RESUMEN
Nano- and microparticles are promising carrier systems for oral delivery of drugs or vaccines, particularly in fish aquaculture. However, the mechanisms of uptake, trans-epithelial transport and immune response to nano/micrometer sized particles, or microorganisms such as bacteria are poorly understood in fish. Here, adult zebrafish were used to study the uptake of different nano- and microparticles and the pathogenic bacteria Mycobacterium marinum in the intestine, and their interactions with epithelial cells and the mucosal immune system. Fluorescent particles or bacteria were delivered directly into the adult zebrafish intestine by oral intubation and their localization was imaged in intestine, liver and spleen sections. Zebrafish do not appear to have M-cells, but both nanoparticles and bacteria were rapidly taken up in the intestine and transported to the liver and spleen. In each tissue, both bacteria and particles largely localized to leukocytes, presumably macrophages.
Asunto(s)
Enterocitos/inmunología , Enfermedades de los Peces/inmunología , Mucosa Intestinal/fisiología , Infecciones por Mycobacterium no Tuberculosas/inmunología , Mycobacterium marinum/fisiología , Nanopartículas/metabolismo , Pez Cebra/inmunología , Animales , Presentación de Antígeno , Traslocación Bacteriana/inmunología , Transporte Biológico , Células Cultivadas , Sistemas de Liberación de Medicamentos , Inmunidad Mucosa , Mucosa Intestinal/microbiología , Migración Transendotelial y TransepitelialRESUMEN
We have previously characterized the response to gonadotropin-releasing hormone (Gnrh) 2 in luteinizing hormone (lhb)-expressing cells from green fluorescent protein (Gfp)-transgenic medaka (Oryzias latipes), with regard to changes in the cytosolic Ca(2+) concentration. In the current study we present the corresponding responses to Gnrh1 and Gnrh3. Ca(2+) imaging revealed three response patterns to Gnrh1 and Gnrh3, one monophasic and two types of biphasic patterns. There were few significant differences in the shape of the response patterns between the three Gnrh forms, although the amplitude of the Ca(2+) signal was considerably lower for Gnrh1 and Gnrh3 than for Gnrh2, and the distribution between the two different biphasic patterns differed. The different putative Ca(2+) sources were examined by depleting intracellular Ca(2+) stores with thapsigargin, or preventing influx of extracellular Ca(2+) by either extracellular Ca(2+) depletion or the L-type Ca(2+)-channel blocker verapamil. Both Gnrh1 and 3 relied on Ca(2+) from both intracellular and extracellular sources, with some unexpected differences in the relative contribution. Furthermore, gene expression of Gnrh-receptors (gnrhr) in whole pituitaries was studied during development from juvenile to adult. Only two of the four identified medaka receptors were expressed in the pituitary, gnrhr1b and gnrhr2a, with the newly discovered gnrhr2a showing the highest expression level at all stages as analyzed by quantitative PCR. While both receptors differed in expression level according to developmental stage, only the expression of gnrhr2a showed a clear-cut increase with gonadal maturation. RNA sequencing analysis of FACS-sorted Gfp-positive lhb-cells revealed that both gnrhr1b and gnrhr2a were expressed in lhb-expressing cells, and confirmed the higher expression of gnrhr2a compared to gnrhr1b. These results show that although lhb-expressing gonadotropes in medaka show similar Ca(2+) response patterns to all three endogenous Gnrh forms through the activation of two different receptors, gnrhr1b and gnrhr2a, the differences observed between the Gnrh forms indicate activation of different Ca(2+) signaling pathways.
Asunto(s)
Hormona Liberadora de Gonadotropina/metabolismo , Oryzias/metabolismo , Receptores LHRH/metabolismo , Animales , Animales Modificados Genéticamente , CalcioRESUMEN
Amicon(®) Ultra centrifugal filters were critically evaluated for various sample preparations, namely (a) proteome fractionation, (b) sample cleanup prior to liquid chromatography mass spectrometry (LC-MS) measurement of small molecules in cell lysate, and (c) separating drug-loaded nanoparticles and released drugs for accurate release profiling in biological samples. (a) Filters of supposedly differing molar mass (MM) selectivity (10, 30, 50 and 100K) were combined to attempt fractionation of samples of various complexity and concentration. However, the products had surprisingly similar MM retentate/filtrate profiles, and the filters were unsuited for proteome fractionation. (b) Centrifugal filtration was the only clean-up procedure in a FDA-guideline validated LC-MS method for determining anti-tuberculosis agents rifampicin and thioridazine in macrophage cell lysate. An additional organic solvent washing step (drug/protein-binding disruption) was required for satisfactory recovery. (c) The centrifugation filters are well suited for separating drugs and nanoparticles in simple aqueous solutions, but significantly less so for biological samples, as common drug-protein binding disruptors can dissolve NPs or be incompatible with LC-MS instrumentation.
