RESUMEN
In mammals, the olfactory sensory neurons are the only ones directly in contact with an aggressive environment. Thus, the olfactory mucosa is one of the few neuronal zones which are continuously renewed during adulthood. We have previously shown that endothelin is locally matured in the olfactory mucosa and that olfactory sensory neurons preferentially express ETB receptors, while ETA receptors are rather present in non neuronal olfactory mucosa cells. In addition to its vasoactive effect, the endothelin system is known for its pleiotropic effects including the modulation of cell population dynamics. We thus examined its potential neuroprotective effect in the olfactory mucosa using a primary culture of olfactory sensory neurons lying on non neuronal cells. While a serum deprivation led to a massive decrease of the density of olfactory sensory neurons in the primary cultures, endothelin 1 (ET-1) rescued part of the neuronal population through both ETA and ETB receptors. This effect was mainly anti-apoptotic as it reduced cleaved caspase-3 signal and nuclear condensation. Furthermore, the olfactory epithelium of ETB-deficient rats displayed increased apoptosis. These results strongly suggest that ET-1 acts as an anti-apoptotic factor on olfactory sensory neurons, directly through ETB and indirectly by limiting non neuronal cells death through ETA.
Asunto(s)
Citoprotección/fisiología , Endotelina-1/fisiología , Mucosa Olfatoria/fisiología , Neuronas Receptoras Olfatorias/fisiología , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Citoprotección/efectos de los fármacos , Citoprotección/genética , Endotelina-1/genética , Técnicas de Inactivación de Genes , Masculino , Fármacos Neuroprotectores/farmacología , Mucosa Olfatoria/citología , Mucosa Olfatoria/efectos de los fármacos , Neuronas Receptoras Olfatorias/efectos de los fármacos , Neuronas Receptoras Olfatorias/metabolismo , Ratas , Ratas Mutantes , Ratas Wistar , Receptor de Endotelina B/deficiencia , Receptor de Endotelina B/genética , Receptor de Endotelina B/fisiologíaRESUMEN
The olfactory system is regulated by several nervous and hormonal factors, and there is a growing body of evidence that some of these modulations already take place in the olfactory mucosa (OM). We recently suggested that, among others, vasoactive peptides might play multifaceted roles in different OM cells. Here we studied the effect of the vasoconstrictive peptide endothelin (ET) in the rat OM. We identified different components of the ET system both in the olfactory mucosa and in long-term primary culture of OM cells, composed of olfactory sensory neurons (OSNs) lying on a blend of non-neuronal OM cells (nNCs). We demonstrated that ET receptors are differentially expressed on OM cells, and that ET might be locally matured by the endothelin-converting enzyme ECE-1 located in OSNs. Using calcium imaging, we showed that ET triggers robust dose-dependent Ca(2+) responses in most OM cells, which consist of a transient phase, followed, in nNCs, by a sustained plateau phase. All transient responses depended on intracellular calcium release, while the sustained plateau phase also depended on subsequent external calcium entry. Using both pharmacology and spotting lethal (sl/sl) mutant rats, lacking functional ET(B) receptors, we finally demonstrated that these effects of ET are mediated through ET(B) receptors in OSNs and ET(A) receptors in nNCs.The present study therefore identifies endothelin as a potent endogenous modulator of the olfactory mucosa; specific endothelin-mediated Ca(2+) signals may serve distinct signaling functions, and thereby suggest differential functional roles of endothelin in both neuronal and non-neuronal OM cells.
