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L Gonzalez
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AI Sotelo
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A Bartke
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D Turyn
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To study the effects of homologous mouse GH (mGH) on the presence and characteristics of serum GH-binding protein (GHBP) we have used transgenic mice expressing GH-releasing hormone (GHRH) as a model. Chromatographic techniques allowed the characterization of GHBP bioactivity, and immunological techniques were used to determine its concentration and molecular components. Chromatographic separation of labeled human GH or mGH cross-linked to serum GHBPs showed two GH-binding serum fractions in normal as well as in transgenic mice serum. SDS-PAGE of this material revealed a specific band of 66 kDa and another higher molecular weight broad band, which, in the presence of 2-mercapto-ethanol, is converted into the 66 kDa fraction.Since normal mice serum has an mGH concentration of 0. 40+/-0.06 nM and a GHBP concentration of 5.7+/-1.1 nM, while the high-affinity site for mGH has a K(d)</+/-27 nM, only a small percentage (2.9%) of total serum mGH is bound to GHBP in the sera of these mice. In transgenic mice serum, where the mGH concentration is 60 times higher (23+/-2.7 nM), 22.5% of total serum mGH is bound to serum GHBP. These values agree with the experimental data (4+/-2% and 17+/-4% for normal and transgenic mice serum respectively).The concentration of GHBP in GHRH transgenic mice was found to be increased four- to tenfold, depending on the technique used. This increment closely resembles the increased concentration of GHBP in the serum of transgenic bovine GH (bGH) mice, in which peripheral bGH levels are grossly elevated. Our results support the idea that the circulating levels of mGH in normal mouse serum are capable of influencing the levels of GHBP in peripheral circulation in a way similar to that of bGH, in spite of the different affinities of these two hormones. The fact that the up-regulation of GHBP occurs, even though a small percentage of mGH is bound in these animals, strongly suggests the existence of a physiological function for GHBP. These results also question some of the assigned or attributed physiological roles of GHBP, at least in the mouse, since only a negligible percentage of total mGH would be prevented from degradation and/or renal filtration by binding to GHBP. This small percentage of bound mGH also invalidates its role as a reservoir or a buffer of mGH concentration during pulses of GH release or rapid changes of mGH levels. Our results also demonstrate the presence of high molecular weight forms of GH-GHBP complexes that could be dissociated by dilution or in the presence of 2-mercapto-ethanol.

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C Bellido
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D Gonzalez
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R Aguilar
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JE Sanchez-Criado
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We have previously shown that administration of antiprogestin (AP) type II RU486 to ovariectomized (OVX) rats on the morning of pro-oestrus decreases the magnitude of preovulatory gonadotrophin surge. This suggests that the effect of RU486 on LHRH-dependent gonadotrophin release may be independent of its ability to block progesterone actions. The aim of the present research was to study the possible site of RU486 action and to determine whether the gonadotrophin suppressive effect of APs RU486 and ZK299 is dependent on the oestrogen background. Intact or OVX rats in the morning of pro-oestrus were injected s.c. with 4 mg of RU486 or ZK299 (AP type I) at 0900 h on pro-oestrus. At 1830 h, serum concentration of FSH and LH and median eminence (ME) content of LHRH were determined. In the second experiment, the effect of RU486 and ZK299 on pituitary responsiveness to LHRH (100 ng, i.p.) and ME content of LHRH at 1830 h pentobarbital-blocked intact or OVX rats was evaluated. In the last study, the anterior pituitary release of FSH and LH from pro-oestrus or metoestrus donors incubated with or without LHRH (1, 10 or 100 nM) in the presence or absence of APs (20 nM) was evaluated. Both APs reduced serum FSH and LH levels at 1830 h on pro-oestrus in intact and OVX rats. The suppressive effect on gonadotrophin release brought about by AP treatment was also evidenced in PB-blocked intact and OVX rats. This suggested that the inhibitory effect of APs occurred, at least in part, at pituitary level. Furthermore, in the absence of the natural ligand, APs significantly reduced basal and LHRH-stimulated FSH and LH release from pro-oestrous but not from metoestrus pituitaries. In conclusion, these experiments have shown, both 'in vivo' and 'in vitro', that APs RU486 and ZK299 have suppressive effects at pituitary level on basal and LHRH-stimulated FSH and LH secretion, regardless of their antiprogestagenic activity, in pro-oestrus but not in metoestrus.

