Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity

Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity were studied. The morphological evaluation of the ovaries was performed by histological paraffin embedded and stained with HE. The immunohistochemical expressions of CDC47, VEGF, Flk-1, angiopoietin, Tie-2 and thyroid receptor (TRα) were performed by the technique of streptavidein-biotin-peroxidase. Apoptosis was assessed using the TUNEL kit. The relative expression of thyroid hormone receptors (TRα and TRβ) was assessed by RT-PCR real time. The nuclear expression of CDC47 increased with the stage of maturation of the oocyte and was observed in the follicle cells. Apoptotic bodies were observed in the follicular cells of atretic follicles and postovulatory follicles from the ovaries of 150g and 350g fish. Expression of VEGF and its receptor Flk-1 was also observed in the follicular cells, and the expression of both increased with the maturity of the oocyte, with a higher intensity observed in the full-grown follicle. The expression of angiopoietin and of its receptor (Tie 2) was discrete and moderate respectively. TRα expression was independent of follicular development. However, the 350 g tilapia exhibited higher expression of TRβ compared with the 50 g tilapia. We conclude that the proliferative activity and the expression of VEGF and its receptor increase with follicular maturation and that the TRs expression increases with ovarian maturity in tilapia (Oreochromis niloticus).

Prolactin inhibits auto- and cross-induction of thyroid hormone and estrogen receptor and vitellogenin genes in adult Xenopus (Amphibia) hepatocytes

It is well known that virtually every tissue of the amphibian larvae is highly sensitive to the mutually antagonistic actions of thyroid hormone (TH) and prolactin (PRL), but it is not known if adult amphibian tissues respond similarly to these two hormones. We have previously shown that very low doses of triiodothyronine (T3) rapidly and strongly potentiate the activation of silent vitellogenin (Vit) genes by estrogen (E2) and the autoinduction of estrogen receptor (ER) transcripts in primary cultures of adult Xenopus hepatocytes. This response to T3 is accompanied by the upregulation of thyroid hormone receptor b (TRb) mRNA. Using Northern blot and RNase protection assays, we now show that ovine PRL added for 12 h along with 2 x 10-9 M T3 will completely prevent potentiation of E2 induction of Vit mRNA in primary cultures of adult Xenopus hepatocytes. PRL also abolished the auto-upregulation of TRb mRNA and the cross-activation of autoinduction of ER mRNA. Thus, we show for the first time that the anti-TH action of PRL that is manifested in Xenopus tadpole tissues during metamorphosis is retained in adult liver, and suggest that the mutually antagonistic actions of the two hormones may be brought about by similar molecular mechanisms in larval and adult amphibian tissues

Interaction between NO and oxytocin: Influence on LHRH release

Nitric oxide synthase (NOS)-containing neurons have been localized in various parts of the CNS. These neurons occur in the hypothalamus, mostly in the paraventricular and supraoptic nuclei and their axons project to the neural lobe of the pituitary gland. We have found that nitric oxide (NO) controls luteinizing hormone-releasing hormone (LHRH) release from the hypothalamus acting as a signal transducer in norepinephrine (NE)-induced LHRH release. LHRH not only releases LH from the pituitary but also induces sexual behavior. On the other hand, it is known that oxytocin also stimulates mating behavior and there is some evidence that oxytocin can increase NE release. Therefore, it occurred to us that oxytocin may also stimulate LHRH release via NE and NO. To test this hypothesis, we incubated medial basal hypothalamic (MBH) explants from adult male rats in vitro. Following a preincubation period of 30 min, MBH fragments were incubated in Krebs-Ringer bicarbonate buffer in the presence of various concentrations of oxytocin. Oxytocin released LHRH at concentrations ranging from 0.1 nM to 1 µM with a maximal stimulatory effect (P<0.001) at 0.1 µM, but with no stimulatory effect at 10 µM. That these effects were mediated by NO was shown by the fact that incubation of the tissues with NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, blocked the stimulatory effects. Furthermore, the release of LHRH by oxytocin was also blocked by prazocin, an a1-adrenergic receptor antagonist, indicating that NE mediated this effect. Oxytocin at the same concentrations also increased the activity of NOS (P<0.01) as measured by the conversion of [14C]arginine to citrulline, which is produced in equimolar amounts with NO by the action of NOS. The release of LHRH induced by oxytocin was also accompanied by a significant (P<0.02) increase in the release of prostaglandin E2 (PGE2), a mediator of LHRH release that is released by NO. On the other hand, incubation of neural lobes with various concentrations of sodium nitroprusside (NP) (300 or 600 µM), a releaser of NO, revealed that NO acts to suppress (P<0.01) the release of oxytocin. Therefore, our results indicate that oxytocin releases LHRH by stimulating NOS via NE, resulting in an increased release of NO, which increases PGE2 release that in turn induces LHRH release. Furthermore, the released NO can act back on oxytocinergic terminals to suppress the release of oxytocin in an ultrashort-loop negative feedback