Asunto(s)
Calcio/metabolismo , Endotelinas/metabolismo , Mucosa Olfatoria/metabolismo , Células Receptoras Sensoriales/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Células Cultivadas , Enzimas Convertidoras de Endotelina , Fluorescencia , Inmunohistoquímica , Espacio Intracelular/metabolismo , Masculino , Metaloendopeptidasas/metabolismo , Ratas , Ratas Mutantes , Ratas Wistar , Receptor de Endotelina A/metabolismo , Receptor de Endotelina B/genética , Receptor de Endotelina B/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Factores de TiempoRESUMEN
Food odours are major determinants for food choice, and their detection depends on nutritional status. The effects of different odour stimuli on both behavioural responses (locomotor activity and sniffing) and Fos induction in olfactory bulbs (OB) were studied in satiated or 48-h fasted rats. We focused on two odour stimuli: isoamyl acetate (ISO), as a neutral stimulus either unknown or familiar, and food pellet odour, that were presented to quiet rats during the light phase of the day. We found significant effects of nutritional status and odour stimulus on both behavioural and OB responses. The locomotor activity induced by odour stimuli was always more marked in fasted than in satiated rats, and food odour induced increased sniffing activity only in fasted rats. Fos expression was quantified in periglomerular, mitral and granular OB cell layers. As a new odour, ISO induced a significant increase in Fos expression in all OB layers, similar in fasted and satiated rats. Significant OB responses to familiar odours were only observed in fasted rats. Among the numerous peptides shown to vary after 48 h of fasting, we focused on orexins (for which immunoreactive fibres are present in the OB) and leptin, as a peripheral hormone linked to adiposity, and tested their effects of food odour. The administration of orexin A in satiated animals partially mimicked fasting, since food odour increased OB Fos responses, but did not induce sniffing. The treatment of fasted animals with either an orexin receptors antagonist (ACT-078573) or leptin significantly decreased both locomotor activity, time spent sniffing food odour and OB Fos induction in all cell layers, thus mimicking a satiated status. We conclude that orexins and leptin are some of the factors that can modify behavioural and OB Fos responses to a familiar food odour.
Asunto(s)
Conducta Animal , Alimentos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Leptina/fisiología , Neuropéptidos/fisiología , Odorantes , Bulbo Olfatorio/metabolismo , Pentanoles , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Animales , Ayuno , Péptidos y Proteínas de Señalización Intracelular/farmacología , Leptina/farmacología , Masculino , Actividad Motora , Neuropéptidos/farmacología , Receptores de Orexina , Orexinas , Ratas , Ratas Wistar , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Leptina/metabolismo , Receptores de Neuropéptido/antagonistas & inhibidores , Receptores de Neuropéptido/metabolismo , SaciedadRESUMEN
Food odours are major determinants for food choice; their detection is influenced by nutritional status. Among different metabolic signals, insulin plays a major role in food intake regulation. The aim of the present study was to investigate a potential role of insulin in the olfactory mucosa (OM), using ex vivo tissues and in vitro primary cultures. We first established the expression of insulin receptor (IR) in rat olfactory mucosa. Transcripts of IR-A and IR-B isoforms, as well as IRS-1 and IRS-2, were detected in OM extracts. Using immunocytochemistry, IR protein was located in olfactory receptor neurones, sustentacular and basal cells and in endothelium of the lamina propria vessels. Moreover, the insulin binding capacity of OM was quite high compared to that of olfactory bulb or liver. Besides the main pancreatic insulin source, we demonstrated insulin synthesis at a low level in the OM. Interestingly 48 h of fasting, leading to a decreased plasmatic insulin, increased the number of IR in the OM. Local insulin concentration was also enhanced. These data suggest a control of OM insulin system by nutritional status. Finally, an application of insulin on OM, aiming to mimic postprandial insulin increase, reversibly decreased the amplitude of electro-olfactogramme responses to odorants by approximately 30%. These data provide the first evidence that insulin modulates the most peripheral step of odour detection at the olfactory mucosa level.
Asunto(s)
Insulina/metabolismo , Mucosa Olfatoria/metabolismo , Receptor de Insulina/metabolismo , Animales , Células Cultivadas , Ingestión de Alimentos , Electrofisiología , Ayuno , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Insulina/genética , Proteínas Sustrato del Receptor de Insulina/genética , Proteínas Sustrato del Receptor de Insulina/metabolismo , Masculino , Estado Nutricional , Odorantes , Mucosa Olfatoria/citología , Neuronas Receptoras Olfatorias/citología , Neuronas Receptoras Olfatorias/metabolismo , Isoformas de Proteínas/metabolismo , Radioinmunoensayo , Ratas , Ratas Wistar , Receptor de Insulina/genética , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Neuroanatomical data show that olfactory mucosa (OM) is a possible place for interactions between nutrition and smell. A combination of differential display mRNA analysis together with a macroarray screening was developed to identify transcripts that are differentially expressed in rat OM following food deprivation. Using this method, backed on a stringent statistical analysis, we identified molecules that fell into several Gene Ontology terms including cellular and physiological process, signal transduction, and binding. Among the 15 most differentially expressed molecules, only one was upregulated, but 14 were downregulated in the fasted state among which was, unexpectedly, odorant-binding protein 1F (OBP-1F). Because of its potential relevance to olfactory physiology, we focused our further analysis on OBP-1F using in situ hybridization, quantitative polymerase chain reaction, and western blot analysis. OBP-1F was highlighted in the lateral nasal glands, but its expression (mRNA and protein) did not change following food deprivation. Only the minor fraction of OBP-1F mRNA expressed by the OM itself was downregulated following 48 h fasting. Altogether, our results suggest that the fine transcriptional control of OBP-1F in the OM following food deprivation could be efficient only at the local level, close to its site of secretion to participate in the perireceptor events of the olfactory signal reception.
