RESUMEN
There are well-recognized sex differences in many pituitary endocrine axes, usually thought to be generated by gonadal steroid imprinting of the neuroendocrine hypothalamus. However, the recognition that growth hormone (GH) cells are arranged in functionally organized networks raises the possibility that the responses of the network are different in males and females. We studied this by directly monitoring the calcium responses to an identical GH-releasing hormone (GHRH) stimulus in populations of individual GH cells in slices taken from male and female murine GH-eGFP pituitary glands. We found that the GH cell network responses are sexually dimorphic, with a higher proportion of responding cells in males than in females, correlated with greater GH release from male slices. Repetitive waves of calcium spiking activity were triggered by GHRH in some males, but were never observed in females. This was not due to a permanent difference in the network architecture between male and female mice; rather, the sex difference in the proportions of GH cells responding to GHRH were switched by postpubertal gonadectomy and reversed with hormone replacements, suggesting that the network responses are dynamically regulated in adulthood by gonadal steroids. Thus, the pituitary gland contributes to the sexually dimorphic patterns of GH secretion that play an important role in differences in growth and metabolism between the sexes.
Asunto(s)
Hormonas Esteroides Gonadales/metabolismo , Hormona del Crecimiento/metabolismo , Caracteres Sexuales , Animales , Calcio/metabolismo , Señalización del Calcio/fisiología , Femenino , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Masculino , Ratones , Ratones TransgénicosRESUMEN
Growth hormone (GH) exerts its actions via coordinated pulsatile secretion from a GH cell network into the bloodstream. Practically nothing is known about how the network receives its inputs in vivo and releases hormones into pituitary capillaries to shape GH pulses. Here we have developed in vivo approaches to measure local blood flow, oxygen partial pressure, and cell activity at single-cell resolution in mouse pituitary glands in situ. When secretagogue (GHRH) distribution was modeled with fluorescent markers injected into either the bloodstream or the nearby intercapillary space, a restricted distribution gradient evolved within the pituitary parenchyma. Injection of GHRH led to stimulation of both GH cell network activities and GH secretion, which was temporally associated with increases in blood flow rates and oxygen supply by capillaries, as well as oxygen consumption. Moreover, we observed a time-limiting step for hormone output at the perivascular level; macromolecules injected into the extracellular parenchyma moved rapidly to the perivascular space, but were then cleared more slowly in a size-dependent manner into capillary blood. Our findings suggest that GH pulse generation is not simply a GH cell network response, but is shaped by a tissue microenvironment context involving a functional association between the GH cell network activity and fluid microcirculation.
Asunto(s)
Hormona del Crecimiento/metabolismo , Microcirculación , Hipófisis/irrigación sanguínea , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Hipófisis/citología , Hipófisis/metabolismoRESUMEN
The pituitary gland adapts the proportion of each of its endocrine cell types to meet differing hormonal demands throughout life. There is circumstantial evidence that multipotent adult progenitor cells contribute to this plasticity, but these cells have not been identified. Here, we describe a small (<0.05%) population of progenitor cells in the adult pituitary gland. We show that these cells express SOX2, a marker of several early embryonic progenitor and stem cell types, and form "pituispheres" in culture, which can grow, form secondary spheres, and differentiate to all of the pituitary endocrine cell types, as well as folliculostellate cells. Differentiation of cells in the pituispheres was associated with the expression of nestin, SOX9, and S100. Cells expressing SOX2 and E-cadherin are found throughout Rathke's pouch (RP) in embryos but persist in the adult gland, mostly in a narrow zone lining the pituitary cleft, but also are scattered throughout the pituitary. However, unlike in embryonic RP, most of these SOX2(+) cells in the adult gland also express SOX9 and S100. We suggest that this SOX2(+)/SOX9(+) population represents transit-amplifying cells, whereas the SOX2(+)/SOX9(-) cells we identify are multipotent progenitor/stem cells persisting in the adult pituitary.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas HMGB/metabolismo , Hipófisis/citología , Células Madre/citología , Factores de Transcripción/metabolismo , Animales , Ratones , Microscopía Confocal , Microscopía Fluorescente , Hipófisis/metabolismo , Factores de Transcripción SOXB1 , Células Madre/metabolismoRESUMEN
Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans.
