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M Tena-Sempere, L Pinilla and E Aguilar

Tena-Sempere M, Pinilla L, Aguilar E. Orchidectomy selectively increases follicle-stimulating hormone secretion in gonadotropin-releasing hormone agonist-treated male rats. Eur J Endocrinol 1995;132: 357–62. ISSN 0804–4643

The pituitary component of the feedback mechanisms exerted by testicular factors on gonadotropin secretion was analyzed in adult male rats treated with a potent gonadotropin-releasing hormone (GnRH) antagonist. In order to discriminate between androgens and testicular peptides, groups of males were orchidectomized (to eliminate androgens and non-androgenic testicular factors) or injected with ethylene dimethane sulfonate (EDS), a selective toxin for Leydig cells (to eliminate selectively androgens) and treated for 15 days with vehicle or the GnRH antagonist Ac-d-pClPhe-d-pClPhe-d-TrpSer-Tyr-d-Arg-Leu-Arg-Pro-d-Ala-NH2CH3COOH (Org.30276, 5 mg/kg/72 hours). Serum concentrations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured 7 and 14 days after the beginning of treatment. We found that: in males treated with GnRH antagonist, orchidectomy or EDS treatment did not induce any increase in LH secretion; and orchidectomy, but not EDS treatment, increased FSH secretion in GnRH-treated males. The present results show that negative feedback of testicular factors on LH secretion is mediated completely through changes in GnRH actions. In contrast, a part of the inhibitory action of the testis on FSH secretion is exerted directly at the pituitary level. It can be hypothesized that non-Leydig cell testicular factor(s) inputs at different levels of the hypothalamic–pituitary axis in controlling LH and FSH secretion.

Manuel Tena-Sempere, Department of Physiology, Faculty of Medicine, University of Córdoba, 14004 Córdoba, Spain

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E Aguilar, M Tena-Sempere and L Pinilla

The effect of different androgens and estradiol on pituitary responsiveness to growth hormone releasing hormone was studied in intact and orchidectomized adult male Wistar rats, by injecting subcutaneously immediately after orchidectomy for two weeks with testosterone, dihydrotestosterone, 5-α androstane, 3-α, 17 β-diol or estradiol dissolved in olive oil (in doses of 0.2 or 2.0 mg·kg−1·day−1) or vehicle. Pituitary responsiveness was tested in pentobarbital anaesthetized rats by measuring growth hormone plasma levels at different times after administration of growth hormone releasing hormone (1-29) NH2. We found that: (a) High doses of testosterone, dihydrotestosterone and 5-α androstane, 3α, 1 7 β-diol restored gonadotropin plasma concentrations and organ weights altered by orchidectomy; (b) both pituitary growth hormone content and concentration remained unaffected after orchidectomy or androgen replacement and decreased significantly after estradiol injection; (c) orchidectomy significantly reduced growth hormone-stimulated growth hormone releasing hormone secretion; (d) treatment with 5-α androstane, 3-α, 1 7 β-diol increased more than testosterone or dihydrotestosterone both the peak concentration and the mean growth hormone secretion after growth hormone releasing hormone stimulation: (e) no differences were observed in the treatment with testosterone or dihydrotestosterone; (f) estradiol given at a dose of 0.2 mg·kg−1·day−1 increased pituitary responsiveness to growth hormone releasing hormone. These results demonstrated that testosterone and 5-α androstane, 3-α, 17 β-diol, which do not differ in their action on pituitary growth hormone content, increased the pituitary responsiveness to growth hormone releasing hormone differently and that the low pituitary responsiveness to growth hormone releasing hormone previously described in prepubertal animals was not due mainly to the secretion of 5-α androstane, 3-α, 1 7 β-diol.

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L Pinilla, LC Gonzalez, M Tena-Sempere and E Aguilar

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.