Asunto(s)
Privación de Alimentos , Perfilación de la Expresión Génica , Mucosa Olfatoria/metabolismo , Receptores Odorantes/genética , Animales , Regulación hacia Abajo , Glándulas Exocrinas/citología , Glándulas Exocrinas/metabolismo , Masculino , Mucosa Nasal/citología , Mucosa Nasal/metabolismo , Mucosa Olfatoria/citología , Ratas , Ratas Wistar , Receptores Odorantes/metabolismoRESUMEN
The authors investigated whether neuromuscular and directional constraints are dissociable limitations that affect learning and transfer of a bimanual coordination pattern. Participants (N = 9) practiced a 45 degrees muscular relative phasing pattern in the transverse plane over 4 days. The corresponding to-be-learned spatial relative phasing was 225 degrees. Before, during, and following practice, the authors administered probe tests in the sagittal plane to assess transfer of learning. In the probe tests, participants performed various patterns characterized by different muscular and spatial relative phasing (45 degrees, 45 degrees, 45 degrees, 225 degrees, 225 degrees, 45 degrees, and 225 degrees, 225 degrees). The acquisition of the to-be-learned pattern in the transverse plane resulted in spontaneous positive transfer of learning only to coordination patterns having 45 degrees of spatial relative phase, irrespective of muscular phasing. Moreover, transfer occurred in the sagittal plane to coordination patterns that had symmetry properties similar to those of the to-be-learned pattern. The authors conclude that learning and transfer of spatial features of coordination patterns from the transverse to the sagittal plane of motion are mediated by mirror-symmetry constraints.
Asunto(s)
Lateralidad Funcional/fisiología , Mano/fisiología , Desempeño Psicomotor/fisiología , Transferencia de Experiencia en Psicología/fisiología , Adolescente , Adulto , Análisis de Varianza , Humanos , Movimiento/fisiología , Patrones de Reconocimiento Fisiológico/fisiología , Práctica Psicológica , Valores de ReferenciaRESUMEN
The present work investigated the effects of spatial and neuromuscular constraints on the mean states and variability of interlimb coordination patterns performed in the para-sagittal plane of motion in a hand-held pendulum oscillation task. Nine right-handed students had to oscillate two pendulums through wrist adduction-abduction movements. Relative movement direction was manipulated by asking participants to perform both isodirectional and non-isodirectional movements. Participants were required to grab the pendulums either with both forearms in the same neutral or supine posture or with one forearm in neutral while the other one was in prone-inversed position. When both forearms were in a similar posture, isodirectional movements were generated predominantly by simultaneous activation of homologous muscle groups whereas non-isodirectional movements mainly resulted from simultaneous activation of non-homologous muscle groups. When forearms were in dissimilar posture, isodirectional movements were generated predominantly by the simultaneous activation of non-homologous muscle groups whereas non-isodirectional movements mainly resulted from simultaneous activation of homologous muscle groups. Standard deviation of relative phase and absolute error of relative phase were analyzed for each forearm posture condition. We hypothesized that neuromuscular and spatial constraints would affect two different aspects of coordination performance, i.e., pattern stability and accuracy, respectively. Comparison of the results obtained for similar and dissimilar postures suggested that changes of pattern stability were mediated by changes in the nature of the muscle activation patterns that gave rise to wrist movement in each condition. On the other hand, the results also showed that movement direction exclusively affected phase shift. The findings are consistent with the conclusion of Park et al. [Park, H., Collins, D. R., & Turvey, M. T. (2001). Dissociation of muscular and spatial constraints on patterns of interlimb coordination. Journal of Experimental Psychology: Human Perception and Performance, 27, 32-47.] that neuromuscular constraints affect variability of relative phase (attractor strength) and spatial constraints affect the shift of relative phase (attractor location).