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JE Sanchez-Criado
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C Bellido
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M Tebar
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A Ruiz
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D Gonzalez
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Administration of 4 mg of the antisteroid RU486 over 8 consecutive days to adult male rats dissociated in vivo and in vitro gonadotrophin secretion, increasing FSH and decreasing LH secretion. In subsequent experiments we evaluated the involvement of testicular or adrenal secretory products, as well as hypothalamic LHRH, in the effects of 4 consecutive days of RU486 treatment on the secretion of gonadotrophins. The first day of RU486 injection was designated day 1, subsequent days being numbered consecutively. Groups of rats injected with oil (0.2 ml) or RU486 (4 mg) were: (i) injected s.c. from day 1 to day 4 with the antiandrogen flutamide (10 mg/kg); (ii) bilateral orchidectomized (ORCH) on day 1; and (iii) bilateral adrenalectomized (ADX) on day 1. Controls were given flutamide vehicle or were sham operated. To ascertain whether the secretion of LHRH is involved in the effects of RU486 on gonadotrophin secretion, we measured the LHRH secretion into the pituitary stalk blood vessels at 1100 h on day 5 in oil- or RU486-treated rats. Additional oil- and RU486-treated rats were injected i.p. with 100 ng LHRH at 1000 h on day 5, or s.c. with 1 mg LHRH antagonist (LHRH-ANT) at 1000 h on days 2 and 4. Controls were given saline. All animals were decapitated at 1100 h on day 5, trunk blood collected and serum stored frozen until FSH, LH and testosterone assays.%While ADX had no effect on FSH and LH secretion in either oil- or RU486-treated rats, the removal of androgen negative feedback with flutamide treatment or by ORCH substantially increased serum levels of FSH and LH in both oil- and RU486-treated rats, and thus annulled the effects of RU486. No differences in pituitary stalk plasma LHRH concentrations were found between oil- and RU486-treated rats. Injection of LHRH increased serum FSH and LH concentrations in oil-treated rats but only, and to a lesser extent, LH concentrations in RU486-treated rats. Treatment with LHRH-ANT decreased serum concentrations of FSH and LH in both oil- and RU486-treated rats. These results suggest that RU486 inhibited LHRH-stimulated LH secretion at the pituitary level, and that FSH secretion increased in response to a reduction in the negative feedback of androgen.

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N Zmora
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D Gonzalez-Martinez
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JA Munoz-Cueto
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T Madigou
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E Mananos-Sanchez
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SZ Doste
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Y Zohar
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O Kah
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A Elizur
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The cDNA sequences encoding three GnRH forms, sea bream GnRH (sbGnRH), salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), were cloned from the brain of European sea bass, Dicentrarchus labrax. Comparison of their deduced amino acid sequences to the same forms in the gilthead sea bream, Sparus aurata, and striped bass, Morone saxatilis, revealed high homology of the prepro-cGnRH II (94% and 98% respectively), and prepro-sGnRH (92% to both species). The sbGnRH exhibited dissimilar identities, with high homology to the striped bass (93%), and lower homology (59%) to the gilthead sea bream. Two transcript types were identified for the GnRH-associated peptide (GAP)-sGnRH as well as for the GAP-cGnRH II, which suggests a possible alternative splicing followed by the addition of an early stop codon. In order to obtain antibodies specific for the three GnRH precursors, recombinant GAP proteins were produced. The differential expression of the three GnRHs previously reported in the brain by means of in situ hybridization, using riboprobes corresponding to the GAP-coding regions, was fully confirmed by immunocytochemistry using antibodies raised against the recombinant GAP proteins, indicating that the transcripts are translated into functional proteins. Moreover, this approach allowed us to follow, for the first time, the specific projections of the different cell groups: sGAP fibers are distributed mainly in the forebrain with few projections reaching the pituitary, sbGAP fibers are mainly present in the preoptic area, mediobasal hypothalamus and predominantly project to the pars distalis of the pituitary, whereas cGnRH II fibers have a widespread distribution primarily in the posterior brain, and do not project to the pituitary. These new tools will be extremely useful to study further the development, regulation and functional significance of three independent GnRH systems in the brain of vertebrate species.