Asunto(s)
Mutación con Ganancia de Función , Hipopituitarismo/genética , Hipotálamo/metabolismo , Hipófisis/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Niño , Preescolar , Corticotrofos/citología , Corticotrofos/metabolismo , Displasia Ectodérmica/genética , Facies , Insuficiencia de Crecimiento/genética , Células HEK293 , Cardiopatías Congénitas/genética , Humanos , Lactante , Sistema de Señalización de MAP Quinasas/genética , Melanotrofos/citología , Melanotrofos/metabolismo , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas B-raf/metabolismo , Secuenciación del Exoma/métodosRESUMEN
Normal childhood growth is determined by ultradian and infradian variations in GH secretion, yet GH treatment of children with short stature is restricted to daily fixed doses. We have used GH-deficient dwarf rats to determine whether variable GH dose regimens promote growth more effectively than fixed doses. Animals were treated with saline or 4.2 mg of recombinant bovine GH given as 1) 700 microg/wk in 100 microg/day doses, 2) alternating weekly doses of 966 (138 microg/day) or 434 microg (62 microg/day), or 3) 700 microg/wk in randomized daily doses (5-250 microg/day). Body weight and length were measured weekly. Femur and tibia lengths and internal organ, fat pad, and muscle weights were recorded at the end of the study (6 wk); blood was collected for IGF axis measurements. GH promoted femur [F(3,60) = 14.67, P < 0.05], tibia [F(3,60) = 14.90, P < 0.05], muscle [F(3,60) = 10.37, P < 0.05], and organ growth [liver: F(3,60) = 9.30, P < 0.05; kidney: F(3,60) = 2.82, P < 0.05] and an increase in serum IGF-I [F(3,60) = 9.18, P < 0.05] and IGFBP-3 [F(3,60) = 6.70, P < 0.05] levels. IGF-I levels correlated with final weight (r = 0.45, P < 0.05) and length (r = 0.284, P < 0.05) in the whole cohort, but within each group, growth parameters correlated with serum IGF-I only in animals treated with random GH doses. The variable regimens promoted femur length (P < 0.05) and muscle (P < 0.05) and kidney (P < 0.05) weight more effectively than treatment with the fixed regimen. This study demonstrates that aspects of growth are improved following introduction of infradian variation to GH treatment in a GH-deficient model. The data suggest that varying the pattern of GH doses administered to children may enhance growth performance without increasing the overall GH dose.
Asunto(s)
Desarrollo Óseo/efectos de los fármacos , Enanismo Hipofisario/fisiopatología , Hormona del Crecimiento/administración & dosificación , Somatomedinas/metabolismo , Animales , Tamaño Corporal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Enanismo Hipofisario/tratamiento farmacológico , Masculino , RatasRESUMEN
The transcription factor SOX2 is expressed most notably in the developing CNS and placodes, where it plays critical roles in embryogenesis. Heterozygous de novo mutations in SOX2 have previously been associated with bilateral anophthalmia/microphthalmia, developmental delay, short stature, and male genital tract abnormalities. Here we investigated the role of Sox2 in murine pituitary development. Mice heterozygous for a targeted disruption of Sox2 did not manifest eye defects, but showed abnormal anterior pituitary development with reduced levels of growth hormone, luteinizing hormone, and thyroid-stimulating hormone. Consequently, we identified 8 individuals (from a cohort of 235 patients) with heterozygous sequence variations in SOX2. Six of these were de novo mutations, predicted to result in truncated protein products, that exhibited partial or complete loss of function (DNA binding, nuclear translocation, or transactivation). Clinical evaluation revealed that, in addition to bilateral eye defects, SOX2 mutations were associated with anterior pituitary hypoplasia and hypogonadotropic hypogonadism, variable defects affecting the corpus callosum and mesial temporal structures, hypothalamic hamartoma, sensorineural hearing loss, and esophageal atresia. Our data show that SOX2 is necessary for the normal development and function of the hypothalamo-pituitary and reproductive axes in both humans and mice.