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M Tena-Sempere, ML Barreiro, E Aguilar and L Pinilla

OBJECTIVE: Raloxifene is a non-steroidal selective estrogen receptor modulator (SERM) that mimics estrogenic activity on bone density and blood lipid concentration without uterotropic actions. Previous data from our laboratory indicated that, as is the case for estrogen, neonatal administration of raloxifene disturbed normal differentiation of the hypothalamic circuitries governing the gonadotropic axis. In contrast, raloxifene did not act in the same way as estrogen does on the neuronal systems controlling sexual receptivity in the female rat. At present, however, the mechanisms for these organizing effects of raloxifene are not completely elucidated. DESIGN AND METHODS: To analyze this phenomenon, female rats were injected daily with raloxifene (50, 100, 250 or 500 microg/rat per day) between days 1 and 5 of age. On day 23, hypothalamic gonadotropin-releasing hormone (LHRH) mRNA expression was assessed, and pituitary and plasma luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels were measured in basal and LHRH-stimulated conditions. In addition, LH and FSH responses to ovariectomy were evaluated in raloxifene-treated females. Finally, we monitored the ability of neonatal administration of a potent LHRH agonist ([d-Ala(6),d-Gly(10)]-LHRH ethylamide; 0.01 microg/kg per 12 h on days 1-5) to counteract the effects of raloxifene. RESULTS: Our analyses demonstrated that prepubertal rats (23-day-old females) treated neonatally with raloxifene showed decreased hypothalamic LHRH mRNA expression levels, reduced pituitary content of LH and FSH, reduced basal and LHRH-stimulated LH secretion in vivo and in vitro, and decreased response to ovariectomy. In addition, adult females treated neonatally with raloxifene showed anovulation and reduced serum LH levels; these effects were not prevented by the simultaneous administration of a LHRH agonist. CONCLUSION: In conclusion, our data demonstrate that neonatal administration of raloxifene can disrupt the programming of hypothalamic-pituitary-ovarian axis function. Reduced LH secretion, under basal and LHRH-stimulated conditions and after ovariectomy, is probably related to decreased LHRH expression, reduced pituitary LH content and/or decreased pituitary responsiveness to hypothalamic LHRH.

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L Pinilla, LC Gonzalez, M Tena-Sempere and E Aguilar

OBJECTIVE: Excitatory amino acids and serotonin are involved in the control of gonadotropin secretion. The actions of these neurotransmitters are interconnected and recently we have reported that 5-HT(1) and 5-HT(2) receptor agonists blunted (+/-)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-stimulated GH secretion in prepubertal rats. The present experiments were carried out to analyze the effects of activation of different 5-hydroxytryptamine (5-HT) receptor subtypes on gonadotropin secretion and their role in the N-methyl-d-aspartate (NMDA)-stimulated LH release. DESIGN AND METHODS: We analyzed the gonadotropin secretion after manipulation of serotoninergic and aminoacidergic systems and their interactions in 5-, 16- and 23-day-old male and female rats. To this end, serum LH and FSH concentrations were measured in rats treated with 5-hydroxytryptophan methyl ester (5-HTP) (a precursor of 5-HT synthesis) plus Fluoxetine (Fx, a blocker of 5-HT reuptake), d,l-p-chlorophenyl-alanine methyl ester (PCPA, a blocker of 5-HT synthesis), R-(+)-8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT, an agonist of 5-HT(1A) receptors), (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) and alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT, agonists of 5-HT(2) receptors), and 1-Phenylbiguanide (1-PHE an agonist of 5-HT(3) receptors). In addition, the effects of 8-OH-DPAT and DOI on NMDA-stimulated LH secretion were analyzed. RESULTS: Neither the activation nor blockade of the serotoninergic system modified LH secretion. Basal gonadotropin secretion remained unchanged in 23-day-old male and female rats after activation of 5-HT(1A), 5-HT(2) and 5-HT(3) receptors. The stimulatory effect of NMDA on LH secretion was blocked in both sexes after activation of the serotoninergic system, through specific 5-HT(1) and 5-HT(2) receptor agonists. CONCLUSIONS: Activation of serotoninergic receptors decreased the stimulatory effect of NMDA on LH secretion in prepubertal male and female rats.

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ML Barreiro, L Pinilla, E Aguilar and M Tena-Sempere