Asunto(s)
Nanopartículas/química , Preparaciones Farmacéuticas/química , Proteínas/química , Centrifugación/métodos , Fraccionamiento Químico/métodos , Cromatografía Liquida/métodos , Peso Molecular , Proteoma/química , Solventes/química , Espectrometría de Masas en Tándem/métodosRESUMEN
Therapeutic nanoparticles (NPs) have great potential to deliver drugs against human diseases. Encapsulation of drugs in NPs protects them from being metabolized, while they are delivered specifically to a target site, thereby reducing toxicity and other side-effects. However, non-specific tissue accumulation of NPs, for example in macrophages, especially in the spleen and liver is a general problem with many NPs being developed for cancer therapy. To address the problem of non-specific tissue accumulation of NPs we describe the development of the zebrafish embryo as a transparent vertebrate system for characterization of NPs against cancer. We show that injection of human cancer cells results in tumor-like structures, and that subsequently injected fluorescent NPs, either made of polystyrene or liposomes can be imaged in real-time. NP biodistribution and general in vivo properties can be easily monitored in embryos having selective fluorescent labeling of specific tissues. We demonstrate in vitro, by using optical tweezer micromanipulation, microscopy and flow cytometry that polyethylene glycol (PEG) coating of NPs decreases the level of adhesion of NPs to macrophages, and also to cancer cells. In vivo in zebrafish embryos, PEG coating resulted in longer NP circulation times, decreased macrophage uptake, and reduced adhesion to the endothelium. Importantly, liposomes were observed to accumulate passively and selectively in tumor-like structures comprised of human cancer cells. These results show that zebrafish embryo is a powerful system for microscopy-based screening of NPs on the route to preclinical testing.
Asunto(s)
Micromanipulación/métodos , Nanopartículas/química , Neoplasias/tratamiento farmacológico , Pez Cebra/embriología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Citometría de Flujo , Colorantes Fluorescentes/química , Células HEK293 , Humanos , Liposomas/química , Macrófagos/metabolismo , Nanopartículas del Metal/química , Microscopía , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Nanomedicina/métodos , Neoplasias/metabolismo , Neoplasias/terapia , Pinzas Ópticas , Polietilenglicoles/química , Polímeros/química , Poliestirenos/química , Distribución TisularRESUMEN
Encapsulating antibiotics such as rifampicin in biodegradable nanoparticles provides several advantages compared to free drug administration, including reduced dosing due to localized targeting and sustained release. Consequently, these characteristics reduce systemic drug toxicity. However, new nanoformulations need to be tested in complex biological systems to fully characterize their potential for improved drug therapy. Tuberculosis, caused by infection with the bacterium Mycobacterium tuberculosis, requires lengthy and expensive treatment, and incomplete therapy contributes to an increasing incidence of drug resistance. Recent evidence suggests that standard therapy may be improved by combining antibiotics with bacterial efflux pump inhibitors, such as thioridazine. However, this drug is difficult to use clinically due to its toxicity. Here, we encapsulated thioridazine in poly(lactic-co-glycolic) acid nanoparticles and tested them alone and in combination with rifampicin nanoparticles, or free rifampicin in macrophages and in a zebrafish model of tuberculosis. Whereas free thioridazine was highly toxic in both cells and zebrafish embryos, after encapsulation in nanoparticles no toxicity was detected. When combined with rifampicin nanoparticles, the nanoparticles loaded with thioridazine gave a modest increase in killing of both Mycobacterium bovis BCG and M. tuberculosis in macrophages. In the zebrafish, the thioridazine nanoparticles showed a significant therapeutic effect in combination with rifampicin by enhancing embryo survival and reducing mycobacterial infection. Our results show that the zebrafish embryo is a highly sensitive indicator of drug toxicity and that thioridazine nanoparticle therapy can improve the antibacterial effect of rifampicin in vivo.