Asunto(s)
Proteínas de Unión al ADN/genética , Anomalías del Ojo/genética , Proteínas HMGB/genética , Hipotálamo/anomalías , Mutación , Hipófisis/anomalías , Transactivadores/genética , Factores de Transcripción/genética , Anomalías Múltiples/genética , Adulto , Animales , Niño , Femenino , Humanos , Lactante , Masculino , Ratones , Factores de Transcripción SOXB1RESUMEN
BACKGROUND: The heterotrimeric GTP-binding protein eIF2 forms a ternary complex with initiator methionyl-tRNA and recruits it to the 40S ribosomal subunit for start codon selection and thereby initiates protein synthesis. Mutations in EIF2S3, encoding the eIF2γ subunit, are associated with severe intellectual disability and microcephaly, usually as part of MEHMO syndrome. METHODS: Exome sequencing of the X chromosome was performed on three related males with normal head circumferences and mild learning difficulties, hypopituitarism (GH and TSH deficiencies), and an unusual form of glucose dysregulation. In situ hybridisation on human embryonic tissue, EIF2S3-knockdown studies in a human pancreatic cell line, and yeast assays on the mutated corresponding eIF2γ protein, were performed in this study. FINDINGS: We report a novel hemizygous EIF2S3 variant, p.Pro432Ser, in the three boys (heterozygous in their mothers). EIF2S3 expression was detectable in the developing pituitary gland and pancreatic islets of Langerhans. Cells lacking EIF2S3 had increased caspase activity/cell death. Impaired protein synthesis and relaxed start codon selection stringency was observed in mutated yeast. INTERPRETATION: Our data suggest that the p.Pro432Ser mutation impairs eIF2γ function leading to a relatively mild novel phenotype compared with previous EIF2S3 mutations. Our studies support a critical role for EIF2S3 in human hypothalamo-pituitary development and function, and glucose regulation, expanding the range of phenotypes associated with EIF2S3 mutations beyond classical MEHMO syndrome. Untreated hypoglycaemia in previous cases may have contributed to their more severe neurological impairment and seizures in association with impaired EIF2S3. FUND: GOSH, MRF, BRC, MRC/Wellcome Trust and NIGMS funded this study.
Asunto(s)
Factor 2 Eucariótico de Iniciación/genética , Genes Ligados a X , Glucosa/metabolismo , Hipopituitarismo/etiología , Hipopituitarismo/metabolismo , Fenotipo , Sustitución de Aminoácidos , Apoptosis , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Línea Celular , Preescolar , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Hipopituitarismo/diagnóstico , Hibridación in Situ , Lactante , Imagen por Resonancia Magnética , Mutación , Linaje , Polimorfismo de Nucleótido Simple , Biosíntesis de ProteínasRESUMEN
The organization of the peptidergic neurons of the hypothalamic arcuate nucleus is not fully understood. These include growth hormone-releasing hormone (GHRH) neurons involved in growth and metabolism. We studied identified GHRH neurons of GHRH-green fluorescent protein transgenic mice using patch-clamp methods and focused on gender differences, which govern the physiological patterns of GHRH release. Both the spontaneous firing rates and the intrinsic properties of GHRH neurons were similar in males and females, although higher glutamatergic currents were noticed in females. Surprisingly, marked gender differences in GHRH neuronal activity were observed in response to the muscarinic agonist carbachol (CCh). In females, CCh enhanced action potential firing in all GHRH neurons. In males, CCh enhanced action potential firing in two-thirds of GHRH neurons, whereas it decreased firing in the remainders. M1 agonist McN-A343 (10 microM) mimicked, and M1 antagonist pirenzepine (3 microM) blocked the effects of CCh. In both genders, CCh did not change the intrinsic properties of GHRH neurons, although it strongly increased the frequency of glutamatergic currents, in the presence or absence of tetrodotoxin. In males only, CCh enhanced the frequency of GABAergic currents, and this modulation was antagonized by tetrodotoxin. Thus, the muscarinic regulation involved differential control of afferent inputs at short and long distances in male and female mice. The dual-level control could be a mechanism whereby the selective modulation of the GHRH system (short-distance control) is adjusted to the integrated regulation of arcuate nucleus activity (long-distance control).
