[3T tractography imaging: anatomy and clinical applications]. / Imagen de tractografía 3T: anatomía y aplicaciones clínicas.

Tractography (TG) is the only non-invasive technique that enables the fibres of the white substance to be dissected. This technique can study the projection, association, and commissural fibres, and is an improvement and an important complement to conventional MR imaging. TG is an important tool for preoperative sub-cortical mapping, and there is a good correlation between TG and the direct sub-cortical stimulation technique. TG can have false positives in regions infiltrated by the tumour or with a mass effect. Furthermore, a negative TG does not exclude functional fibre persistence. TG is capable of demonstrating changes in other pathologies (congenital malformations, ischaemic disease and demyelinating diseases).

nSec1 binds a closed conformation of syntaxin1A.

The Sec1 family of proteins is proposed to function in vesicle trafficking by forming complexes with target membrane SNAREs (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein [SNAP] receptors) of the syntaxin family. Here, we demonstrate, by using in vitro binding assays, nondenaturing gel electrophoresis, and specific neurotoxin treatment, that the interaction of syntaxin1A with the core SNARE components, SNAP-25 (synaptosome-associated protein of 25 kD) and VAMP2 (vesicle-associated membrane protein 2), precludes the interaction with nSec1 (also called Munc18 and rbSec1). Inversely, association of nSec1 and syntaxin1A prevents assembly of the ternary SNARE complex. Furthermore, using chemical cross-linking of rat brain membranes, we identified nSec1 complexes containing syntaxin1A, but not SNAP-25 or VAMP2. These results support the hypothesis that Sec1 proteins function as syntaxin chaperons during vesicle docking, priming, and membrane fusion.

A 13-year molecular epidemiological analysis of tuberculosis in San Francisco.

BACKGROUND: We examined the molecular epidemiology of tuberculosis (TB) in San Francisco during a 13-year period encompassing the peak of TB resurgence and subsequent decline to historic low levels. OBJECTIVE: To compare rates of TB caused either by rapid progression of recent Mycobacterium tuberculosis infection or by reactivation of latent infection. METHODS: All TB cases reported from 1991 to 2003 were included. Genotyping was performed to identify clustered cases. RESULTS: The annual TB case rate decreased significantly from 50.8 to 28.8 cases/100000 persons from 1992 to 1999 (P < 0.0001). After 1999, no significant decrease was observed for the population as a whole or in any subgroup examined. Similarly, the rate of clustered cases decreased significantly from 1992 to 1999 (11.4 to 3.1 cases/100000, P < 0.0001). Although the rate of non-clustered cases also declined significantly (25.6 to 17.6 cases/100,000, P < 0.0001), there was a disproportionate reduction in clustered cases (94.7% vs. 50.8%, P < 0.0001). Neither clustered nor non-clustered cases decreased significantly after 1999. CONCLUSIONS: TB case rates reached a plateau despite ongoing application of control measures implemented in 1993. These data suggest that intensification of measures designed to identify and treat persons with latent TB infection will be necessary to further reduce TB incidence.

Neuroactive steroids influence peripheral myelination: a promising opportunity for preventing or treating age-dependent dysfunctions of peripheral nerves.

The process of aging deeply influences morphological and functional parameters of peripheral nerves. The observations summarized here indicate that the deterioration of myelin occurring in the peripheral nerves during aging may be explained by the fall of the levels of the major peripheral myelin proteins [e.g., glycoprotein Po (Po) and peripheral myelin protein 22 (PMP22)]. Neuroactive steroids, such as progesterone (PROG), dihydroprogesterone (5alpha-DH PROG), and tetrahydroprogesterone (3alpha,5alpha-TH PROG), are able to stimulate the low expression of these two myelin proteins present in the sciatic nerve of aged male rats. Since Po and PMP22 play an important physiological role in the maintenance of the multilamellar structure of PNS myelin, we have evaluated the effect of PROG and its neuroactive derivatives, 5alpha-DH PROG and 3alpha,5alpha-TH PROG, on the morphological alterations of myelinated fibers in the sciatic nerve of 22-24-month-old male rats. Data obtained clearly indicate that neuroactive steroids are able to reduce aging-associated morphological abnormalities of myelin and aging-associated myelin fiber loss in the sciatic nerve.

Male sexual behavior and human papillomavirus DNA: key risk factors for cervical cancer in Spain.