Clinical and molecuar characterization of Brazilian patients with growth hormone gene deletions

Genomic DNA from 23 patients with isolated growth hormone (GH) deficiency (12 males and 11 females: heights -4.9 ± 1.4 SDS) was screened for GH gene deletions by restriction endonuclease analysis of polymerase chain reaction amplification products. Three unrelated patients had typical features of severe GH deficiency and deletions (6.7 kb in two and 7.6 kb in one) of the GH gene. The two patients with 6.7-kb deletions developed growth-attenuating anti-GH antibodies whereas the patient with the 7.6-kb deletion continued to grow with GH replacement therapy. Our finding that 3/23 (~13%) Brazilian subjects had GH gene deletions agrees with previous studies of severe isolated GH deficiency subjects in other populations. Two of three subjects (67%) with deletions developed blocking antibodies despite administration of exogenous GH at low doses. Interestingly, only 1/10 of cases with affected relatives or parental consanguinity had GH-1 gene deletions

Antinociception induced by stimulating amygdaloid nuclei in rats: changes produced by systemically administered antagonists

The antinociceptive effects of stimulating the medial (ME) and central (CE) nuclei of the amygdala in rats were evaluated by the changes in the latency for the tail withdrawal reflex to noxious heating of the skin. A 30-s period of sine-wave stimulation of the ME or CE produced a significant and short increase in the duration of tail flick latency. A 15-s period of stimulation was ineffective. Repeated stimulation of these nuclei at 48-h intervals produced progressively smaller effects. The antinociception evoked from the ME was significantly reduced by the previous systemic administration of naloxone, methysergide, atropine, phenoxybenzamine, and propranolol, but not by mecamylamine, all given at the dose of 1.0 mg/kg. Previous systemic administration of naloxone, atropine, and propranolol, but not methysergide, phenoxybenzamine, or mecamylamine, was effective against the effects of stimulating the CE. We conclude that the antinociceptive effects of stimulating the ME involve at least opioid, serotonergic, adrenergic, and muscarinic cholinergic descending mechanisms. The effects of stimulating the CE involve at least opioid, ß-adrenergic, and muscarinic cholinergic descending mechanisms.

Thyroid hormone regulates protein expression in C6 glioma cells

Thyroid hormone (T3) is essential to normal brain development. Previously, we have shown that T3 induces cerebellar astrocyte proliferation. This effect is accompanied by alteration in glial fibrillary acidic protein (GFAP) and fibronectin organization. In the present study, we report that the C6 glioma cell line, which expresses GFAP and is classified as an undifferentiated astrocytic cell type, is a target for T3 action. The C6 monolayers were treated with 50 nM T3 for 3 days, after which the cells were maintained for 2 days without medium changes. In C6 cells, T3 induced the expression of proteins of 107, 73 and 62 kDa. The hormone also up-regulated protein bands of 100 (+50%), 37 (+50%) and 25.5 kDa (+50%) and down-regulated proteins of 94 (-100%), 86.5 (-100%), 68 (-100%), 60 (-100%), 54 (-33%), 51 (-33%) and 43.5 kDa (-33%). We suggest, on the basis of molecular mass, that the 54-, 51- and 43.5-kDa proteins could be the cytoskeletal proteins vimentin, GFAP and actin, respectively. The down-regulation of these proteins may be involved in the effects of thyroid hormone on C6 differentiation.