Asunto(s)
Vías Aferentes/fisiología , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Neuronas/fisiología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Análisis de Varianza , Animales , Núcleo Arqueado del Hipotálamo/citología , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Potenciales Postsinápticos Excitadores/efectos de la radiación , Femenino , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hormona Liberadora de Hormona del Crecimiento/genética , Inmunohistoquímica/métodos , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Factores Sexuales , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Splicing mutations in the human GH (hGH) gene (GH-1) that cause skipping of exon 3 result in a form of GH deficiency termed isolated GH deficiency type II (IGHD II). The GH-1 gene contains five exons; constitutive splicing produces the wild-type 22-kDa hormone, whereas skipping of exon 3 results in transcripts encoding a 17.5-kDa isoform that acts as a dominant-negative to block secretion of the wild-type hormone. Common characteristics of IGHD II include short stature due to impaired bone elongation, growth, and, in severe cases, anterior pituitary hypoplasia. Typically, IGHD II is treated by sc delivery of hGH, which can rescue stature but, unfortunately, does not inhibit pituitary hypoplasia. Direct destruction of transcripts encoding the dominant-negative 17.5-kDa isoform should both rescue stature and prevent hypoplasia. Here, we have used delivery of short hairpin RNAs to rescue a murine model of IGHD II by specifically targeting transcripts encoding the 17.5-kDa isoform using RNA interference. To our knowledge, this is the first example where a short hairpin RNA has been expressed to specifically degrade an incorrectly spliced transcript and rescue a dominant-negative disease phenotype in vivo.
Asunto(s)
Enanismo Hipofisario/terapia , Terapia Genética/métodos , Hormona de Crecimiento Humana/genética , Adenohipófisis/fisiología , Interferencia de ARN , Animales , Modelos Animales de Enfermedad , Enanismo Hipofisario/patología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica , Adenohipófisis/patología , Adenohipófisis/ultraestructura , Recuperación de la FunciónRESUMEN
CONTEXT: Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus. OBJECTIVE: We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues. RESULTS: All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit beta-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathke's pouch, the developing anterior pituitary, and the eye. CONCLUSIONS: Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-beta-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Ojo/embriología , Proteínas HMGB/fisiología , Hipófisis/embriología , Prosencéfalo/embriología , Factores de Transcripción/fisiología , Adolescente , Adulto , Niño , Proteínas de Unión al ADN/genética , Anomalías del Ojo/etiología , Anomalías del Ojo/genética , Femenino , Proteínas HMGB/genética , Humanos , Hipopituitarismo/etiología , Hipopituitarismo/genética , Mutación , ARN Mensajero/análisis , Factores de Transcripción SOXB1 , Transducción de Señal , Factores de Transcripción/genética , beta Catenina/fisiologíaRESUMEN
Growth hormone (GH) secretion decreases spontaneously during lifespan, and the resulting GH deficiency participates in aging-related morbidity. This deficiency appears to involve a defect in the activity of hypothalamic GH-releasing hormone (GHRH) neurons. Here, we investigated this hypothesis, as well as the underlying mechanisms, in identified GHRH neurons from adult ( approximately 13 weeks old) and aged ( approximately 100 weeks old) transgenic GHRH-green fluorescent protein mice, using morphological, biochemical and electrophysiological methods. Surprisingly, the spontaneous action potential frequency was similar in adult and aged GHRH neurons studied in brain slices. This was explained by a lack of change in the intrinsic excitability, and simultaneous increases in both stimulatory glutamatergic- and inhibitory GABAergic-synaptic currents of aged GHRH neurons. Aging did not decrease GHRH and enhanced green fluorescent protein contents, GHRH neuronal number or GHRH-fibre distribution, but we found a striking enlargement of GHRH-positive axons, suggesting neuropeptide accumulation. Unlike in adults, autophagic vacuoles were evident in aged GHRH-axonal profiles using electron microscopy. Thus, GHRH neurons are involved in aging of the GH axis. Aging had a subtle effect at the nerve terminal level in GHRH neurons, contrasting with the view that neuronal aging is accompanied by more widespread damage.