BACKGROUND: It is now established that certain types of human papillomaviruses (HPVs) are the sexually transmitted agents etiologically linked to cervical cancer. Studies assessing the contribution of the male's sexual behavior and genital HPV DNA status to the risk of development of cervical neoplasia in sexual partners have yielded inconsistent results. PURPOSE: This study evaluates the role of men's sexual behavior and the presence of HPV DNA in the penis on the development of cervical cancer in their sexual partners in Spain, a low-risk area for cervical neoplasia. METHODS: Husbands (n = 633) of women participating in two case-control studies of cervical neoplasia were interviewed to obtain information on lifestyle habits, including sexual practices. Cytologic samples were taken from the distal urethra and the surface of the glans penis of 183 husbands of case women and of 171 husbands of control women. These samples were analyzed by a polymerase chain reaction-based system using a generic probe and 25 type-specific probes for the detection and typing of HPV DNA. Serologic specimens were also obtained and analyzed for antibodies to Chlamydia trachomatis, Treponema pallidum, herpes simplex virus type II, and Neisseria gonorrhoeae. RESULTS: The presence of HPV DNA in the husbands' penis conveyed a fivefold risk of cervical cancer to their wives (adjusted odds ratio [OR] for HPV DNA positivity = 4.9; 95% confidence interval [CI] = 1.9-12.6). The risk of cervical cancer was strongly related to HPV type (adjusted OR for HPV type 16 = 9.0; 95% CI = 1.1-77.5), to the husbands' number of extramarital partners (adjusted OR = 11.0; 95% CI = 3.0-40.0; for > or = 21 women versus one), and to the number of prostitutes as extramarital sexual partners (adjusted OR = 8.0; 95% CI = 2.9-22.2; for > or = 10 women versus none). Presence of antibodies to C. trachomatis (adjusted OR = 2.6; 95% CI = 1.4-4.6) and an early age at first sexual intercourse of the husband (adjusted OR = 3.2; 95% CI = 1.7-5.9; for < or = 15 years versus > or = 21 years) were also associated with cervical neoplasia in the wife. After adjustment for these variables and for the wife's pack-years of smoking, the husband's smoking was moderately associated with cervical cancer in his wife (adjusted OR = 2.5; 95% CI = 1.4-4.4; for > or = 26.2 pack-years versus none). CONCLUSIONS: The study supports the role of men as vectors of the HPV types that are related to cervical cancer. Life-time number of female sexual partners, number of female prostitutes as sexual partners, and detection of HPV DNA in the penis of husbands are all surrogate markers of exposure to HPV during marriage. IMPLICATIONS: Men who report multiple sexual partners or who are carriers of HPV DNA may be vectors of high-risk HPV types and may place their wives at high risk of developing cervical cancer. Prostitutes are an important reservoir of high-risk HPVs.

[Migraine and evolutionary theory: paths for a clinical approach]. / Migraña y teoria evolutiva: vias para un acercamiento clinico.

INTRODUCTION: Migraine is a very common disorder with a raising incidence. The theory of evolution allow us to explain the emergence of the disorder, due to the advantages that the overreactivity to stimulus provided to ancestral groups of Homo sapiens, and a greater presence of the disorder in modern societies, based in the interactions with external factors. Herein we analyze these points. DEVELOPMENT: Design of organisms and their responses to environmental factors emerge to improve survival. Thus pain and headache can be contemplated as homeostatic and adaptative responses. Below 10% of the population has no experience with headache and the migrainous phenotype is quite frequent in secondary headaches and in syndromic forms of migraine. These features can be understood under the next undergrounds: specific neurophysiological data (lack of habituation, sensibilization and low preactivation), genetic features (polygenic disorder with the implication of many gens with a low penetrance, that interact with the environment and are shared with comorbid disorders such as depression and anxiety); and environmental interactions in modern societies (increase in the number of estrogenic cycles and particularly overexposition to stress). CONCLUSIONS: A feature that was an evolutionary advantage has been transformed in a highly prevalent and disabling disorder in modern societies. It is the result of the interaction with internal (estrogenic cycles) and external (stress) stimuli. As a consequence, it becomes a mismatch disorder. The effects appear in childhood through epigenetics. Therefore, therapeutic interventions would yield greater benefits if whole populations were included in educative interventions incorporating these aspects.