Low levels of sex hormone-binding globulin and hyperproinsulinemia as markers of increased pancreatic ß-cell demand in men

Low levels of sex hormone-binding globulin (SHBG) are considered to be an indirect index of hyperinsulinemia, predicting the later onset of diabetes mellitus type 2. In the insulin resistance state and in the presence of an increased pancreatic ß-cell demand (e.g. obesity) both absolute and relative increases in proinsulin secretion occur. In the present study we investigated the correlation between SHBG and pancreatic ß-cell secretion in men with different body compositions. Eighteen young men (30.0 ± 2.4 years) with normal glucose tolerance and body mass indexes (BMI) ranging from 22.6 to 43.2 kg/m2 were submitted to an oral glucose tolerance test (75 g) and baseline and 120-min blood samples were used to determine insulin, proinsulin and C-peptide by specific immunoassays. Baseline SHBG values were significantly correlated with baseline insulin (r = -0.58, P<0.05), proinsulin (r = -0.47, P<0.05), C-peptide (r = -0.55, P<0.05) and also with proinsulin at 120 min after glucose load (r = -0.58, P<0.05). Stepwise regression analysis revealed that proinsulin values at 120 min were the strongest predictor of SHBG (r = -0.58, P<0.05). When subjects were divided into obese (BMI >28 kg/m2, N = 8) and nonobese (BMI £25 kg/m2, N = 10) groups, significantly lower levels of SHBG were found in the obese subjects. The obese group had significantly higher baseline proinsulin, C-peptide and 120-min proinsulin and insulin levels. For the first time using a specific assay for insulin determination, a strong inverse correlation between insulinemia and SHBG levels was confirmed. The finding of a strong negative correlation between SHBG levels and pancreatic ß-cell secretion, mainly for the 120-min post-glucose load proinsulin levels, reinforces the concept that low SHBG levels are a suitable marker of increased pancreatic ß-cell demand.

Pituitary glycoprotein hormone a-subunit secretion by cirrhotic patients

Secretion of the a-subunit of pituitary glycoprotein hormones usually follows the secretion of intact gonadotropins and is increased in gonadal failure and decreased in isolated gonadotropin deficiency. The aim of the present study was to determine the levels of the a-subunit in the serum of patients with cirrhosis of the liver and to compare the results obtained for eugonadal cirrhotic patients with those obtained for cirrhotic patients with hypogonadotropic hypogonadism. Forty-seven of 63 patients with cirrhosis (74.6%) presented hypogonadism (which was central in 45 cases and primary in 2), 7 were eugonadal, and 9 women were in normal menopause. The serum a-subunit was measured by the fluorimetric method using monoclonal antibodies. Cross-reactivity with LH, TSH, FSH and hCG was 6.5, 1.2, 4.3 and 1.1%, respectively, with an intra-assay coefficient of variation (CV) of less than 5% and an interassay CV of 5%, and sensitivity limit of 4 ng/l. The serum a-subunit concentration ranged from 36 to 6253 ng/l, with a median of 273 ng/l. The median was 251 ng/l for patients with central hypogonadism and 198 ng/l for eugonadal patients. The correlation between the a-subunit and basal LH levels was significant both in the total sample (r = 0.48, P<0.01) and in the cirrhotic patients with central hypogonadism (r = 0.33, P = 0.02). Among men with central hypogonadism there was a negative correlation between a-subunit levels and total testosterone levels (r = 0.54, P<0.01) as well as free testosterone levels (r = -0.53, P<0.01). In conclusion, although the a-subunit levels are correlated with LH levels, at present they cannot be used as markers for hypogonadism in patients with cirrhosis of the liver.