Asunto(s)
Senescencia Celular/fisiología , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Neuronas/fisiología , Terminales Presinápticos/ultraestructura , Potenciales de Acción , Vías Aferentes/fisiología , Animales , Potenciales Postsinápticos Excitadores , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Hormona del Crecimiento/fisiología , Hormona Liberadora de Hormona del Crecimiento/genética , Masculino , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Técnicas de Placa-Clamp , Terminales Presinápticos/metabolismoRESUMEN
The majority of mutations that cause isolated GH deficiency type II (IGHD II) affect splicing of GH-1 transcripts and produce a dominant-negative GH isoform lacking exon 3 resulting in a 17.5-kDa isoform, which further leads to disruption of the GH secretory pathway. A clinical variability in the severity of the IGHD II phenotype depending on the GH-1 gene alteration has been reported, and in vitro and transgenic animal data suggest that the onset and severity of the phenotype relates to the proportion of 17.5-kDa produced. The removal of GH in IGHD creates a positive feedback loop driving more GH expression, which may itself increase 17.5-kDa isoform productions from alternate splice sites in the mutated GH-1 allele. In this study, we aimed to test this idea by comparing the impact of stimulated expression by glucocorticoids on the production of different GH isoforms from wild-type (wt) and mutant GH-1 genes, relying on the glucocorticoid regulatory element within intron 1 in the GH-1 gene. AtT-20 cells were transfected with wt-GH or mutated GH-1 variants (5'IVS-3 + 2-bp T->C; 5'IVS-3 + 6 bp T->C; ISEm1: IVS-3 + 28 G->A) known to cause clinical IGHD II of varying severity. Cells were stimulated with 1 and 10 mum dexamethasone (DEX) for 24 h, after which the relative amounts of GH-1 splice variants were determined by semiquantitative and quantitative (TaqMan) RT-PCR. In the absence of DEX, only around 1% wt-GH-1 transcripts were the 17.5-kDa isoform, whereas the three mutant GH-1 variants produced 29, 39, and 78% of the 17.5-kDa isoform. DEX stimulated total GH-1 gene transcription from all constructs. Notably, however, DEX increased the amount of 17.5-kDa GH isoform relative to the 22- and 20-kDa isoforms produced from the mutated GH-1 variants, but not from wt-GH-1. This DEX-induced enhancement of 17.5-kDa GH isoform production, up to 100% in the most severe case, was completely blocked by the addition of RU486. In other studies, we measured cell proliferation rates, annexin V staining, and DNA fragmentation in cells transfected with the same GH-1 constructs. The results showed that that the 5'IVS-3 + 2-bp GH-1 gene mutation had a more severe impact on those measures than the splice site mutations within 5'IVS-3 + 6 bp or ISE +28, in line with the clinical severity observed with these mutations. Our findings that the proportion of 17.5-kDa produced from mutant GH-1 alleles increases with increased drive for gene expression may help to explain the variable onset progression, and severity observed in IGHD II.
Asunto(s)
Apoptosis/fisiología , Trastornos del Crecimiento/genética , Hormona de Crecimiento Humana/genética , Hipófisis/citología , Sitios de Empalme de ARN/fisiología , Animales , División Celular/fisiología , Células Cultivadas , Fragmentación del ADN , Dexametasona/farmacología , Exones/genética , Genes Dominantes , Glucocorticoides/farmacología , Trastornos del Crecimiento/fisiopatología , Hormona de Crecimiento Humana/deficiencia , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Mutación/fisiología , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , TransfecciónRESUMEN
In the present work, we took advantage of a recently described model of GHRH-enhanced green fluorescent protein (eGFP) transgenic mice to evaluate the extent of co-localization of GHRH neurons with galanin (GAL), neurotensin (NT) and tyrosine hydroxylase (TH) in 3- and 8-month-old male and female mice. The total number of GHRH-eGFP neurons along the rostro-caudal axis of the arcuate nucleus did not differ according to gender or age. GAL-immunoreactivity was present in 40-44% of 3-month-old GHRH-eGFP neurons in male and female arcuate nucleus, respectively, but only 25-22% in 8-month-old mice. TH immunoreactivity occurred in 36-35% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice and these proportions increased to 40 and 45% in 8-month-old mice. NT immunoreactivity was present in 14 and 24% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice up to 28 and 26% in 8-month-old mice. Thus, co-localization of peptides and enzyme in GHRH-eGFP neurons displays a sexual dimorphism at 3-month of age for NT, and at 8-month for TH, while the total number of GHRH-eGFP neurons does not exhibit gender difference at either age. In summary, it appears that changes in co-localized (and presumably co-released) peptides, rather than GHRH per se, may contribute to the changes in sexually dimorphic GH secretion with aging in the mouse.