Asunto(s)
Mano/fisiología , Movimiento (Física) , Movimiento/fisiología , Músculo Esquelético/fisiología , Conducta Espacial/fisiología , Transmisión Sináptica/fisiología , Adulto , Humanos , Contracción Muscular/fisiología , Periodicidad , Postura/fisiologíaRESUMEN
In the present study, we investigated the contributions of motor and perceptual processes to directional constraints as observed during hand-foot coordination. Participants performed cyclical flexion-extension movements of the right hand and foot under two coordination modes: in-phase (isodirectional) and antiphase (non-isodirectional). Those tasks were performed either with full vision or no vision of the limbs. Depending on the position of the forearm (prone or supine), the coordination patterns were performed with similar and dissimilar neuro-muscular coupling with respect to their phylogenetic origin as antigravity muscles. Results showed that the antiphase pattern was more difficult to maintain than the in-phase pattern and that neuro-muscular coupling significantly influenced the coordination dynamics. Moreover, the effect of vision differed as a function of both neuro-muscular coupling and coordination mode. Under dissimilar neuro-muscular coupling, the presence of visual feedback stabilized the in-phase pattern and destabilized the antiphase pattern. In contrast, visual feedback did not influence pattern stability during conditions of similar neuro-muscular coupling. These results shed light on the complex interactions between motor and perceptual (visual) constraints during the production of hand-foot coordination patterns.
Asunto(s)
Pie/fisiología , Mano/fisiología , Desempeño Psicomotor/fisiología , Percepción Espacial/fisiología , Conducta Espacial/fisiología , Percepción Visual/fisiología , Adulto , HumanosRESUMEN
Understanding how multiple constraints contribute to the emergence of coordinated behavior has been the topic of considerable debate in cognitive sciences. The present experiment addressed the issue of the effects of visual motion structures (iso- and non-isodirectionality) on the stability of hand-foot coordination patterns. Visuo-motor transformations--decorrelating the perceived movement direction from the actually generated direction--were applied to both in-phase and anti-phase patterns. Two mutually exclusive hypotheses--the "visual grouping hypothesis" and the "incongruency hypothesis"--were tested. The results indicated that both conditions of transformed visual feedback destabilized the actual performed coordination patterns. Thus, despite the existence of common underlying principles that govern both the perceived motion pattern and the generation of hand-foot coordination patterns, it appeared that perceptual grouping principles were not exploited to monitor the production of coordination. These results strongly suggest that the congruency between the performed pattern and the perceived visual feedback is the primary factor determining the (in)stability of hand-foot coordination patterns.
Asunto(s)
Retroalimentación/fisiología , Pie/fisiología , Mano/fisiología , Movimiento/fisiología , Desempeño Psicomotor/fisiología , Adulto , Femenino , Lateralidad Funcional/fisiología , Humanos , Cinestesia/fisiología , Masculino , Modelos Neurológicos , Percepción de Movimiento/fisiología , Propiocepción/fisiología , Percepción Visual/fisiologíaRESUMEN
G-protein-coupled receptors (GPCRs) constitute the largest but the most divergent class of cell surface proteins. Although they are thought to share a common 3D-structure composed of seven transmembrane helical domains, they can be activated by extracellular signals as diverse as light, peptides, proteins, lipids, organic odorants, taste molecules, nucleotides or nucleosides. They are involved in an extraordinarily large number of physiological functions and are therefore potential drug targets for many human diseases. During the last decade various GPCRs have been successfully expressed in S. cerevisiae. Yeast is an attractive expression system because it offers the genetic engineering tools typical of a microorganism while possessing an eukaryotic type of secretory pathway and post-translational machinery. This host is particularly attractive for in-vivo manipulation of these receptors due to the high homology between the yeast pheromone signaling pathway and that of mammalian GPCRs. When expressed in yeast, mammalian GPCRs have been shown to couple functionally to either the endogenous yeast Galpha (Gpa1), or co-expressed mammalian Galpha subunits (wild-type or chimeric), and are characterized by a similar pharmacology in response to agonists or antagonists as in native cells. Heterologous expression of wild type or mutant GPCRs in S. cerevisiae allows a rapid assessment of their ability to detect and transduce extracellular stimulations, through the use of a reporter system. Furthermore, this approach is amenable to high-throughput screening of new drugs, which would provide a determinant advantage in the field of therapeutic research, and also for investigation of the still unknown ligands of orphan receptors. This review will focus on the latest developments of yeast-based technology to screen for potential GPCR agonists/antagonists.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Receptores Acoplados a Proteínas G/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Animales , Técnicas Biosensibles , Evaluación Preclínica de Medicamentos/instrumentación , Humanos , Feromonas/metabolismo , Saccharomyces cerevisiae/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Active immunization of proven fertile adult male bonnet monkeys (Macaca radiata) with phage-expressed follicle-stimulating hormone receptor (FSHR)-specific peptides from the extracellular domain resulted in a progressive drop in sperm count with all animals becoming azoospermic by day 100. However, serum testosterone concentrations were unaltered during the entire course of study and animals exhibited normal mating behaviour. Breeding studies with proven fertile female monkeys revealed that all the immunized males were infertile. Following interruption of immunization on day 225, sperm counts returned to normal with restoration of fertility. These results indicate that infertility can be induced in adult male monkeys by interfering with the action of FSH using specific peptides of the extracellular domain of FSHR as antigens, without the risk of producing cross-reacting antibodies to the other glycoprotein hormones.
