 microg/ml), or their combination. The release of prostaglandins F (PGF) and E (PGE) were determined by RIA, PKA (alpha-cat subunit), the proliferation-associated peptide PCNA and ERK-1, -2 expression in cell lyzates were analysed by Western-blotting. OT stimulated the release of PGF and PGE, and accumulation of PKA, ERK-1/-2, and PCNA in cell lysate. PD98059 decreased the basal PGF and PGE output, as well as reduced both ERK-1 and ERK-2 accumulation in cell lysates. Rp-cAMPS decreased PKA accumulation in cell lysates. Rp-cAMPS prevented the OT-induced stimulation of PKA, ERK-1, ERK-2, PGF, and PGE, PD98059 did so for PKA, PGF, and PGE. However, PD98059 reduced either basal or OT-induced p-ERK level. OT-stimulated PCNA accumulation was only slightly modified by these blockers. These observations suggest that OT, PKA, and ERKs MAPK can be involved in the control of PGs release and proliferation of ovarian cells. The influence of OT on both PKA and MAPK, and the ability of PKA and MAPK blockers to prevent completely or partially OT effects suggest, that effects of OT on PGF and PGE can be mediated by both PKA and MAPK. The role of MAPK and PKA in mediating the proliferative effects of OT seems to be minor assuming the involvement of other intracellular messengers.