TITLE: Migraña y teoria evolutiva: vias para un acercamiento clinico.Introduccion. La migraña es un trastorno muy comun, con incidencia en aumento. La teoria evolutiva permite explicar su aparicion, dadas las ventajas que aportaba a grupos originarios de Homo sapiens una mayor reactividad a estimulos, y la presencia creciente de interaccion con factores ambientales. Analizamos estos aspectos a traves de los mecanismos potenciales que los explican. Desarrollo. El diseño de los organismos y sus respuestas ambientales surgen para mejorar la supervivencia. Asi, el dolor y la cefalea pueden entenderse como respuestas homeostaticas y adaptativas. Menos del 10% de la poblacion no tiene experiencia de cefalea, y el fenotipo migrañoso es una repuesta dolorosa comun en formas secundarias y sindromicas de cefalea. Estas caracteristicas se entienden segun rasgos neurofisiologicos especificos (falta de habituacion, sensibilizacion y baja preactivacion), caracteristicas geneticas (trastorno poligenico con multiples genes de baja penetrancia, que interaccionan con el ambiente y que son comunes a los de los trastornos comorbidos, como depresion-ansiedad) e interaccion ambiental en las sociedades modernas (aumento del numero de ciclos estrogenicos, y especialmente sobreexposicion al estres). Conclusiones. Lo que fue una ventaja evolutiva se ha transformado en la sociedad moderna en un trastorno muy prevalente y frecuentemente incapacitante. Es el resultado de una interaccion con sobrecarga de estimulos externos (estres) e internos (hormonales), lo que, de acuerdo con la teoria evolutiva, convertiria a la migraña en una enfermedad por desajuste. Los efectos ocurririan precozmente, en la infancia, a traves de mecanismos de epigenetica. Se obtendria un gran beneficio terapeutico mediante intervenciones poblacionales y educativas que incorporen estos aspectos.

Regulation of growth hormone secretion by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in infantile, prepubertal, and adult male rats.

Excitatory amino acids, such as glutamate, constitute a major transmitter system in the control of hypothalamic-pituitary function. Different subtypes of glutamate receptors, such as N-methyl-D-aspartic acid and kainate receptors, have been involved in the control of GH secretion. Other excitatory amino acid receptor subtypes, as alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), amino-4-phosphobutyric acid, and metabotropic receptors, have been identified, yet their role in the control of neuroendocrine function remains to be completely characterized. The purpose of this study was to assess the potential involvement of AMPA receptors in the control of GH secretion. In a first set of experiments, neonatal (5 and 10 days) and prepubertal (23 days) male rats were injected with AMPA (1, 2.5, or 5 mg/kg) or the antagonist of AMPA receptors, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo(f)quinoxaline-7-sulfonamide (NBQX; 0.25 or 0.50 mg/kg). Serum GH concentrations significantly increased 15 min after i.p. administration of AMPA in both neonatal and prepubertal male rats. In addition, serum GH concentrations decreased after NBQX treatment. The stimulatory effect of AMPA was abolished by pretreatment with the blocker of nitric oxide synthase, nitro(w)-arginine-methyl ester (40 mg/kg), and was partially counteracted by the simultaneous administration of GH-releasing hormone (500 microg/kg). Moreover, AMPA was unable to elicit in vitro GH secretion by hemipituitaries from prepubertal males, pointing out that the hypothalamus is probably the site of action for the reported stimulatory action of AMPA on GH release. In a second set of experiments, the effects of AMPA and NBQX were tested in adult male rats. As in prepubertal animals, AMPA significantly increased GH secretion in adult males, whereas NBQX (20 or 40 nmol), administered through intracerebroventricular injection, induced a significant decrease in the amplitude of GH pulses. In conclusion, our data indicate that AMPA receptors have a physiological stimulatory role in the control of GH secretion in male rats throughout the life span. This effect depends on appropriate nitric oxide synthesis during the prepubertal age. In addition, AMPA receptors appear to modulate pulsatile GH secretion in adulthood.

Novel expression and functional role of ghrelin in rat testis.

Ghrelin, the endogenous ligand for the GH-secretagogue receptor (GHS-R), is a recently cloned peptide, primarily expressed in the stomach and hypothalamus, that acts at central levels to elicit GH release and, notably, to regulate food intake. However, the possibility of additional, as yet unknown, peripheral effects of ghrelin cannot be ruled out. In the present communication, we provide evidence for the novel expression of ghrelin and its functional receptor in rat testis. Testicular ghrelin gene expression was demonstrated throughout postnatal development, and ghrelin protein was detected in Leydig cells from adult testis specimens. Accordingly, ghrelin mRNA signal became undetectable in rat testis following selective Leydig cell elimination. In addition, testicular expression of the gene encoding the cognate ghrelin receptor was observed from the infantile period to adulthood, with the GHS-R mRNA being persistently expressed after selective withdrawal of mature Leydig cells. From a functional standpoint, ghrelin, in a dose-dependent manner, induced an average 30% inhibition of human CG- and cAMP-stimulated T secretion in vitro. This inhibitory effect was associated with significant decreases in human CG-stimulated expression levels of the mRNAs encoding steroid acute regulatory protein, and P450 cholesterol side-chain cleavage, 3beta-hydroxy steroid dehydrogenase, and 17beta-hydroxy steroid dehydrogenase type III enzymes. Overall, our data are the first to provide evidence for a possible direct action of ghrelin in the control of testicular function. Furthermore, the present results underscore an unexpected role of ghrelin as signal with ability to potentially modulate not only growth and body weight homeostasis but also reproductive function, a phenomenon also demonstrated recently for the adipocyte-derived hormone, leptin.