Asunto(s)
Inmunización , Infertilidad Masculina/inmunología , Receptores de HFE/inmunología , Animales , Bacteriófagos/genética , Bacteriófagos/metabolismo , Biopsia , Femenino , Humanos , Infertilidad Masculina/patología , Macaca radiata , Masculino , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Estructura Terciaria de Proteína/genética , Receptores de HFE/genética , Testículo/patologíaRESUMEN
Despite the recent advances in the field of coordination dynamics addressing the interplay of constraints of different natures in the emergence of human coordination, F. Mechsner (2004) invites us to revive hierarchical and dichotomous thinking b y offering again his exclusive position that coordinated movements are (purely) perceptual-cognitive/psychological in nature. In this comment, the authors address a number of theoretical and methodological issues that might potentially puzzle the readers of Mechsner's article. They contend that the dichotomy proposed by Mechsner (i.e., perceptual-cognitive vs. motor) constitutes a restrictive framework for understanding human coordination.
Asunto(s)
Mano/fisiología , Movimiento/fisiología , Desempeño Psicomotor/fisiología , Humanos , Percepción EspacialRESUMEN
Splice variants of mRNA encoding the LH receptor (LHR) during follicular development were characterized in cyclic and non-cyclic ewes. Granulosa and theca cells were collected from individual follicles. After amplification by RT-PCR of a region situated between exon 9 and exon 11 of the LHR gene, three distinct bands, LHR1 (full length), LHR2 (deletion of exon 10), LHR3 (deletion of 262 bp in exon 11), were observed in the granulosa and theca cells of ovine antral follicles of various sizes (2.5-6.0 mm). Expression of LHR mRNA in theca cells varied with the annual cycle of reproduction (P < 0.001), and was highly expressed in all classes of follicle collected from anoestrous ewes (1.3 +/- 0.1, n = 8 in small follicles; 1.8 +/- 0.2, n = 8 in medium follicles; 1.7 +/- 0.3, n = 4 in large follicles; arbitrary units) compared with follicles collected from oestrous ewes (0.19 +/- 0.06, n = 8 in small follicles; 0.2 +/- 0.04, n = 9 in medium follicles; 0.18 +/- 0.04, n = 5 in large follicles). During the breeding season, no differences in the relative expression of the different splice variants were observed according to follicle size. In contrast, during anoestrus, LHR3 mRNA was significantly more abundant in large (6.0-6.5 mm) and medium (4.0-5.5 mm) than it was in small (2.5-3.5 mm) follicles. These results indicate that RNA alternative splicing plays a role in the seasonal and physiological control of LH receptor expression in theca cells.
Asunto(s)
Empalme Alternativo , ARN Mensajero/análisis , Receptores de HL/genética , Estaciones del Año , Ovinos/metabolismo , Células Tecales/metabolismo , Análisis de Varianza , Animales , Southern Blotting/métodos , Cruzamiento , Femenino , Células de la Granulosa/metabolismo , Folículo Ovárico/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Receptors for the luteotropin/human chorionogonadotropin hormone belong to the G-protein-coupled receptor family by their membrane-anchoring domains. They also possess a large extracellular domain (ECD) responsible for most of the hormone-receptor interactions. Structure-function studies identified several contacts between hormone and receptor ECD, but the precise topology of the complex is still unknown because of the lack of suitable heterologous expression means. Receptor ECDs exhibit leucine repeats and have been modelized on the basis of the three-dimensional structure of the porcine ribonuclease inhibitor, the first structurally known leucine-rich repeats protein. Here we report overexpression (up to 20 mg per liter) and purification to homogeneity of a soluble human chorionogonadotropin-ECD receptor complex secreted by stably cotransfected Chinese hamster ovary cells. Biochemical analysis and surface plasmon resonance data were in favor of a unique dimer with a 1:1 ligand-receptor stoichiometry. Immunopurified complex was submitted to circular dichroism characterization; CD spectra deconvolution indicated more than 25% alpha helices contributed by the receptor, in agreement with the porcine ribonuclease inhibitor-based modelization.