Insect juvenile hormone: from "status quo" to high society

Juvenile hormone (JH) exerts pleiotropic functions during insect life cycles. The regulation of JH biosynthesis by neuropeptides and biogenic amines, as well as the transport of JH by specific binding proteins is now well understood. In contrast, comprehending its mode of action on target organs is still hampered by the difficulties in isolating specific receptors. In concert with ecdysteroids, JH orchestrates molting and metamorphosis, and its modulatory function in molting processes has gained it the attribute "status quo" hormone. Whereas the metamorphic role of JH appears to have been widely conserved, its role in reproduction has been subject to many modifications. In many species, JH stimulates vitellogenin synthesis and uptake. In mosquitoes, however, this function has been transferred to ecdysteroids, and JH primes the ecdysteroid response of developing follicles. As reproduction includes a variety of specific behaviors, including migration and diapause, JH has come to function as a master regulator in insect reproduction. The peak of pleiotropy was definitely reached in insects exhibiting facultative polymorphisms. In wing-dimorphic crickets, differential activation of JH esterase determines wing length. The evolution of sociality in Isoptera and Hymenoptera has also extensively relied on JH. In primitively social wasps and bumble bees, JH integrates dominance position with reproductive status. In highly social insects, such as the honey bee, JH has lost its gonadotropic role and now regulates division of labor in the worker caste. Its metamorphic role has been extensively explored in the morphological differentiation of queens and workers, and in the generation of worker polymorphism, such as observed in ants.

Oxytocin is a cardiovascular hormone

Oxytocin (OT), a nonapeptide, was the first hormone to have its biological activities established and chemical structure determined. It was believed that OT is released from hypothalamic nerve terminals of the posterior hypophysis into the circulation where it stimulates uterine contractions during parturition, and milk ejection during lactation. However, equivalent concentrations of OT were found in the male hypophysis, and similar stimuli of OT release were determined for both sexes, suggesting other physiological functions. Indeed, recent studies indicate that OT is involved in cognition, tolerance, adaptation and complex sexual and maternal behaviour, as well as in the regulation of cardiovascular functions. It has long been known that OT induces natriuresis and causes a fall in mean arterial pressure, both after acute and chronic treatment, but the mechanism was not clear. The discovery of the natriuretic family shed new light on this matter. Atrial natriuretic peptide (ANP), a potent natriuretic and vasorelaxant hormone, originally isolated from rat atria, has been found at other sites, including the brain. Blood volume expansion causes ANP release that is believed to be important in the induction of natriuresis and diuresis, which in turn act to reduce the increase in blood volume. Neurohypophysectomy totally abolishes the ANP response to volume expansion. This indicates that one of the major hypophyseal peptides is responsible for ANP release. The role of ANP in OT-induced natriuresis was evaluated, and we hypothesized that the cardio-renal effects of OT are mediated by the release of ANP from the heart. To support this hypothesis, we have demonstrated the presence and synthesis of OT receptors in all heart compartments and the vasculature. The functionality of these receptors has been established by the ability of OT to induce ANP release from perfused heart or atrial slices. Furthermore, we have shown that the heart and large vessels like the aorta and vena cava are sites of OT synthesis. Therefore, locally produced OT may have important regulatory functions within the heart and vascular beds. Such functions may include slowing down of the heart or the regulation of local vascular tone.

Role of the hypothalamic pituitary adrenal axis in the control of the response to stress and infection

The release of adrenocorticotropin (ACTH) from the corticotrophs is controlled principally by vasopressin and corticotropin-releasing hormone (CRH). Oxytocin may augment the release of ACTH under certain conditions, whereas atrial natriuretic peptide acts as a corticotropin release-inhibiting factor to inhibit ACTH release by direct action on the pituitary. Glucocorticoids act on their receptors within the hypothalamus and anterior pituitary gland to suppress the release of vasopressin and CRH and the release of ACTH in response to these neuropeptides. CRH neurons in the paraventricular nucleus also project to the cerebral cortex and subcortical regions and to the locus ceruleus (LC) in the brain stem. Cortical influences via the limbic system and possibly the LC augment CRH release during emotional stress, whereas peripheral input by pain and other sensory impulses to the LC causes stimulation of the noradrenergic neurons located there that project their axons to the CRH neurons stimulating them by alpha-adrenergic receptors. A muscarinic cholinergic receptor is interposed between the alpha-receptors and nitric oxidergic interneurons which release nitric oxide that activates CRH release by activation of cyclic guanosine monophosphate, cyclooxygenase, lipoxygenase and epoxygenase. Vasopressin release during stress may be similarly mediated. Vasopressin augments the release of CRH from the hypothalamus and also augments the action of CRH on the pituitary. CRH exerts a positive ultrashort loop feedback to stimulate its own release during stress, possibly by stimulating the LC noradrenergic neurons whose axons project to the paraventricular nucleus to augment the release of CRH.