Asunto(s)
Antituberculosos/uso terapéutico , Nanopartículas/química , Rifampin/uso terapéutico , Tioridazina/uso terapéutico , Tuberculosis/tratamiento farmacológico , Pez Cebra , Animales , Antituberculosos/química , Antituberculosos/toxicidad , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Quimioterapia Combinada , Femenino , Humanos , Ácido Láctico/química , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Masculino , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/efectos de los fármacos , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Rifampin/química , Rifampin/toxicidad , Tioridazina/química , Tioridazina/toxicidad , Tuberculosis/microbiología , Pez Cebra/microbiologíaRESUMEN
Nanoparticles (NPs) enclosing antibiotics have provided promising therapy against Mycobacterium tuberculosis (Mtb) in different mammalian models. However, the NPs were not visualized in any of these animal studies. Here, we introduce the transparent zebrafish embryo as a system for noninvasive, simultaneous imaging of fluorescent NPs and the fish tuberculosis (TB) agent Mycobacterium marinum (Mm). The study was facilitated by the use of transgenic lines of macrophages, neutrophils, and endothelial cells expressing fluorescent markers readily visible in the live vertebrate. Intravenous injection of Mm led to phagocytosis by blood macrophages. These remained within the vasculature until 3 days postinfection where they started to extravasate and form aggregates of infected cells. Correlative light/electron microscopy revealed that these granuloma-like structures had significant access to the vasculature. Injection of NPs induced rapid uptake by both infected and uninfected macrophages, the latter being actively recruited to the site of infection, thereby providing an efficient targeting into granulomas. Rifampicin-loaded NPs significantly improved embryo survival and lowered bacterial load, as shown by quantitative fluorescence analysis. Our results argue that zebrafish embryos offer a powerful system for monitoring NPs in vivo and rationalize why NP therapy was so effective against Mtb in earlier studies; bacteria and NPs share the same cellular niche.
Asunto(s)
Portadores de Fármacos/química , Embrión no Mamífero/microbiología , Mycobacterium marinum/efectos de los fármacos , Nanopartículas/química , Imagen Óptica , Pez Cebra/embriología , Pez Cebra/microbiología , Animales , Antibacterianos/química , Antibacterianos/farmacología , Transporte Biológico , Cumarinas/química , Portadores de Fármacos/metabolismo , Granuloma/microbiología , Ácido Láctico/química , Macrófagos/microbiología , Infecciones por Mycobacterium no Tuberculosas/tratamiento farmacológico , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium marinum/fisiología , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Rodaminas/química , Rifampin/química , Rifampin/farmacología , Tiazoles/química , Tuberculosis/microbiología , Tuberculosis/veterinariaRESUMEN
Pituitary gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), are key regulators of vertebrate reproduction. The differential regulation of these hormones, however, is poorly understood and little is known about gonadotrope embryonic development. The different cell types in the vertebrate pituitary develop from common progenitor cells just after gastrulation. Proper development and merging of the anterior and posterior pituitary is dependent upon carefully regulated cell-to-cell interactions, and a suite of signaling pathways with precisely organized temporal and spatial expression patterns, which include transcription factors and their co-activators and repressors. Among the pituitary endocrine cell types, the gonadotropes are the last to develop and become functional. Although much progress has been made during the last decade regarding details of gonadotrope development, the coordinated program for their maturation is not well described. FSH and LH form an integral part of the hypothalamo-pituitary-gonad axis, the main regulator of gonad development and reproduction. Besides regulating gonad development, pre- and early post-natal activity in this axis is thought to be essential for proper development, especially of the central nervous system in mammals. As a means to investigate early functions of FSH and LH in more detail, we have developed a stable transgenic line of medaka with the LH beta subunit gene (lhb) promoter driving green fluorescent protein (Gfp) expression to characterize development of lhb-expressing gonadotropes. The lhb gene is maternally expressed early during embryogenesis. lhb-Expressing cells are initially localized outside the primordial pituitary in the developing gut tube as early as 32 hpf. At hatching, lhb-Gfp is clearly detected in the gut epithelium and in the anterior digestive tract. lhb-Gfp expression later consolidates in the developing pituitary by 2 weeks post-fertilization. This review discusses status of knowledge regarding pituitary morphology and development, with emphasis on gonadotrope cells and gonadotropins during early development, comparing main model species like mouse, zebrafish and medaka, including possible developmental functions of the observed extra pituitary expression of lhb in medaka.