Human papillomavirus and cervical intraepithelial neoplasia grade III/carcinoma in situ: a case-control study in Spain and Colombia.

A case-control study of 525 cases of cervical intraepithelial neoplasia grade III (CIN III) and 512 controls was conducted in Spain and Colombia between 1985 and 1988 to assess the role of human papillomavirus (HPV) in the etiology of CIN III. HPV DNA in cytological scrapes from the cervix was assessed by Virapap and by polymerase chain reaction (PCR) based on the L1 consensus primers. A subsample of 268 specimens was also tested for HPV DNA using Southern hybridization. In Spain, the PCR-based prevalences of HPV DNA were 70.7% among cases and 4.7% among controls. Odds ratio (OR) and 95% confidence interval (numbers in parentheses) for HPV DNA were 56.9 (24.8-130.6). In Columbia HPV DNA was detected by PCR in 63.2% of the cases and in 10.5% of the controls. The OR was 15.5 (8.2-29.4). The estimated fractions of CIN III attributable to HPV were 72.4% in Spain and 60.3% in Colombia. HPV 16 was the predominant viral type and showed the strongest association with CIN III; in Spain the OR was 295.5 (44.8-1946.4) and in Colombia the OR was 27.1 (10.6-69.5). HPV DNA of unknown type was frequent in HPV-positive cases (18.3% in Spain and 38.0% in Colombia) and controls (66.7% in Spain and 47.4% in Colombia). The comparison of results from Virapap and PCR indicated that PCR is the method of choice for epidemiological studies. These data strongly support the hypothesis of the viral origin of CIN III, the common etiology of CIN III and invasive cervical cancer, and the causal nature of the association between HPV and CIN III.

Risk factors for progression of cervical intraepithelial neoplasm grade III to invasive cervical cancer.

Data from four case-control studies on invasive cervical cancer and on cervical intraepithelial neoplasia grade III (CIN III) that were concurrently conducted in Spain and Colombia were used to look for factors that might favor the progression from CIN III to the invasive stage. These studies were compared in two ways; a case-case comparison and an estimation of the ratio of odds ratios with the use of of special logistic regression model that took into account the different design of each study and possible confounding factors. Variables studied were human papillomavirus status, viral load, viral types, sexual behavior, sexually transmitted diseases, reproductive patterns, oral contraceptives, and smoking. Both CIN III and invasive cervical cancer have a very similar profile of risk factors and none of them was different in a consistent way to suggest a role in the progression from CIN III to invasive cervical cancer. Some methodological problems such as cohort-specific differences and some selection biases could be adjusted for with a careful statistical analysis. Other problems derived from the cross-sectional nature of the design are unavoidable and should be considered in the interpretation of the results.

Regulation of prolactin secretion by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in male rats.