Asunto(s)
Gonadotropina Coriónica/metabolismo , Receptores de HL/metabolismo , Secuencia de Bases , Gonadotropina Coriónica/química , Gonadotropina Coriónica/aislamiento & purificación , Cromatografía de Afinidad , Cromatografía en Gel , Reactivos de Enlaces Cruzados , Cartilla de ADN , Humanos , Conformación Proteica , Receptores de HL/química , Receptores de HL/aislamiento & purificaciónRESUMEN
Follicle-stimulating hormone (FSH) via interaction with G-protein coupled specific receptors plays a central role in the control of gametogenesis in mammals of both sexes. In females, FSH is crucial for follicle growth, follicle maturation and ovulation. FSH receptors, together with luteinizing hormone-chorionic gonadotropin and thyrotropin receptors belong to a subfamily of structurally related receptors within the seven transmembrane receptor family. Among several other regions, the N-terminus of these receptors is believed to be responsible for important specific hormone-receptor contact sites. Recombinant filamentous phages displaying at their surface three overlapping N-terminal decapeptides of the FSH receptor, peptides A18-27, B25-34 and C29-38 were constructed. Ewes and female mice were immunized against the three FSH receptor (FSHR) recombinant phages. Immunoglobulins purified from immunized animals were analyzed for their biochemical properties on a Chinese hamster ovary cell line expressing the porcine FSH receptor. AntiA and antiB immunoglobulins (IgGs) behave as antagonists for 125I-FSH binding and for FSH-dependent cAMP production, while antiC IgGs did not compete for hormone binding. By contrast, antibodies against the C29-38 peptide displayed FSH agonist activity and stimulated the FSH receptor, whereas antiA and antiB IgGs did not. Furthermore, when the FSHR phages were used as peptidic vaccines, they induced a reversible inhibition of ovulation rate in ewes, and impaired fertility in female mice.
Asunto(s)
Hormona Folículo Estimulante/agonistas , Hormona Folículo Estimulante/antagonistas & inhibidores , Receptores de HFE/inmunología , Secuencia de Aminoácidos , Animales , Bacteriófagos/genética , Células CHO , Cricetinae , AMP Cíclico/biosíntesis , Femenino , Fertilidad , Hormona Folículo Estimulante/metabolismo , Inmunización , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Ovulación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Embarazo , Receptores de HFE/química , Receptores de HFE/genética , Ovinos , PorcinosRESUMEN
The lutropin receptor ectodomain overexpressed under the control of the powerful polyhedrin promoter in baculovirus-infected Sf9 insect cells, is mainly found in an inactive, intracellularly-aggregated form. It is secreted in an active form under the control of the P10 promoter, a somewhat weaker and earlier promoter, at the price of a lower production. The apparent molecular masses of the two species encoded by the same cDNA are 48 kDa and 60-68 kDa, respectively. The relationship between the extent and type of glycosylation and the extracellular targeting for the recombinant lutropin receptor ectodomains was investigated precisely with endoglycosidases, lectins of various specificities, and a glycosylation inhibitor, and tested with monoclonal and polyclonal antibodies. The results indicate that the strong polyhedrin promoter probably overwhelms the processing capacity of the ER in Sf9 cells, so that only a high-mannose precursor is expressed in large amounts. Only a minute amount of protein is secreted, which has been processed by Sf9 exoglycosidases/glycosyltransferases and bears complex/hybrid oligosaccharides. The weaker P10 promoter allows secretion of a mature and active receptor ectodomain, bearing complex glycosylation. An important O-linked glycosylation is also added post-translationally on this species. In particular, beta-galactose and sialic acid residues were specifically detected in the secreted species, evidence of the induction of the corresponding glycosyltransferases or of their genes. These results suggest that Sf9 cells should eventually be engineered with chaperones and glycosyltransferases in order to improve the production of demanding glycoproteins such as the porcine lutropin ectodomain, so as to open the way to resolution of the three-dimensional structures of these receptors.
