RESUMEN
The spatiotemporal pattern of distribution of the sulfhydryl oxidase QSOX throughout ontogeny was mapped in rat brain using immunohistochemistry. The enzyme was detected on embryonic day (E) 12 in the dawning mantle layer, but the adult-like pattern was acquired postnatally around day 30 (P30). Throughout ontogenesis, rQSOX was detected in immature and mature neurons, but not in glial cells. The rQSOX developmental pattern can be divided into four periods: on E12 the enzyme was detected in the brainstem, more precisely in motoneurons; later (E16), rQSOX-positive cells were also observed in the forebrain, in the caudoputamen, and the subventricular zone. During late embryogenesis (E18-20), the amount of rQSOX cells considerably increased throughout the brain; they initially appeared in the hippocampus, then in the isocortex. From birth onwards, complex modifications of the rQSOX distribution occurred leading to the adult pattern by P30. Although rQSOX exhibits an overall increasing spatiotemporal pattern of distribution, different expression strategies were distinguished depending on the cell type or brain area. By comparing the rQSOX ontogeny with data on neurogenesis and brain histogenesis, we hypothesize that the enzyme could play a role in guiding migrating cells, their settling, and neuronal maturation, e.g., during outgrowth and synaptogenesis.
Asunto(s)
Encéfalo/embriología , Encéfalo/enzimología , Neuronas/citología , Neuronas/enzimología , Oxidorreductasas/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Embrión de Mamíferos , Inmunohistoquímica , Proteínas del Tejido Nervioso/metabolismo , Ratas , Ratas Sprague-Dawley , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Recent advances in the study of melanin-concentrating hormone (MCH) have depended largely on molecular biological techniques. In mammals, which have attracted the most attention, novel findings concern (i) the MCH gene, which can yield several peptides by either posttranslational cleavage or alternative splicing, as well as bidirectional transcription; (ii) the identification of two G protein-coupled MCH receptors in the brain and peripheral tissues; and (iii) the evidence for subpopulations of MCH neurons in the central nervous system, characterized by their chemical phenotypes, connections, and individual physiological responses to different physiological paradigms. The involvement of central MCH in various functions, including feeding, reproduction, stress, and behavior patterns, is reviewed. The stage during evolution at which MCH may have acquired hypophysiotrophic and hormonal functions in lower vertebrates is considered in light of morphological data. Evidence that MCH also has peripheral paracrine/autocrine effects in mammals is provided.
Asunto(s)
Sistema Nervioso Central/metabolismo , Hormonas Hipotalámicas/metabolismo , Melaninas/metabolismo , Neuronas/metabolismo , Sistemas Neurosecretores/metabolismo , Hormonas Hipofisarias/metabolismo , Receptores de la Hormona Hipofisaria/metabolismo , Animales , Sistema Nervioso Central/citología , Peces/anatomía & histología , Peces/metabolismo , Humanos , Hormonas Hipotalámicas/genética , Melaninas/genética , Neuronas/citología , Sistemas Neurosecretores/citología , Hormonas Hipofisarias/genética , Receptores de la Hormona Hipofisaria/genética , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Vísceras/metabolismoRESUMEN
Mammalian Augmenter of Liver Regeneration protein (ALR) was first identified as a secondary growth factor involved in liver regeneration. Its sulfhydryl oxidase activity and involvement in iron homeostasis have been recently demonstrated. ALR is expressed in a broad range of peripheral organs, and initial experiments gave also evidence for the occurrence of this protein in brain. In the present study, we investigated in detail the expression of ALR in rat brain sections and determined its cellular and subcellular localizations using biomolecular and immunohistochemical procedures. As shown by Northern blot, ALR is differentially expressed throughout the rat brain, with the highest mRNA levels in the cerebellum and diencephalon. High protein levels were also detected in the brain and cerebellum by Western blot. ALR immunoreactivity was found in neurons and glial cells throughout brain rostrocaudal extent. Labeled astrocytes were particularly abundant in the white matter, and immunoreactive neurons were observed in several regions including the olfactory bulb, isocortex, hippocampal formation, amygdala, thalamus, hypothalamus, some nuclei of the brainstem and cerebellum. In neurons, immunoelectron microscopy showed the protein in the nucleus and mainly in mitochondria. These subcellular localizations may correlate with the occurrence of two ALR protein isoforms in the brain. In the central nervous system, the enzyme might be of importance in heavy metal homeostasis whose dysregulation can induce neurodegenerative disorders.
Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica/fisiología , Proteínas/metabolismo , Animales , Northern Blotting/métodos , Western Blotting/métodos , Encéfalo/citología , Encéfalo/ultraestructura , Inmunohistoquímica/métodos , Microscopía Inmunoelectrónica/métodos , Neuronas/metabolismo , Neuronas/ultraestructura , Proteínas/genética , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
The distribution of the sulfhydryl oxidase QSOX in the rat brain was mapped using immunohistochemistry. QSOX is specifically expressed by neurons throughout the rostrocaudal extent of the brain as well as in the spinal cord. Although a majority of neurons express QSOX, different intensities of labeling were observed depending on the area: the strongest labeling was observed in the olfactory bulbs, isocortex, hippocampus, basal telencephalon, several thalamic and hypothalamic nuclei, cerebellum, and numerous brainstem nuclei. This study also describes the ultrastructural localization of QSOX in neuronal cells and demonstrates that the enzyme is associated with the Golgi apparatus. Finally, selected double immunohistochemistry showed that in the hypothalamus the highest levels of QSOX labeling were colocalized in neuron populations that express disulfide-bounded neuropeptides. These observations are consistent with a role of the enzyme in secreted peptide/protein folding. Data presented herein will serve as a basis for further investigations of the physiological function of QSOX in the central nervous system.
Asunto(s)
Sistema Nervioso Central/enzimología , Flavina-Adenina Dinucleótido , Oxidorreductasas/metabolismo , Animales , Western Blotting/métodos , Mapeo Encefálico , Sistema Nervioso Central/ultraestructura , Aparato de Golgi/enzimología , Aparato de Golgi/ultraestructura , Inmunohistoquímica/métodos , Microscopía Inmunoelectrónica/métodos , Neuropéptido Y/metabolismo , Oxidorreductasas/inmunología , Proopiomelanocortina/metabolismo , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/enzimologíaRESUMEN
Sulfhydryl oxidases belonging to the FAD-dependent sulfhydryl oxidase/quiescin Q6 family were previously reported in rat peripheral organs but they were not detected in brain. In the present study, by using reverse transcription-polymerase chain reaction and northern blot analysis, we clearly show an ubiquitous expression of the gene in brain; moreover, while only one transcript was present in peripheral organs, at least two transcripts were detected in brain, suggesting a tissue-specific splicing of its mRNA. The shorter one, likely corresponding to the mRNA identified from rat seminal vesicles, was highly expressed in diencephalon and telencephalon. The finding of gene expression in brain is relevant, since its dysregulation could lead to oxidative stress, a causative factor in the pathogenesis of neurodegenerative diseases.
Asunto(s)
Encéfalo/enzimología , Oxidorreductasas/metabolismo , Tiorredoxinas/metabolismo , Animales , Northern Blotting , Encéfalo/metabolismo , Regulación Enzimológica de la Expresión Génica , Masculino , Enfermedades Neurodegenerativas/enzimología , Estrés Oxidativo , Oxidorreductasas/genética , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro , Empalme del ARN , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiorredoxinas/genéticaRESUMEN
We showed earlier that a specific neuron population of the rat lateral hypothalamus, differing from the codistributed melanin-concentrating hormone (MCH) neurons, express both dynorphin (DYN) and secretogranin II (SgII) genes. We demonstrated later that this population corresponds in fact to the newly identified orexin/hypocretin (OX/Hcrt) neurons. In the present study, by revisiting the chemical phenotype of these neurons, we confirm that all of them contain DYN B- and SgII-immunoreactive materials. The roles played by these peptide/protein in OX/Hcrt neurons are still unclear. Double immunocytochemical stainings highlight putative somasomatic, axosomatic and axodendritic contacts between OX/Hcrt and MCH neurons. Adding OX/Hcrt to the culture medium of hypothalamic slices from 8-day-old rats results either in a significant increase of MCH mRNA after 24 h survival or a strong fall after 10 days culture. These results taken together suggest that OX/Hcrt can directly and/or indirectly affect MCH expression, and that both OX/Hcrt and MCH neuron populations interact to respond in a coordinated manner to central and peripheral signals.