The secretion of PRL is controlled by different hypothalamic signals. Depending on the experimental model, PRL secretion increases or decreases after activation of N-methyl-d -aspartic acid and kainate receptors. Recently we have described that activation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors inhibits PRL secretion in prepubertal male rats. The aim of present study was to examine (1) the physiological relevance of this finding, (2) the possible age-related changes observed after activation or blockade of AMPA receptors, (3) the specificity of the AMPA effect, (4) the hypothalamic and/or pituitary localization of AMPA action, and (5) the mechanism(s) of action of AMPA agonists. In a first set of experiments, neonatal males (5 and 10 days old) and prepubertal (23 days old) male rats were injected with AMPA (1, 2.5 or 5 mg/kg) or the antagonist of AMPA receptors 1,2,3, 4-tetrahydro-6-nitro-2,3-dioxo-! benzo (f) quinoxaline-7-sulfonamide (NBQX; 0.25 or 0.50 mg/kg). Serum PRL concentrations decreased significantly 15 and 30 min after i.p. administration of AMPA in prepubertal male rats, while the inhibitory effect of AMPA was not observed in 5- and 10-day-old males. The effect of AMPA was abolished by NBQX but not by MK-801 (a selective antagonist of NMDA receptors). NBQX alone (0.25 or 0.50 mg/kg) had no effect on PRL release. In vitro, AMPA slightly stimulated PRL secretion by hemipituitaries from prepubertal males, suggesting that the hypothalamus is likely the site of action for the reported inhibitory action of AMPA on PRL release. In this sense, the blockade of AMPA effects in animals pretreated with domperidone (a dopaminergic antagonist) or alpha-methyl-p-tyrosine (an inhibitor of dopamine synthesis) suggests that an increase in the release of hypothalamic dopamine is probably the mechanism i! nvolved in the effect of AMPA. In a second set of experiments, the effects of AMPA (2.5 mg/kg i.p.) and NBQX (0.5 mg/kg i.p. and 20 or 40 nmol i.c.v.) were tested in freely moving adult male rats sampled during periods of 2, 3 or 6 h. In contrast with data obtained in prepubertal rats, neither AMPA nor NBQX affected PRL secretion. In conclusion, these data indicate that activation of AMPA receptors inhibits PRL secretion in prepubertal male rats. This effect probably involves the release of dopamine from the hypothalamus and disappears in adulthood.

Role of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors in the control of prolactin, growth hormone and gonadotropin secretion in prepubertal rats.

Excitatory amino acids, such as glutamate, constitute a major transmitter system in the control of hypothalamic-pituitary secretion. Different subtypes of glutamate receptors, such as NMDA (N-methyl-d-aspartic acid) and KA (kainate) receptors, are involved in the control of anterior pituitary secretion. Other receptor subtypes, such as AMPA (activated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and metabotropic receptors, have been identified, although their role in the control of neuroendocrine function remains largely unknown. Recent reports have demonstrated the involvement of AMPA receptors in the control of the steroid-induced luteinizing hormone (LH) surge in female and growth hormone (GH) secretion in male rats. The aim of this study was to assess the potential role of AMPA receptors in the control of GH, prolactin (PRL), LH and follicle-stimulating hormone (FSH) secretion in prepubertal 23-day-old rats. To this end, prepubertal female rats were injected with AMPA (2.5 or 5 mg/kg i.p.) or the antagonist of AMPA receptors 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo-benzo (f) quinoxaline-7-sulfonamide (NBQX; 0.25 or 0.50 mg/kg i.p.). Serum LH and FSH concentrations and hypothalamic LH-releasing hormone (LHRH) content remained unchanged after AMPA or NBQX administration. In contrast, serum PRL levels significantly decreased 15, 30 and 60 min after i.p. administration of AMPA and increased 120 min after NBQX treatment, whereas serum GH levels increased after AMPA treatment and decreased after NBQX administration. Considering that AMPA has been shown to activate a subset of kainate receptors, its effects were compared with those elicited by 2.5 mg/kg KA in prepubertal female rats. At this age, however, KA was unable to reproduce the effects of AMPA on PRL and GH secretion, thus suggesting that the actions observed after AMPA administration were carried out specifically through AMPA receptors. In addition, as the effects of AMPA on LH secretion in adult females have been proved to be steroid-dependent, the effects of AMPA (2.5 mg/kg) and NBQX (0.5 mg/kg) were tested in prepubertal animals with different gonadal backgrounds, i.e. intact males, and intact and ovariectomized (OVX) females. The effects of AMPA in prepubertal females appeared to be modulated by ovarian secretion, as the inhibition of PRL secretion disappeared and LH secretion was partially suppressed by AMPA in OVX animals whereas the stimulatory effect on GH release was enhanced by ovariectomy. Furthermore, in male rats, AMPA administration significantly decreased PRL secretion and increased serum GH levels, the amplitude of the GH response being higher than in prepubertal females. To ascertain the pituitary component for the reported actions of AMPA, hemi-pituitaries of male rats were incubated in the presence of AMPA (10(-8)-10(-6) M). The results obtained showed no effect of AMPA on PRL, GH and gonadotropin secretion in vitro. Finally, we investigated the involvement of the dopaminergic (DA) system in the inhibitory action of AMPA on PRL secretion. Pre-treatment of prepubertal female rats with a dopamine receptor antagonist (domperidone: 1 mg/kg) resulted in the blockage of AMPA-mediated inhibition of PRL secretion, thus suggesting that this action is probably mediated by an increase in DA activity. In conclusion, we provide evidence for the physiological role of AMPA receptors in the control of PRL and GH secretion in prepubertal rats. In contrast, our data cast doubts on the involvement of AMPA receptors in the regulation of gonadotropin secretion at this age. The effects of AMPA reported herein were not mediated through activation of kainate receptors and were probably exerted at the hypothalamic or suprahypothalamic levels. In addition, we show that ovarian secretion actively modulates the effects of AMPA receptor activation on anterior pituitary secretion in prepubertal female rats.