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Shiao Y Chan School of Clinical and Experimental Medicine, Department of Pathology, Fetal Medicine Centre, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

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Laura A Hancox School of Clinical and Experimental Medicine, Department of Pathology, Fetal Medicine Centre, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

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Azucena Martín-Santos School of Clinical and Experimental Medicine, Department of Pathology, Fetal Medicine Centre, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

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Laurence S Loubière School of Clinical and Experimental Medicine, Department of Pathology, Fetal Medicine Centre, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

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Merlin N M Walter School of Clinical and Experimental Medicine, Department of Pathology, Fetal Medicine Centre, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

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Ana-Maria González School of Clinical and Experimental Medicine, Department of Pathology, Fetal Medicine Centre, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

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Phillip M Cox School of Clinical and Experimental Medicine, Department of Pathology, Fetal Medicine Centre, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

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Ann Logan School of Clinical and Experimental Medicine, Department of Pathology, Fetal Medicine Centre, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

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Christopher J McCabe School of Clinical and Experimental Medicine, Department of Pathology, Fetal Medicine Centre, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

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Jayne A Franklyn School of Clinical and Experimental Medicine, Department of Pathology, Fetal Medicine Centre, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

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Mark D Kilby School of Clinical and Experimental Medicine, Department of Pathology, Fetal Medicine Centre, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
School of Clinical and Experimental Medicine, Department of Pathology, Fetal Medicine Centre, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

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The importance of the thyroid hormone (TH) transporter, monocarboxylate transporter 8 (MCT8), to human neurodevelopment is highlighted by findings of severe global neurological impairment in subjects with MCT8 (SLC16A2) mutations. Intrauterine growth restriction (IUGR), usually due to uteroplacental failure, is associated with milder neurodevelopmental deficits, which have been partly attributed to dysregulated TH action in utero secondary to reduced circulating fetal TH concentrations and decreased cerebral thyroid hormone receptor expression. We postulate that altered MCT8 expression is implicated in this pathophysiology; therefore, in this study, we sought to quantify changes in cortical MCT8 expression with IUGR. First, MCT8 immunohistochemistry was performed on occipital and parietal cerebral cortex sections obtained from appropriately grown for gestational age (AGA) human fetuses between 19 weeks of gestation and term. Secondly, MCT8 immunostaining in the occipital cortex of stillborn IUGR human fetuses at 24–28 weeks of gestation was objectively compared with that in the occipital cortex of gestationally matched AGA fetuses. Fetuses demonstrated widespread MCT8 expression in neurons within the cortical plate and subplate, in the ventricular and subventricular zones, in the epithelium of the choroid plexus and ependyma, and in microvessel wall. When complicated by IUGR, fetuses showed a significant fivefold reduction in the percentage area of cortical plate immunostained for MCT8 compared with AGA fetuses (P<0.05), but there was no significant difference in the proportion of subplate microvessels immunostained. Cortical MCT8 expression was negatively correlated with the severity of IUGR indicated by the brain:liver weight ratios (r 2=0.28; P<0.05) at post-mortem. Our results support the hypothesis that a reduction in MCT8 expression in the IUGR fetal brain could further compromise TH-dependent brain development.

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Alejandro Ibáñez-Costa Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain
Department of Cell Biology, Physiology and Immunology, Universidad de Córdoba, Córdoba, Spain
Hospital Universitario Reina Sofía, Córdoba, Spain
CIBER Fisiopatología de la Obesidad y Nutrición (CIBERObn), Córdoba, Spain

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Esther Rivero-Cortés Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain
Department of Cell Biology, Physiology and Immunology, Universidad de Córdoba, Córdoba, Spain
Hospital Universitario Reina Sofía, Córdoba, Spain
CIBER Fisiopatología de la Obesidad y Nutrición (CIBERObn), Córdoba, Spain

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Mari C Vázquez-Borrego Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain
Department of Cell Biology, Physiology and Immunology, Universidad de Córdoba, Córdoba, Spain
Hospital Universitario Reina Sofía, Córdoba, Spain
CIBER Fisiopatología de la Obesidad y Nutrición (CIBERObn), Córdoba, Spain