The neuroimmune-endocrine axis: pathophysiological implications for the central nervous system cytokines and hypothalamus-pituitary-adrenal hormone dynamics

Cytokines are molecules that were initially discovered in the immune system as mediators of communication between various types of immune cells. However, it soon became evident that cytokines exert profound effects on key functions of the central nervous system, such as food intake, fever, neuroendocrine regulation, long-term potentiation, and behavior. In the 80's and 90's our group and others discovered that the genes encoding various cytokines and their receptors are expressed in vascular, glial, and neuronal structures of the adult brain. Most cytokines act through cell surface receptors that have one transmembrane domain and which transduce a signal through the JAK/STAT pathway. Of particular physiological and pathophysiological relevance is the fact that cytokines are potent regulators of hypothalamic neuropeptidergic systems that maintain neuroendocrine homeostasis and which regulate the body's response to stress. The mechanisms by which cytokine signaling affects the function of stress-related neuroendocrine systems are reviewed in this article.

The diversity of abnormal hormone receptors in adrenal Cushing's syndrome allows novel pharmacological therapies

Recent studies from several groups have indicated that abnormal or ectopic expression and function of adrenal receptors for various hormones may regulate cortisol production in ACTH-independent hypercortisolism. Gastric inhibitory polypeptide (GIP)-dependent Cushing's syndrome has been described in patients with either unilateral adenoma or bilateral macronodular adrenal hyperplasia; this syndrome results from the large adrenal overexpression of the GIP receptor without any activating mutation. We have conducted a systematic in vivo evaluation of patients with adrenal Cushing's syndrome in order to identify the presence of abnormal hormone receptors. In macronodular adrenal hyperplasia, we have identified, in addition to GIP-dependent Cushing's syndrome, other patients in whom cortisol production was regulated abnormally by vasopressin, ß-adrenergic receptor agonists, hCG/LH, or serotonin 5HT-4 receptor agonists. In patients with unilateral adrenal adenoma, the abnormal expression or function of GIP or vasopressin receptor has been found, but the presence of ectopic or abnormal hormone receptors appears to be less prevalent than in macronodular adrenal hyperplasia. The identification of the presence of an abnormal adrenal receptor offers the possibility of a new pharmacological approach to control hypercortisolism by suppressing the endogenous ligands or by using specific antagonists for the abnormal receptors.

Sex hormone modulation of serotonin-induced coronary vasodilation in isolated heart

The present study was designed to evaluate the differences in the coronary vasodilator actions of serotonin (5-HT) in isolated heart obtained from naive or castrated male and female rats that were treated with either estrogen or testosterone. Hearts from 12 groups of rats were used: male and female naive animals, castrated, castrated and treated with 17ß-estradiol (0.5 µg kg-1 day-1) for 7 or 30 days, and castrated and treated with testosterone (0.5 mg kg-1 day-1) for 7 or 30 days. After treatment, the vascular reactivity of the coronary bed was evaluated. Baseline coronary perfusion pressure (CPP) was determined and dose-response curves to 5-HT were generated. Baseline CPP differed between male (70 ± 6 mmHg, N = 10) and female (115 ± 6 mmHg, N = 12) naive rats. Maximal 5-HT-induced coronary vasodilation was higher (P<0.05) in naive female than in naive male rats. In both sexes, 5-HT produced endothelium-dependent coronary vasodilation. After castration, there was no significant difference in baseline CPP between hearts obtained from male and female rats (75 ± 7 mmHg, N = 8, and 83 ± 5 mmHg, N = 8, respectively). Castration reduced the 5-HT-induced maximal vasodilation in female and male rats (P<0.05). Estrogen treatment of castrated female rats restored (P<0.05) the vascular reactivity. In castrated male rats, 30 days of estrogen treatment increased (P<0.05) the responsiveness to 5-HT. The endothelium-dependent coronary vasodilator actions of 5-HT are greater in female rats and are modulated by estrogen. A knowledge of the mechanism of action of estrogen on coronary arteries could aid in the development of new therapeutic strategies and potentially decrease the incidence of cardiovascular disease in both sexes.