Asunto(s)
Proteínas Portadoras/farmacología , Dinorfinas/biosíntesis , Endorfinas/biosíntesis , Área Hipotalámica Lateral/efectos de los fármacos , Hormonas Hipotalámicas/biosíntesis , Péptidos y Proteínas de Señalización Intracelular , Melaninas/biosíntesis , Neuronas/metabolismo , Neuropéptidos/farmacología , Hormonas Hipofisarias/biosíntesis , Biosíntesis de Proteínas , Proteínas , Animales , Proteínas Portadoras/biosíntesis , Comunicación Celular/fisiología , Cromograninas , Área Hipotalámica Lateral/citología , Inmunohistoquímica/métodos , Masculino , Neuronas/fisiología , Neuropéptidos/biosíntesis , Orexinas , Ratas , Ratas Sprague-Dawley , Distribución Tisular/fisiologíaRESUMEN
Neurotropic viruses are involved in pathologies of the central nervous system, triggering transient or irreversible disorders, such as neurological diseases or homeostasis imbalance. In experimental animals, viruses have been shown to cause obesity, a complex disease depending on multiple factors, including genetic susceptibility and environmental components. Using a mouse model of virally induced obesity following brain infection by the Canine Distemper Virus (CDV), a morbillivirus closely related to the human measles virus, we investigated the modulation of expression of several hypothalamic neuropeptides known to intervene in the regulation of body weight and energy expenditure, both during the acute and late stages of infection. During the acute stage, while viral replication occurs, we found a dramatic decrease of expressions of neuropeptides, in particular neuropeptide Y, melanin-concentrating hormone (MCH), hypocretin, vasopressin and tachykinins, the magnitude of which seemed to be linked to the viral burden and the individual susceptibility. The effect of the virus, however, varied with the hypothalamic nucleus and neuropeptide involved, suggesting that certain circuits were affected while others remained intact. During the late stage of infection, marked recovery to the initial hypothalamic levels of peptide expression was seen in a number of lean animals, suggesting recovery of homeostasis equilibrium. Interestingly, some neuropeptidergic systems remained disturbed in mice exhibiting obese phenotype, arguing for their involvement in triggering/maintaining obesity. Even though our data could not fully explain the viral-induced obesity, they may be helpful in understanding the molecular events associated with obesity and in investigating therapeutic alternatives.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Hipotálamo/metabolismo , Hipotálamo/virología , Infecciones por Morbillivirus/metabolismo , Neuropéptidos/metabolismo , Animales , Recuento de Células/métodos , Virus del Moquillo Canino/patogenicidad , Femenino , Hormonas Hipotalámicas/metabolismo , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Melaninas/metabolismo , Ratones , Infecciones por Morbillivirus/etiología , Neuropéptido Y/metabolismo , Neuropéptidos/clasificación , Neuropéptidos/genética , Orexinas , Hormonas Hipofisarias/metabolismo , ARN Mensajero/metabolismo , Taquicininas/metabolismo , Factores de Tiempo , Vasopresinas/metabolismoRESUMEN
Rat quiescin/sulphydryl oxidase (rQSOX) introduces disulphide bridges into peptides and proteins with the reduction of molecular oxygen to hydrogen peroxide. Its occurrence has been previously highlighted in a wide range of organs by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses, methods that have provided information concerning its expression in whole organs but that do not reveal the cell types expressing this enzyme. In this report, in addition to RT-PCR and Western blot experiments, the cell-specific localization of rQSOX has been investigated in a wide range of male and female adult rat tissues by using in situ hybridization and immunohistochemistry. Labelling was detected in most organs and systems including the immune, endocrine and reproductive systems, the respiratory, digestive and urinary tracts and the skin. No labelling was observed in the heart, blood vessel endothelium, liver or smooth and skeletal muscles. rQSOX expression was mainly localized in epithelial cells specialized in secretion, strengthening the hypothesis that QSOX enzymes play an important role in the mechanism of secretion, notably in the folding of secreted proteins. The intracellular patterns of immunolabelling indicate that the protein usually follows the secretory pathway, which is in accordance with its secreted nature and its presumed involvement in the elaboration of the extracellular matrix. In seminiferous tubules, where a high level of expression was noticed, QSOX might play an important physiological role in sperm function and serve as a marker for the diagnosis of male infertility.