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Manuel D Gahete Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain
Department of Cell Biology, Physiology and Immunology, Universidad de Córdoba, Córdoba, Spain
Hospital Universitario Reina Sofía, Córdoba, Spain
CIBER Fisiopatología de la Obesidad y Nutrición (CIBERObn), Córdoba, Spain

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Luis Jiménez-Reina Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain
Department of Morphological Sciences, Universidad de Córdoba, Córdoba, Spain

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Eva Venegas-Moreno Metabolism and Nutrition Unit, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS), Seville, Spain

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Andrés de la Riva Service of Neurosurgery, Hospital Universitario Reina Sofía, Córdoba, Spain

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Miguel Ángel Arráez Neurosurgical Department, Carlos Haya Hospital, Málaga, Spain

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Inmaculada González-Molero Department of Endocrinology and Nutrition, Carlos Haya Hospital, Málaga, Spain

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Herbert A Schmid Novartis Pharma AG, Novartis Institutes for Biomedical Research, Oncology, CH-4057 Basel, Switzerland

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Silvia Maraver-Selfa Service of Endocrinology and Nutrition, Hospital Clínico Universitario Virgen de la Victoria, Málaga, Spain

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Inmaculada Gavilán-Villarejo Endocrinology and Nutrition Unit, Hospital Universitario Puerta del Mar, Cádiz, Spain

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Juan Antonio García-Arnés Department of Endocrinology and Nutrition, Carlos Haya Hospital, Málaga, Spain

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Miguel A Japón Department of Pathology, Hospital Universitario Virgen del Rocío, Seville, Spain

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Alfonso Soto-Moreno Metabolism and Nutrition Unit, Hospital Universitario Virgen del Rocío, Instituto de Biomedicina de Sevilla (IBIS), Seville, Spain

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María A Gálvez Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain
Service of Endocrinology and Nutrition, Hospital Universitario Reina Sofía, Córdoba, Spain

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Raúl M Luque Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain
Department of Cell Biology, Physiology and Immunology, Universidad de Córdoba, Córdoba, Spain
Hospital Universitario Reina Sofía, Córdoba, Spain
CIBER Fisiopatología de la Obesidad y Nutrición (CIBERObn), Córdoba, Spain

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Justo P Castaño Instituto Maimónides de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, Spain
Department of Cell Biology, Physiology and Immunology, Universidad de Córdoba, Córdoba, Spain
Hospital Universitario Reina Sofía, Córdoba, Spain
CIBER Fisiopatología de la Obesidad y Nutrición (CIBERObn), Córdoba, Spain

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Somatostatin analogs (SSA) are the mainstay of pharmacological treatment for pituitary adenomas. However, some patients escape from therapy with octreotide, a somatostatin receptor 2 (sst2)-preferring SSA, and pasireotide, a novel multi-sst-preferring SSA, may help to overcome this problem. It has been proposed that correspondence between sst1-sst5 expression pattern and SSA-binding profile could predict patient’s response. To explore the cellular/molecular features associated with octreotide/pasireotide response, we performed a parallel comparison of their in vitro effects, evaluating sst1-sst5 expression, intracellular Ca2+ signaling ([Ca2+]i), hormone secretion and cell viability, in a series of 85 pituitary samples. Somatotropinomas expressed sst5>sst2, yet octreotide reduced [Ca2+]i more efficiently than pasireotide, while both SSA similarly decreased growth hormone release/expression and viability. Corticotropinomas predominantly expressed sst5, but displayed limited response to pasireotide, while octreotide reduced functional endpoints. Non-functioning adenomas preferentially expressed sst3 but, surprisingly, both SSA increased cell viability. Prolactinomas mainly expressed sst1 but were virtually unresponsive to SSA. Finally, both SSA decreased [Ca2+]i in normal pituitaries. In conclusion, both SSA act in vitro on pituitary adenomas exerting both similar and distinct effects; however, no evident correspondence was found with the sst1-sst5 profile. Thus, it seems plausible that additional factors, besides the simple abundance of a given sst, critically influence the SSA response.

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