Asunto(s)
Hormona Folículo Estimulante/metabolismo , Gonadotrofos/metabolismo , Sistema Hipotálamo-Hipofisario/embriología , Hormona Luteinizante/metabolismo , Oryzias/embriología , Sistema Hipófiso-Suprarrenal/embriología , Pez Cebra/embriología , Animales , Regulación del Desarrollo de la Expresión Génica/fisiología , Gonadotrofos/citología , Ratones , Sistema Hipófiso-Suprarrenal/citología , Proteínas de Pez Cebra/biosíntesisRESUMEN
Gonadotropin-releasing hormone receptor (GnRH-R) activation stimulates synthesis and release of gonadotropins in the vertebrate pituitary and also mediates other processes both in the brain and in peripheral tissues. To better understand the differential function of multiple GnRH-R paralogs, three GnRH-R genes (gnrhr1a, 1b, and 2) were isolated and characterized in the European eel. All three gnrhr genes were expressed in the brain and pituitary of pre-pubertal eels, and also in several peripheral tissues, notably gills and kidneys. During hormonally induced sexual maturation, pituitary expression of gnrhr1a (female) and gnrhr2 (male and female) was up-regulated in parallel with gonad development. In the brain, a clear regulation during maturation was seen only for gnrhr2 in the midbrain, with highest levels recorded during early vitellogenesis. These data suggest that GnRH-R2 is the likely hypophysiotropic GnRH-R in male eel, while both GnRH-R1a and GnRH-R2 seems to play this role in female eels.
Asunto(s)
Anguilla/metabolismo , Proteínas de Peces/metabolismo , ARN Mensajero/metabolismo , Receptores LHRH/metabolismo , Maduración Sexual/genética , Secuencia de Aminoácidos , Anguilla/genética , Animales , Encéfalo/metabolismo , Clonación Molecular , Femenino , Proteínas de Peces/química , Proteínas de Peces/genética , Regulación del Desarrollo de la Expresión Génica , Branquias/metabolismo , Riñón/metabolismo , Masculino , Datos de Secuencia Molecular , Filogenia , Hipófisis/metabolismo , Receptores LHRH/química , Receptores LHRH/genética , Alineación de Secuencia , Análisis de Secuencia de Proteína , Regulación hacia ArribaRESUMEN
The onset of puberty and reproduction are tightly controlled by extrinsic and intrinsic inputs combined with genetically determined biological blueprints. Environmental inputs are then mediated by the brain-pituitary-gonad endocrine axis resulting in a unified output. In fish, one of the primary factors controlling the timing of sexual maturation is light, although how these signals are mediated in the brain and pituitary is not well understood. We therefore aimed to elucidate the molecular basis of the control of reproduction during the first spawning season in two year old female Atlantic cod. To this end, we measured GnRH and GnRH-R variant gene expression in brains and pituitaries collected from cod kept under four different photoperiod regimes: natural light (NL), continuous light (LL) and combined treatment of NL-LL and LL-NL. LL inhibited sexual development and spawning and LL-NL delayed sexual development and spawning. LL inhibited the spawning-related increase in brain GnRH3 and pituitary GnRH-R2a gene expression found under NL conditions, and the expression of these genes were delayed in concert with spawning of LL-NL cod. This study indicates that regulation of brain GnRH3 and pituitary GnRH-R2a genes likely mediates photoperiod induced changes in cod gonadal maturation.
Asunto(s)
Encéfalo/metabolismo , Encéfalo/efectos de la radiación , Gadus morhua/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Luz , Hipófisis/metabolismo , Hipófisis/efectos de la radiación , Animales , Femenino , FotoperiodoRESUMEN
BACKGROUND: Luteinizing hormone (LH) and follicle stimulating hormone (FSH), produced in gonadotrope cells in the adenohypophysis are key regulators of vertebrate reproduction. The differential regulation of these hormones, however, is poorly understood and little is known about gonadotrope embryonic development. We developed a stable transgenic line of medaka with the LH beta subunit gene (lhb) promotor driving green fluorescent protein (gfp) expression to characterize development of LH-producing gonadotropes in whole larvae and histological sections. Additionally, developmental and tissue-specific gene expression was examined. RESULTS: The lhb gene is maternally expressed during early embryogenesis. Transcript levels increase by stage 21 (36 hours post fertilization [hpf]) and then decrease during continued larval development. Examination of the expression of pituitary marker genes show that LH-producing cells are initially localized outside the primordial pituitary, and they were localized to the developing gut tube by 32 hpf. At hatching, lhb-GFP is clearly detected in the gut epithelium and in the anterior digestive tract. lhb-GFP expression later consolidate in the developing pituitary by 2 weeks postfertilization. CONCLUSIONS: During embryonic development, lhb is primarily expressed outside the central nervous system and pituitary. The novel expression of lhb in the embryonic gut suggests that LH has a hitherto unidentified developmental function.