Asunto(s)
Núcleo Arqueado del Hipotálamo/metabolismo , Galanina/metabolismo , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Neuronas/metabolismo , Neurotensina/metabolismo , Envejecimiento , Animales , Mapeo Encefálico/métodos , Femenino , Proteínas Fluorescentes Verdes , Masculino , Ratones , Ratones Transgénicos , Factores Sexuales , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
The pulsatile pattern of GH secretion exhibits sexual dimorphism that is likely to depend on somatostatin (SRIH) effects on somatoliberin (GHRH) neurons in the hypothalamus. Using transgenic GHRH-enhanced green fluorescent protein (eGFP) mice, no difference in the total number of GHRH-eGFP neurons or change in somatostatin receptor sst2 and SRIH mRNA levels in ventromedial hypothalamic nucleus-arcuate nucleus and periventricular nucleus regions and GHRH mRNA levels in the ventromedial hypothalamic-arcuate region were observed between male and female mice. However, the percentage of GHRH-eGFP neurons bearing sst2A receptors reached 78% in female vs. 26% in male GHRH-eGFP mice (P < 0.02). This sex difference in sst2A distribution on GHRH neurons may play an important role in the sexually differentiated pattern of GH secretion.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/análisis , Hipotálamo/química , Receptores de Somatostatina/análisis , Caracteres Sexuales , Animales , Femenino , Hormona del Crecimiento/metabolismo , Hormona Liberadora de Hormona del Crecimiento/genética , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/análisis , Somatostatina/genéticaRESUMEN
BACKGROUND: Type II isolated GH deficiency (IGHD type II) is caused by dominant negative splicing or point mutations of the GH-1 gene. Studies have suggested that dominant mutant GH forms prevent the secretion of wild-type GH, resulting in eventual cell death; surprisingly, some patients with these GH mutations develop other hormonal deficiencies (ACTH, TSH). SUBJECTS: The proband presented at the age of 2.3 years with IGHD. His father, also known to have been treated for IGHD as a child, had subsequently been lost to follow-up, having remained without treatment during this time. At re-evaluation at the age of 38 years, he complained of lack of stamina and poor libido. Clinical and biochemical assessment confirmed severe GHD, borderline ACTH insufficiency, suboptimal basal and stimulated gonadotropins, and a poor prolactin response to provocation. The basal testosterone concentration was low, and he complained of secondary infertility. Magnetic resonance imaging revealed anterior pituitary hypoplasia in both patients. Genetic testing revealed a heterozygous splicing mutation in GH-1 (intervening sequence-3 + 1G>A) in both patients, known to cause IGHD type II. INTERVENTIONS: The proband showed an excellent growth response to recombinant human GH (rhGH). His father, also treated with rhGH, showed improved quality of life on rhGH, but testosterone concentrations continued to decline, necessitating treatment with testosterone with symptomatic benefit but no improvement in semen quality. CONCLUSIONS: This case supports recent experimental and clinical observations suggesting that the cytotoxicity associated with accumulation of dominant negative mutant 17.5 kDa GH causes a form of GHD that can evolve into multiple hormone deficiencies. Hence, patients diagnosed initially with IGHD type II require continued long-term clinical follow-up.
Asunto(s)
Enanismo Hipofisario/genética , Gonadotropinas/deficiencia , Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/genética , Hormona Adrenocorticotrópica/deficiencia , Adulto , Preescolar , Femenino , Genes Dominantes , Humanos , Masculino , Linaje , Fenotipo , Sitios de Empalme de ARN/genéticaRESUMEN
Animal and clinical models of GHRH excess suggest that GHRH provides an important trophic drive to pituitary somatotrophs. We have adopted a novel approach to silence or ablate GHRH neurons, using a modified H37A variant of the influenza virus M2 protein ((H37A)M2). In mammalian cells, (H37A)M2 forms a high conductance monovalent cation channel that can be blocked by the antiviral drug rimantadine. Transgenic mice with (H37A)M2 expression targeted to GHRH neurons developed postweaning dwarfism with hypothalamic GHRH transcripts detectable by RT-PCR but not by in situ hybridization and immunocytochemistry, suggesting that expression of (H37A)M2 had silenced or ablated virtually all the GHRH cells. GHRH-M2 mice showed marked anterior pituitary hypoplasia with GH deficiency, although GH cells were still present. GHRH-M2 mice were also deficient in prolactin but not TSH. Acute iv injections of GHRH in GHRH-M2 mice elicited a significant GH response, whereas injections of GHRP-6 did not. Twice daily injections of GHRH (100 microg/d) for 7 d in GHRH-M2 mice doubled their pituitary GH but not PRL contents. Rimantadine treatment failed to restore growth or pituitary GH contents. Our results show the importance of GHRH neurons for GH and prolactin production and normal growth.