Asunto(s)
Receptores de HL/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae , Unión Competitiva , Biotinilación , Células Cultivadas , Clonación Molecular , Retículo Endoplásmico/metabolismo , Ensayo de Inmunoadsorción Enzimática , Glicósido Hidrolasas/metabolismo , Glicosilación , Insectos , Lectinas/metabolismo , Datos de Secuencia Molecular , Proteínas de la Matriz de Cuerpos de Oclusión , Polisacáridos/metabolismo , Regiones Promotoras Genéticas , Pliegue de Proteína , Receptores de HL/genética , Receptores de HL/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Porcinos , Proteínas Virales/genética , Proteínas Estructurales ViralesRESUMEN
Expression of gonadotropin receptors and granulosa cell sensitivity to gonadotropin hormones by small (1-3 mm) and large (3.5-7 mm) follicles were compared in Romanov (ROM, ovulation rate = 3) and Ile-de-France (IF, ovulation rate = 1) ewes in the early and late follicular phase. In healthy follicles, LH receptor levels in granulosa cells increased with increasing follicular size (p < 0. 001) while FSH receptor levels decreased (p < 0.05). In granulosa cells of large follicles, LH receptor (LHR) mRNA levels were greater in the late than in the early follicular phase (p < 0.001, p < 0.05, for ROM and IF, respectively). In the early follicular phase, LHR levels in granulosa (p < 0.001) and theca cells (p < 0.05) of small follicles were greater in ROM than in IF ewes. FSH receptor mRNA levels in granulosa cells of small and large ROM follicles were greater than in the corresponding IF follicles (p < 0.05). Finally, a greater responsiveness (increase in cAMP secretion) to both FSH and hCG was observed by granulosa cells collected during the early follicular phase from ROM vs. IF ewes. Data provide evidence that the greater ovulation rate in the ROM as compared to the IF breed is associated with a greater gonadotropin responsiveness during the early follicular phase.
Asunto(s)
Expresión Génica , Folículo Ovárico/metabolismo , Ovulación/genética , Receptores de HFE/genética , Receptores de HL/genética , Ovinos/genética , Animales , AMP Cíclico/biosíntesis , Femenino , Tamaño de la Camada/genética , ARN Mensajero/análisis , Receptores de HFE/análisis , Receptores de HFE/metabolismo , Receptores de HL/análisis , Receptores de HL/metabolismo , Especificidad de la EspecieRESUMEN
In porcine Leydig cells in primary culture, 95% of the internalization of [125I]porcine lutropin ([125I]pLH, which bears sulfated GalNAc) could not be ascribed to the high-affinity LH receptor (LHR). In contrast, >40% of [125I]human choriogonadotropin (hCG, with sialylated sugar chains) uptake was performed by the LHR itself. When the LHR was down-regulated by excess unlabeled hormone, the LHR-independent incorporation of [125I]pLH could be inhibited in a dose-dependent fashion by sulfated polysaccharides such as fucoidan or chondroitin-(4 or 6)-sulfate, but not by other polyanionic compounds, nor by sulfated chondroitin disaccharides. Endocytosis occurred through a clathrin-dependent pathway and was inhibited by low temperature, endocytosis inhibitors, increased ionic strength, or by EDTA and dithiothreitol. Taken together, these results suggest that a Leydig cell membrane protein (possibly a lectin, or a glycosaminoglycan receptor) could perform specific LH clearance in the testis via recognition of its sulfated sugars.
Asunto(s)
Endocitosis , Células Intersticiales del Testículo/metabolismo , Hormona Luteinizante/metabolismo , Receptores de HL/metabolismo , Animales , Células Cultivadas , Humanos , Células Intersticiales del Testículo/citología , Masculino , Ensayo de Unión Radioligante , PorcinosRESUMEN
An active recombinant glycoprotein hormone, porcine follicle-stimulating hormone (recFSH), has been produced for the first time in the methylotrophic yeast, Pichia pastoris. The yield of secreted recFSH (10 mg/l) was the highest ever reached. RecFSH displayed an apparent molecular mass of 41 kDa by SDS-PAGE and was found to bear only N-linked carbohydrates of the high-mannose type. Its in vitro binding and cell-stimulating activities were identical to those of pituitary porcine FSH. The large availability and the noncharged N-glycans of FSHrec should render it highly valuable for structural studies.