OBJECTIVE: GH secretagogues (GHSs) elicit a variety of biological effects in several endocrine and non-endocrine target tIssues, including activation of the hypothalamic-pituitary-adrenal axis. The latter is mainly carried out through a central hypothalamic action; yet the possibility of additional effects directly at the adrenal level cannot be ruled out. The aims of this study were to evaluate the expression and homologous regulation of the GHS-receptor (GHS-R) gene in rat adrenal and to assess the effects of synthetic (GH releasing peptide-6 - GHRP-6) and natural (ghrelin) ligands of GHS-R upon basal and ACTH-stimulated corticosterone secretion in vitro. DESIGN AND METHODS: Analysis of adrenal expression of target mRNAs (GHS-R, GHS-R1a, ghrelin, and several steroidogenic factors) was conducted by means of primer-specific, semi-quantitative RT-PCR. Evaluation of corticosterone secretion by incubated adrenal tIssue was carried out by specific RIA. RESULTS: RT-PCR analysis demonstrated expression of the GHS-R gene, but not of the gene encoding the cognate ligand ghrelin, in rat adrenal. Moreover, expression of the mRNA coding for the type 1a GHS-R (GHS-R1a), i.e. the biologically active receptor form, was demonstrated. The adrenal expression of the GHS-R message appeared under the regulation of homologous signals in vitro, as short-term incubation of adrenal samples in serum-free medium induced a significant increase in GHS-R mRNA levels that was inhibited by exposure to different doses of GHRP-6 (10(-9)-10(-5) mol/l) or ghrelin (10(-7) mol/l). Notably, an opposite pattern of homologous regulation of GHS-R gene expression was observed at the pituitary. Finally, short-term stimulation with increasing concentrations of GHRP-6 (10(-9)-10(-5) mol/l) or ghrelin (10(-7) mol/l) failed to alter basal and ACTH-stimulated corticosterone secretion in vitro, neither did it modify ACTH-stimulated mRNA expression levels of several upstream elements in the steroidogenic route: the steroidogenic acute regulatory (StAR) protein, and the enzymes P450 cholesterol side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD). CONCLUSIONS: Our study provides novel evidence for the expression and homologous regulation of the GHS-R gene in rat adrenal. However, our results cast doubts on the possibility of direct adrenal actions of ligands of the GHS-R in the regulation of corticosterone secretion in the rat.

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L Pinilla, M Tena-Sempere, R Aguilar and E Aguilar

OBJECTIVE: The stimulatory and inhibitory effects of N-methyl-D-aspartic acid (NMDA) and kainic acid on prolactin (PRL) secretion have been correlated with the serum prolactin concentrations before drug administration. In the present experiments, we analysed the role of NMDA and kainic acid in PRL secretion in females with different serum concentrations of PRL. METHODS: Hypoprolactinaemic females were obtained by ovariectomy or after administration of diethyldithiocarbamate (an inhibitor of dopamine-beta-hydroxylase). Chronic hyperprolactinaemia was induced by neonatal administration of testosterone or oestradiol and acute hyperprolactinaemia was induced either by administration of alpha-methyl-p-tyrosine (an inhibitor of tyrosine hydroxylase) or by ether exposure. To analyse the role of dopamine in the effects of NMDA, we measured pituitary concentrations of dopamine after NMDA treatment and the effects of pretreatment with domperidone. RESULTS: (1) NMDA, but not kainic acid, stimulated PRL release in cyclic females. This effect was independent of serum PRL concentrations and was not accompanied by a decrease in pituitary concentrations of dopamine. (2) NMDA did not change PRL secretion in neonatally androgenized females, whereas NMDA and kainic acid inhibited PRL release in neonatally oestrogenized females. The inhibitory effects of NMDA and kainic acid were blocked by domperidone. (3) Kainic acid inhibited PRL secretion in prepubertal hyper- and hypoprolactinaemic rats. (4) Hyperprolactinaemia induced by ether stress was counteracted by administration of NMDA and kainic acid. CONCLUSIONS: (a) NMDA has a dual effect on prolactin secretion that is independent of prior prolactin concentrations and of dopamine activity, but kainic acid is only inhibitory. (b) The stimulatory or inhibitory effects of NMDA and kainic acid on PRL secretion were not strictly related to basal PRL concentrations and necessarily involved a change in the secretion of prolactin releasing factors, as no correlations were observed between changes in pituitary concentrations of dopamine and serum PRL concentrations. (c) Females rendered hyperprolactinaemic by neonatal administration of testosterone or oestradiol responded differently after NMDA administration. (d) NMDA and kainic acid blocked the mechanisms involved in stress-induced PRL secretion.