Asunto(s)
Hormona Liberadora de Hormona del Crecimiento/deficiencia , Hipotálamo/metabolismo , Neuronas/metabolismo , Proteínas de la Matriz Viral/genética , Animales , Antivirales/farmacología , Citomegalovirus/genética , Citomegalovirus/metabolismo , Femenino , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Transgénicos , Técnicas de Placa-Clamp , Enfermedades de la Hipófisis/metabolismo , Adenohipófisis/metabolismo , Rimantadina/farmacología , Factores de Tiempo , Proteínas de la Matriz Viral/metabolismoRESUMEN
Hypothalamic growth hormone-releasing hormone (GHRH) neurons orchestrate body growth/maturation and have been implicated in feeding responses and ageing. However, the electrical patterns that dictate GHRH neuron functions have remained elusive. Since the inhibitory neuropeptide somatostatin (SST) is considered to be a primary oscillator of the GH axis, we examined its acute effects on GHRH neurons in brain slices from male and female GHRH-GFP mice. At the cellular level, SST irregularly suppressed GHRH neuron electrical activity, leading to slow oscillations at the population level. This resulted from an initial inhibitory action at the GHRH neuron level via K(+) channel activation, followed by a delayed, sst1/sst2 receptor-dependent unbalancing of glutamatergic and GABAergic synaptic inputs. The oscillation patterns induced by SST were sexually dimorphic, and could be explained by differential actions of SST on both GABAergic and glutamatergic currents. Thus, a tripartite neuronal circuit involving a fast hyperpolarization and a dual regulation of synaptic inputs appeared sufficient in pacing the activity of the GHRH neuronal population. These "feed-forward loops" may represent basic building blocks involved in the regulation of GHRH release and its downstream sexual specific functions.
Asunto(s)
Potenciales de Acción/fisiología , Hormona Liberadora de Hormona del Crecimiento/metabolismo , Hipotálamo/fisiología , Somatostatina/fisiología , Animales , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/antagonistas & inhibidores , Hipotálamo/metabolismo , Masculino , Ratones , Ratones Noqueados , Técnicas de Placa-ClampRESUMEN
Four distinct familial types of isolated GH deficiency have been described so far, of which type II is the autosomal dominant inherited form. It is mainly caused by mutations within the first 6 bp of intervening sequence 3. However, other splice site and missense mutations have been reported. Based on in vitro experiments and transgenic animal data, there is strong evidence that there is a wide variability in phenotype in terms of the severity of GH deficiency. Therefore, we studied a total of 57 subjects belonging to 19 families suffering from different splice site as well as missense mutations within the GH-1 gene. The subjects presenting with a splice site mutation within the first 2 bp of intervening sequence 3 (5'IVS +1/+2 bp) leading to a skipping of exon 3 were found to be more likely to present in the follow-up with other pituitary hormone deficiencies. In addition, although the patients with missense mutations have previously been reported to be less affected, a number of patients presenting with the P89L missense GH form, showed some pituitary hormone impairment. The development of multiple hormonal deficiencies is not age dependent, and there is a clear variability in onset, severity, and progression, even within the same families. The message of clinical importance from these studies is that the pituitary endocrine status of all such patients should continue to be monitored closely over the years because further hormonal deficiencies may evolve with time.