Asunto(s)
Regulación de la Expresión Génica , Oxidorreductasas/metabolismo , Túbulos Seminíferos/metabolismo , Animales , Femenino , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Sensibilidad y Especificidad , Distribución TisularRESUMEN
The Scrg1 gene was initially discovered as one of the genes upregulated in transmissible spongiform encephalopathies (TSE). Scrg1 encodes a highly conserved, cysteine-rich protein expressed principally in the central nervous system. The protein is targeted to the Golgi apparatus and large dense-core vesicles/secretory granules in neurons. We have recently shown that the Scrg1 protein is widely induced in neurons of scrapie-infected mice, suggesting that Scrg1 is involved in the host response to stress and/or the death of neurons. At the ultrastructural level, Scrg1 is associated with dictyosomes of the Golgi apparatus and autophagic vacuoles of degenerative neurons. It is well known that apoptosis plays a major role in the events leading to neuronal cell death in TSE. However, autophagy was identified in experimentally induced scrapie a long time ago and was recently reevaluated as a possible cell death program in prion diseases. The consistent association of Scrg1 with autophagic structures typical of scrapie is in agreement with the recruitment of Golgi-specific proteins in this degradation process and we suggest that Scrg1 might be used as a specific probe to identify neuronal autophagy in TSE.
Asunto(s)
Autofagia , Proteínas del Tejido Nervioso/análisis , Enfermedades por Prión/patología , Animales , Biomarcadores/análisis , Ratones , Neuronas/química , Neuronas/ultraestructura , Enfermedades por Prión/metabolismoRESUMEN
We have previously identified Scrg1, a gene with increased cerebral mRNA levels in transmissible spongiform encephalopathies (TSE) such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. In this study, Scrg1-immunoreactive cells, essentially neurons, were shown to be widely distributed throughout the brain of scrapie-infected mice, while only rare and weakly immunoreactive cells could be detected in the brain of non-infected normal mice. Induction of the protein was confirmed by Western blot analysis. At the ultrastructural level, Scrg1 protein was associated with dictyosomes of the Golgi apparatus and autophagic vacuoles in the central neurons of the scrapie-infected mice. These results suggested a role for Scrg1 in the pathological changes observed in TSE. We have generated transgenic mice specifically expressing Scrg1 in neurons. No significant differences in the time course of the disease were detected between transgenic and non-transgenic mice infected with scrapie prions. However, tight association of Scrg1 with autophagic vacuoles was again observed in brain neurons of infected transgenic mice. High levels of the protein were also detected in degenerating Purkinje cells of Ngsk Prnp 0/0 mice overexpressing the Prnd gene coding for doppel, a neurotoxic paralogue of the prion protein. Furthermore, induction of Scrg1 protein was observed in the brain of mice injured by canine distemper virus or gold thioglucose treatment. Taken together, our results indicate that Scrg1 is associated with neurodegenerative processes in TSE, but is not directly linked to dysregulation of prion protein.
Asunto(s)
Autofagia/genética , Encéfalo/metabolismo , Degeneración Nerviosa/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Enfermedades por Prión/metabolismo , Priones/metabolismo , Animales , Aurotioglucosa/farmacología , Encéfalo/patología , Encéfalo/ultraestructura , Modelos Animales de Enfermedad , Virus del Moquillo Canino/genética , Virus del Moquillo Canino/metabolismo , Proteínas Ligadas a GPI , Aparato de Golgi/patología , Aparato de Golgi/ultraestructura , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Degeneración Nerviosa/genética , Degeneración Nerviosa/fisiopatología , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Neuronas/patología , Neuronas/ultraestructura , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Enfermedades por Prión/genética , Enfermedades por Prión/fisiopatología , Priones/genética , Vacuolas/patología , Vacuolas/ultraestructuraRESUMEN
InViviparus viviparus, the differences between the gonads of young males and young females appear first at the morphological level. The ovary is organised as a poorly branched tubule, whereas the testis is very compact and highly ramified. In the ovary, the non-germ cells (future follicular cells) surround the germ cells and the premeiotic processes begin early. During the morphogenesis of the testis contrary, several successive stages are observed. After a period of intensive cell proliferation concomitant with the ramification of the gonad, the lumen is formed by the death of the central cells. The non-germ cells become nurse cells close to the basal lamina, while the spermatogonia differentiate. The male gametes appear late.
RESUMEN
The origin and evolution of the embryonic gonad ofViviparus viviparus were studied ultrastructurally. The gonad is formed by migration and multiplication of pericardial cells.There are several successive stages during gonad organogenesis: during the sexually undifferentiated stage with one cellular type, the gonad is made up of primordial cells which seem to be identical. The sexually undifferentiated stage with two cell types is characterized by the differentiation of germinal and non-germinal cells. The non-germinal cells are linked to one another by septate junctions and their long extensions completely surround the germinal cells which remain isolated and do not form junctions with their neighbours. The gonad sexualisation does not appear until after organogenesis, towards the end of embryonic development, or even after birth.