Homologous and heterologous down-regulation of leptin receptor messenger ribonucleic acid in rat adrenal gland.

Leptin, the adipocyte-produced hormone that plays a key role in body weight homeostasis, has recently been found to be involved in the regulation of the hypothalamic-pituitary-adrenal axis. Moreover, reciprocal interactions between leptin and glucocorticoids have been described. In the present communication, two different strategies were undertaken to explore the mode of action of leptin in the direct control of rat adrenal function. First, a synthetic peptide approach demonstrated that the inhibitory effect of leptin on basal and ACTH-stimulated corticosterone secretion in vitro is, at least partially, mapped to a domain of the native protein between amino acids 116 and 130, i.e. an area of the molecule also relevant in terms of regulation of food intake and endocrine control. Secondly, semi-quantitative RT-PCR analysis indicated a complex pattern of adrenal leptin receptor (Ob-R) mRNA expression, with predominant expression of the Ob-Ra and Ob-Rb isoforms, as well as moderate levels of the Ob-Rc and Ob-Rf variants, whereas negligible signals for the Ob-Re isoform were detected. Interestingly, such an expression pattern appeared hormonally regulated as exposure to human recombinant leptin (10(-7 )M) or ACTH (10(-7 )M) significantly decreased Ob-R isoform mRNA expression. Indeed, dose-dependent ligand-induced Ob-Ra and Ob-Rb mRNA down-regulation was further confirmed by adrenal stimulation with increasing concentrations (10(-9)-10(-5 )M) of the active leptin fragment, leptin 116-130 amide. Overall, our results provide evidence for a novel regulatory step at the level of Ob-R mRNA expression in the interplay between ACTH and leptin for the tuning of rat adrenal corticosterone secretion. Furthermore, our data showing down-regulation of Ob-R mRNA expression by its cognate ligand may well be relevant to leptin physiology and its alteration in various disease states.

Neonatal exposure to estrogen differentially alters estrogen receptor alpha and beta mRNA expression in rat testis during postnatal development.

The biological actions of estrogens on target cells are mediated by two nuclear receptors: the estrogen receptor (ER) alpha and the recently characterized ER beta. In the male rat, the physiological role of estrogens involves multiple actions, from masculinization of brain areas related to reproductive function and sexual behavior to regulation of testicular development and function. Paradoxically, however, administration of high doses of estrogen during the critical period of neonatal differentiation results in an array of defects in the reproductive axis that permanently disrupt male fertility. The focus of this study was to characterize the effects and mechanism(s) of action of neonatal estrogenization on the pattern of testicular ER alpha and beta gene expression during postnatal development. To this end, groups of male rats were treated at day 1 of age with estradiol benzoate (500 microg/rat), and testicular ER alpha and ER beta mRNA levels were assayed by semi-quantitative RT-PCR from the neonatal period until puberty (days 1-45 of age). Furthermore, the expression of androgen receptor (AR) mRNA was evaluated, given the partially overlapping pattern of tissue distribution of ER alpha, ER beta and AR messages in the developing rat testis. In addition, potential mechanisms for neonatal estrogen action were explored. Thus, to discriminate between direct effects and indirect actions through estrogen-induced suppression of serum gonadotropins, the effects of neonatal estrogenization were compared with those induced by blockade of gonadotropin secretion with a potent LHRH antagonist in the neonatal period. Our results indicate that neonatal exposure to estrogen differentially alters testicular expression of alpha and beta ER messages: ER alpha mRNA levels, as well as those of AR, were significantly decreased, whereas relative and total expression levels of ER beta mRNA increased during postnatal/prepubertal development after neonatal estrogen exposure, a phenomenon that was not mimicked by LHRH antagonist treatment. It is concluded that the effect of estrogen on the expression levels of ER alpha and beta mRNAs probably involves a direct action on the developing testis, and cannot be attributed to estrogen-induced suppression of gonadotropin secretion during the neonatal period.

Leptin inhibits testosterone secretion from adult rat testis in vitro.