Asunto(s)
Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/genética , Hipófisis/patología , Adolescente , Niño , Preescolar , Femenino , Estudios de Seguimiento , Humanos , Lactante , Masculino , Mutación , Empalme del ARNRESUMEN
OBJECTIVE: Four distinct familial types of isolated GH deficiency (IGHD) are classified, of which type II, IGHD II, is the autosomal dominant inherited form. Based on clinical data, it became evident that there is a wide variability in phenotype among the various GH-1 gene alterations leading to the disorder. As subjects suffering from IGHD II caused by the specific missense mutated P89L GH (C6129T) have never been reported in detail, the aim was to analyse the impact of this mutated GH form on its clinical follow-up as well as to study its effect at the cellular level in comparison with the most common missense mutation R183H GH (G6664A). METHODS: Twelve subjects belonging to four families presenting with P89L GH were clinically compared with 17 subjects from 5 families with the R183H GH missense mutation. Further, co-localization of the wild-type (wt-type) and mutant GH forms was studied in AtT-20 cells, mouse pituitary gland, applying quantitative confocal microscopy analysis. Using immunofluorescent techniques, cells were double stained for GH and one of the following organelles: endoplasmic reticulum (anti-Grp94), Golgi (anti-betaCOP) and secretory granules (anti-Rab3a). In addition, GH secretion and cell viability was analysed in detail. RESULTS: Importantly, as well as growth hormone deficiency, eight out of twelve subjects with the P89L mutated GH form developed other endocrine deficits and the pituitary gland became smaller over time (P < 0.05). At the cellular level, quantitative analysis of the variable mutants expressed in AtT-20 cells revealed a different extent of co-localization, different effects on GH secretion, and, therefore, a different impact on the secretory pathway which might be caused by different folding or aggregation problems necessary for sorting, packaging and/or secretion through the regulated secretory pathway. CONCLUSIONS: Our results show that specific and detailed analyses of the different mutations identified in IGHD II may shed light on the different mechanisms of secretory pathophysiology, and may provide a better explanation of the range of clinical features associated with GH missense isoforms. Importantly, the findings in patients with P89L GH extend beyond classical IGHD and stress the need for continued clinical vigilance in IGHD II patients for the development of other hormonal deficiencies.
Asunto(s)
Hormona de Crecimiento Humana/deficiencia , Hormona de Crecimiento Humana/genética , Mutación Missense/fisiología , Hormona Adrenocorticotrópica/metabolismo , Adulto , Animales , Células Cultivadas , Niño , Preescolar , Colforsina/farmacología , Femenino , Hormona del Crecimiento/metabolismo , Hormona de Crecimiento Humana/metabolismo , Hormona de Crecimiento Humana/uso terapéutico , Humanos , Masculino , Microscopía Confocal , Persona de Mediana Edad , Linaje , Hipófisis/citología , Hipófisis/efectos de los fármacos , Hipófisis/metabolismoRESUMEN
Growth hormone (GH) has direct effects on the growth plate to stimulate longitudinal growth, but it is not clear which chondrocyte populations GH acts on. The dual effector theory suggests that GH would act primarily on the "stem cells." However, staining with a GH receptor (GHR) antibody is found in all layers of the growth plate in rabbits and humans. We now have investigated the localization and regulation of GHR and the related GH binding protein (GHBP) in the rat growth plate using a sensitive immunohistochemical method involving tyramide signal amplification (TSA) and antibodies specific for GHR or GHBP. Both GHR and GHBP were shown in the germinal and proliferative chondrocytes, but most clearly in early maturing chondrocytes at the interface between proliferative and hypertrophic cells. Staining for GHR and GHBP was located in both the cytoplasm and the nucleus. Expression of GHR mRNA and GHBP mRNA in the growth plate was confirmed by reverse-transcription polymerase chain reaction (RT-PCR). Immunohistochemical staining for GHR and GHBP decreased with age; in 12-week-old normal rats, only the early maturing chondrocytes were stained. In GH-deficient dwarf rats, staining seemed less than in normal rats, and in hypophysectomized (Hx) rats, staining for GHBP was clearly reduced. Treatment of Hx rats with thyroid hormones (T3 + T4), via subcutaneously (sc) implanted osmotic minipumps, induced little growth and induced a small layer of GHR-positive and GHBP-positive early maturing chondrocytes. Treatment with GH and thyroid hormones (TH) resulted in greater growth and a broader layer of GHR-positive and GHBP-positive cells, indistinguishable from normal rats. In contrast, dexamethasone treatment of normal rats inhibited their growth and reduced GHR and GHBP staining in the growth plate. These results show that GHR and GHBP in the growth plate are under hormonal control. The localization of GHR/GHBP suggests that in addition to actions on germinal and proliferative cells in young rats, GH also has effects on early maturing chondrocytes and may be involved in their differentiation to a fully hypertrophic chondrocyte.