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M Tena-Sempere, L Pinilla, LC Gonzalez, J Navarro, C Dieguez, FF Casanueva and E Aguilar

The obese gene (ob) product, leptin, has recently emerged as a key element in body weight homeostasis, neuroendocrine function and fertility. Identification of biologically active, readily synthesized fragments of the leptin molecule has drawn considerable attention, as they may provide a powerful tool for detailed characterization of the biological actions of leptin in different experimental settings. Recently, a fragment of mouse leptin protein comprising amino acids 116-130, termed leptin(116-130) amide, was shown to mimic the effects of the native molecule in terms of body weight gain and food intake, and to elicit LH and prolactin (PRL) secretion in vivo. As a continuation of our previous experimental work, the present study reports on the effects of leptin(116-130) amide on basal and stimulated testosterone secretion by adult rat testis in vitro. In addition, a comparison of the effects of human recombinant leptin and leptin(116-130) amide at the pituitary level on the patterns of LH, FSH, PRL and GH secretion is presented. As reported previously by our group, human recombinant leptin(10(-9)-10(-7)M) significantly inhibited both basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion in vitro. Similarly, incubation of testicular tissue in the presence of increasing concentrations of leptin(116-130) amide (10(-9)-10(-5)M) resulted in a dose-dependent inhibition of basal and hCG-stimulated testosterone secretion; a reduction that was significant from a dose of 10(-7)M upwards. In addition, leptin(116-130) amide, at all doses tested (10(-9)-10(-5)M), significantly decreased LH and FSH secretion by incubated hemi-pituitaries from adult male rats. In contrast, in the same experimental protocol, recombinant leptin(10(-9)-10(-7)M) was ineffective in modulating LH and FSH release. Finally, neither recombinant leptin nor leptin(116-130) amide were able to change basal PRL and GH secretion in vitro. Our results confirm the ability of leptin, acting at the testicular level, to inhibit testosterone secretion, and map the effect to a domain of the leptin molecule that lies between amino acid residues 116 and 130. In addition, we provide evidence for a direct inhibitory action of leptin(116-130) amide on pituitary LH and FSH secretion, a phenomenon not observed for the native leptin molecule, in the adult male rat.

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E Aguilar, M Tena-Sempere, R Aguilar, D Gonzalez and L Pinilla

The role of N-methyl-D-aspartate (NMDA) in the control of prolactin (PRL) secretion was analysed in prepubertal male rats. In experiment 1, males of different ages were decapitated after administration of NMDA or vehicle. In experiment 2, 30-day-old males were killed at different times after administration of vehicle, NMDA, MK-801 (a non-competitive NMDA antagonist) or NMDA plus MK-801. In experiment 3, 23-day-old males were sham-orchidectomized or orchidectomized. Orchidectomized males were or were not implanted with Silastic capsules containing different amounts of testosterone. On day 30, the animals were decapitated after administration of vehicle, NMDA or MK-801. In experiment 4, 30-day-old male rats were decapitated after being injected with vehicle, NMDA, Nw-nitro-L-arginine methyl ester (NAME) (an inhibitor of nitric oxide (NO) synthase), or NMDA plus NAME. Serum PRL concentrations, and dopamine pituitary and hypothalamic content were measured. In experiment 5, males pretreated with vehicle or NAME were killed after administration of the precursor of serotonin synthesis 5-hydroxytryptophan (5-HTP), the 5-HT1 receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) or the 5-HT2 agonist (+/-) 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI). Finally, the effects of NMDA, NAME and sodium nitroprusside (SNP) were tested in dispersed adenohypophyseal cells. We found that: (1) antagonism of NMDA receptors with MK-801 decreased PRL secretion in intact, orchidectomized and orchidectomized-testosterone treated male rats; (ii) NMDA inhibited PRL release in vivo through an increase in dopamine release and this effect was potentiated by NAME and prevented by testosterone; (iii) NMDA inhibited PRL, secretion in vitro and this effect was observed in presence of both SNP and NAME; (iv) NAME blocked the stimulatory effects of 5-HTP and DOI on PRL secretion. We conclude that endogenous glutamate stimulates PRL release and that NO might have a pivotal role in the mechanisms involved in the control of PRL release, inhibiting the release of dopamine and modulating the effects of NMDA and 5-HT.

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A C Gore, J Balthazart, D Bikle, D O Carpenter, D Crews, P Czernichow, E Diamanti-Kandarakis, R M Dores, D Grattan, P R Hof, A N Hollenberg, C Lange, A V Lee, J E Levine, R P Millar, R J Nelson, M Porta, M Poth, D M Power, G S Prins, E C Ridgway, E F Rissman, J A Romijn, P E Sawchenko, P D Sly, O Söder, H S Taylor, M Tena-Sempere, H Vaudry, K Wallen, Z Wang, L Wartofsky and C S Watson