Leptin, the product of the ob gene, has emerged recently as a pivotal signal in the regulation of fertility. Although the actions of leptin in the control of reproductive function are thought to be exerted mainly at the hypothalamic level, the potential direct effects of leptin at the pituitary and gonadal level have been poorly characterised. In the present study, we first assessed the ability of leptin to regulate testicular testosterone secretion in vitro. Secondly, we aimed to evaluate whether leptin can modulate basal gonadotrophin and prolactin (PRL) release by incubated hemi-pituitaries from fasted male rats. To attain the first goal, testicular slices from prepubertal and adult rats were incubated with increasing concentrations (10(-9)-10(-7) M) of recombinant leptin. Assuming that in vitro testicular responsiveness to leptin may be dependent on the background leptin levels, testicular tissue from both food-deprived and normally-fed animals was used. Furthermore, leptin modulation of stimulated testosterone secretion was evaluated by incubation of testicular samples with different doses of leptin in the presence of 10 IU human chorionic gonadotrophin (hCG). In addition, analysis of leptin actions on pituitary function was carried out using hemi-pituitaries from fasted adult male rats incubated in the presence of increasing concentrations (10(-9)-10(-7) M) of recombinant leptin. Serum testosterone levels, and basal and hCG-stimulated testosterone secretion by incubated testicular tissue were significantly decreased by fasting in prepubertal and adult male rats. However, a significant reduction in circulating LH levels was only evident in adult fasted rats. Doses of 10(-9)-10(-7) M leptin had no effect on basal or hCG-stimulated testosterone secretion by testes from prepubertal rats, regardless of the nutritional state of the donor animal. In contrast, leptin significantly decreased basal and hCG-induced testosterone secretion by testes from fasted and fed adult rats. In addition, 10(-9) M leptin inhibited LH and FSH secretion by incubated hemi-pituitaries from fasted adult males, whereas, at all doses tested, it was ineffective in modulating PRL release. Our results show that leptin, depending on the state of sexual maturation, is able to inhibit testosterone secretion acting at the testicular level. Furthermore, the present data suggest that the actions of leptin on the reproductive system are complex and are probably carried out at different levels of the hypothalamic-pituitary-gonadal axis.

Comparative effects of testosterone propionate, oestradiol benzoate, ICI 182,780, tamoxifen and raloxifene on hypothalamic differentiation in the female rat.

Hypothalamic differentiation in the female rat during the neonatal period is critically dependent on the steroid milieu, as permanent changes in reproductive function are observed after administration of oestradiol and testosterone during such a critical stage. Selective oestrogen modulators (SERMs) constitute a family of drugs that, depending on the tissue, are able to exert oestrogenic or antioestrogenic actions. The present experiments were conducted to analyse whether the SERMs, tamoxifen and raloxifene, can cause oestrogenic actions during the hypothalamic differentiation period. Postnatal female rats were injected between days 1 and 5 with 100 microg/day tamoxifen, raloxifene or ICI 182,780 (a pure antioestrogen). Other groups of animals were injected on day 1 of age with 100 microg oestradiol benzoate (OeB) or 1.25 mg testosterone propionate (TP) alone or in combination with raloxifene (500 microg/day between days 1 and 5). In all experimental groups, the age, body weight and concentrations of serum gonadotrophins at vaginal opening were recorded, whereas vaginal cyclicity and the negative and positive feedback between oestradiol and LH were monitored in adulthood. The results obtained confirmed the ability of high doses of OeB or TP to alter the normal differentiation of the brain permanently. They also reinforced the hypothesis that oestrogens are also necessary for normal brain differentiation in female rats because administration of a pure antioestrogen, such as ICI 182,780 permanently altered the function of the reproductive axis. In addition, our data provided evidence for different actions of the two SERMs under analysis (raloxifene and tamoxifen) upon peripheral targets, as raloxifene advanced vaginal opening whereas tamoxifen did not. In contrast, their actions on brain differentiation appeared similar and analogous to those obtained after neonatal administration of oestradiol, as evidenced by vaginal acyclicity, ovarian atrophy, sterility and abolition of negative and positive feedback between oestradiol and LH, thus suggesting an oestrogenic action of these SERMs on hypothalamic differentiation. Moreover, the oestrogenic activity of raloxifene was supported by its inability to block the effects of OeB and TP administered neonatally. In conclusion, the present results indicated that the SERMs, tamoxifen and raloxifene, exert an oestrogen-like effect upon hypothalamic differentiation of the neonatal female rat.