RESUMEN
The study of gonadal organogenesis and differentiation by means of light and electron microscopy suggested the following in Helix aspersa: (1) the distal parts of the acini have components of mesodermal origin, whereas the neck and efferent duct comprise ectodermal elements; (2) a segregation of a germinal line occurs early, during the embryonic life; (3) in juvenile and adult animals, male and female cells arise from a germinal ring located at the base of the acinar neck. Apart from developing oocytes, the epithelium lining the distal region of the acini consists of somatic cells (Sertoli and follicle cells).
RESUMEN
The total soluble protein content in crude extracts of gonads ofHelix aspersa Müller from different stages of its life cycle was quantified and then resolved into different protein fractions using polyacrylamide gel electrophoresis. It was found that both the protein content and wet weight of the gonads increased during growth but decreased markedly during the copulation and egg-laying phases. Electrophoretic analysis of the extracts from different stages revealed that additional fractions progressively appeared and that in adults, the number and comcentration of the bands changed in response to physiological conditions (activity, copulation, egg-laying or hibernation) so that the maximum number of 27 fractions was observed during the active phase. We have attempted to correlate these biochemical observations with previously published descriptions of histological changes that occur during the development of the gonad.
RESUMEN
Scrapie responsive gene one (Scrg1) is a novel transcript discovered through identification of the genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies. Scrg1 mRNA is distributed principally in the central nervous system and the cDNA sequence predicts a small cysteine-rich protein 98 amino acids in length, with a N-terminal signal peptide. In this study, we have generated antibodies against the predicted protein and revealed expression of a predominant immunoreactive protein of 10 kDa in mouse brain by Western blot analysis. We have established CAD neuronal cell lines stably expressing Scrg1 to determine its subcellular localization. Several lines of evidence show that the protein is targeted to dense-core vesicles in these cells. (i) Scrg1 is detected by immunocytochemistry as very punctate signals especially in the Golgi apparatus and tips of neurites, suggesting a vesicular localization for the protein. Moreover, Scrg1 exhibits a high degree of colocalization with secretogranin II, a dense-core vesicle marker and a very limited colocalization with markers for small synaptic vesicles. (ii) Scrg1 immunoreactivity is associated with large secretory granules/dense-core vesicles, as indicated by immuno-electron microscopy. (iii) Scrg1 is enriched in fractions of sucrose density gradient where synaptotagmin V, a dense-core vesicle-associated protein, is also enriched. The characteristic punctate immunostaining of Scrg1 is observed in N2A cells transfected with Scrg1 and for the endogenous protein in cultured primary neurons, attesting to the generality of the observations. Our findings strongly suggest that Scrg1 is associated with the secretory pathway of neuronal cells.
Asunto(s)
Proteínas de Unión al Calcio , Proteínas del Tejido Nervioso/análisis , Neuronas/química , Vesículas Sinápticas/química , Secuencia de Aminoácidos , Animales , Anticuerpos , Western Blotting , Línea Celular , Sistema Nervioso Central/química , Cromograninas , ADN Complementario , Inmunohistoquímica , Glicoproteínas de Membrana/análisis , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Proteínas/análisis , Proteínas Recombinantes/análisis , SinaptotagminasRESUMEN
Neurons producing melanin-concentrating hormone (MCH) are involved in a large array of functions. Some of these functions may be mediated by specific subpopulations. One such subpopulation was characterized by the expression of the neurokinin 3 receptor and the 'cocaine- and amphetamine-regulated transcript' (CART) peptide, while another expresses neither one of these two molecules. MCH+/CART+ axons were traced throughout the brain and showed a strikingly different pattern of distribution than that of MCH+/CART- axons. Particularly, many MCH+/CART+ axons are observed in the telencephalon, while MCH+/CART- projections are mostly directed toward the brainstem. Calbindin, a protein involved in calcium homeostasis, has been largely used in many structures of the brain for the identification of neuronal phenotypes. However, few MCH neurons were labeled for this protein. On the other hand, neurons producing the peptides hypocretins (Hcrt), and codistributed with the MCH neurons, were all labeled for calbindin. Thus, at least two subpopulations of MCH neurons can be distinguished on the basis of neuronal phenotypes and connections. These neurons may be involved in distinct circuitry and in distinct functions.