Molecular mechanisms of leptin action in adult rat testis: potential targets for leptin-induced inhibition of steroidogenesis and pattern of leptin receptor messenger ribonucleic acid expression.

Leptin, the product of the ob gene, is a pivotal signal in the regulation of neuroendocrine function and fertility. Although much of the action of leptin in the control of the reproductive axis is exerted at the hypothalamic level, some direct effects of leptin on male and female gonads have also been reported. Indeed, recent evidence demonstrated that leptin is able to inhibit testosterone secretion at the testicular level. However, the molecular mechanisms behind this effect remain unclear. The focus of this study was twofold: (1) to identify potential targets for leptin-induced inhibition of steroidogenesis, and (2) to characterize in detail the pattern of expression and cellular distribution of leptin receptor (Ob-R) mRNA in adult rat testis. In pursuit of the first goal, slices of testicular tissue from adult rats were incubated with increasing concentrations of recombinant leptin (10(-9)--10(-7 )M) in the presence of human chorionic gonadotropin (hCG; 10 IU/ml). In this setting, testosterone secretion in vitro was monitored, and expression levels of mRNAs encoding steroidogenic factor 1 (SF-1), steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450 scc) and 17 beta-hydroxysteroid dehydrogenase type III (17 beta-HSD) were assessed by Northern hybridization. In pursuit of the second goal, the pattern of cellular expression of the Ob-R gene in adult rat testis was evaluated by in situ hybridization using a riboprobe complementary to all Ob-R isoforms. In addition, testicular expression levels of the different Ob-R isoforms, previously identified in the hypothalamus, were analyzed by means of semi-quantitative RT-PCR. In keeping with our previous data, recombinant leptin significantly inhibited hCG-stimulated testosterone secretion. In this context, leptin, in a dose-dependent manner, was able to co-ordinately decrease the hCG-stimulated expression levels of SF-1, StAR and P450 scc mRNAs, but it did not affect those of 17 beta-HSD type III. In situ hybridization analysis showed a scattered pattern of cellular expression of the Ob-R gene within the adult rat testis, including Leydig and Sertoli cells. In addition, assessment of the pattern of expression of Ob-R subtypes revealed that the long Ob-Rb isoform was abundantly expressed in adult rat testis. However, variable levels of expression of Ob-Ra, Ob-Re, and Ob-Rf mRNAs were also detected, whereas those of the Ob-Rc variant were nearly negligible. In conclusion, our results indicate that decreased expression of mRNAs encoding several up-stream elements in the steroidogenic pathway may contribute, at least partially, to leptin-induced inhibition of testicular steroidogenesis. In addition, our data on the pattern of testicular expression of Ob-R isoforms and cellular distribution of Ob-R mRNA may help to further elucidate the molecular mechanisms of leptin action in rat testis.

5-HT1 and 5-HT2 receptor agonists blunt +/- -alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-stimulated GH secretion in prepubertal male rats.

OBJECTIVE: Excitatory amino acids, gamma-amino butyric acid (GABA), serotonin and catecholamines are involved in the control of GH secretion. The actions of these neurotransmitters are interconnected, and recently we showed that the stimulatory effect of GABA was blocked by MK-801, an antagonist of N-methyl-D-aspartate receptors. The present experiments were carried out to analyze the interrelationships between +/- -alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and serotoninergic and catecholaminergic pathways in the control of GH secretion in prepubertal (16-23-day-old) male rats. DESIGN AND METHODS: The GH response to AMPA was analyzed in animals pretreated with 5-hydroxytryptophan methyl ester (5-HTP) plus fluoxetine (a precursor of 5-hydroxytryptamine (5-HT) synthesis and a blocker of 5-HT re-uptake), R (+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT, an agonist of the 5-HT1 receptors), +/- -2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) and alpha-methyl-5-hydroxytryptamine (agonists of 5-HT2 receptors), I-phenylbiguanide (an agonist of 5-HT3 receptors), or alpha-methyl-p-tyrosine (alpha-MPT) and diethyldithiocarbamate (DDC) (blockers of catecholamine synthesis). RESULTS: Basal GH secretion remained unchanged in prepubertal rats after activation of the serotoninergic system or blockade of catecholamine synthesis. The stimulatory effect of AMPA on GH secretion was blocked after activation of the serotoninergic system, through specific 5-HT1 and 5-HT2 receptor agonists. In contrast, activation of 5-HT3 receptors potentiated AMPA-stimulated GH secretion. CONCLUSIONS: Serotoninergic receptors modulate the stimulatory effect of AMPA on GH secretion in prepubertal male rats.