Asunto(s)
Hormona Luteinizante/metabolismo , Oryzias/metabolismo , Animales , Animales Modificados Genéticamente , Regulación del Desarrollo de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hibridación in Situ , Hormona Luteinizante/genética , Oryzias/genética , Reacción en Cadena de la PolimerasaRESUMEN
Gonadotropin releasing hormones (GnRH) are an important part of the brain-pituitary-gonad axis in vertebrates. GnRH binding to its receptors (GnRH-R) stimulates synthesis and release of gonadotropins in the pituitary. GnRH-Rs also mediate other processes in the central nervous system such as reproductive behavior and neuromodulation. As many as five GnRH-R genes have been identified in two teleost fish species, but the function and phylogenetic relationship of these receptors is not fully understood. To gain a better understanding of the functional relationship between multiple GnRH-Rs in an important aquaculture species, the Atlantic cod (Gadus morhua), we identified four GnRH-Rs (gmGnRH-R) by RT-PCR, followed by full-length cloning and sequencing. The deduced amino acid sequences were used for phylogenetic analysis to identify conserved functional motifs and to clarify the relationship of gmGnRH-Rs with other vertebrate GnRH-Rs. The function of GnRH-R variants was investigated by quantitative PCR gene expression analysis in the brain and pituitary of female cod during a full reproductive cycle and in various peripheral tissues in sexually mature fish. Phylogenetic analysis revealed two types of teleost GnRH-Rs: Type I including gmGnRH-R1b and Type II including gmGnRH-R2a, gmGnRH-R2b and gmGnRH-R2c. All four gmGnRH-Rs are expressed in the brain, and gmGnRH-R1b, gmGnRH-R2a and gmGnRH-R2c are expressed in the pituitary. The only GnRH-R differentially expressed in the pituitary during the reproductive cycle is gmGnRH-R2a such that its expression is significantly increased during spawning. These data suggest that gmGnRH-R2a is the most likely candidate to mediate the hypophysiotropic function of GnRH in Atlantic cod.
Asunto(s)
Encéfalo/metabolismo , Gadus morhua/metabolismo , Hipófisis/metabolismo , Receptores LHRH/química , Receptores LHRH/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Receptores LHRH/clasificación , Receptores LHRH/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
Gonadotropin releasing hormone (GnRH) is a key regulator of sexual development and reproduction in vertebrates. Fish have either two or three pre-pro-GnRH genes, encoding structurally distinct peptides. We identified three pre-pro-GnRH genes in Atlantic cod (Gadus morhua, gmGnRH) using RT-PCR, RACE-PCR and BAC DNA library clone sequencing based on synteny searching. Gene identity was confirmed by sequence alignment and subsequent phylogenetic analysis. The expression of these genes was measured by quantitative PCR in the brain and pituitary of female cod throughout their reproductive cycle and in peripheral tissues. All three gmGnRH genes have highly conserved deduced decapeptide sequences, but sequence and phylogenetic data for gmGnRH1 suggest that this is a pseudogene. gmGnRH1 shares low identity with all fish GnRH variants and grouped with the GnRH3 clade. Although gmGnRH1 is a putative pseudogene, it is transcribed in multiple tissues but at low levels in the brain, indicating the loss of conserved hypophysiotrophic function. Phylogenetic analysis reveals that gmGnRH2 and gmGnRH3 variants are located in variant-specific clades. Both gmGnRH2 and gmGnRH3 transcripts are most abundant in the brain, with lower expression in pituitaries and ovaries. Brain gmGnRH3 gene expression increases in spawning fish and is expressed in the pituitary during puberty. Brain gmGnRH2 transcripts are highly expressed relative to gmGnRH3 before and during spawning. Sequence and expression data suggest that gmGnRH1 is a pseudogene and that gmGnRH3 is likely the hypophysiotrophic form of GnRH in Atlantic cod.
Asunto(s)
Proteínas de Peces/genética , Gadus morhua/genética , Eliminación de Gen , Hormona Liberadora de Gonadotropina/genética , Maduración Sexual , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , Clonación Molecular , Secuencia Conservada , Femenino , Proteínas de Peces/metabolismo , Gadus morhua/crecimiento & desarrollo , Gadus morhua/metabolismo , Perfilación de la Expresión Génica , Biblioteca de Genes , Hormona Liberadora de Gonadotropina/metabolismo , Datos de Secuencia Molecular , Ovario/metabolismo , Filogenia , Hipófisis/metabolismo , Seudogenes , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de SecuenciaRESUMEN
The role of growth hormone (GH) and insulin-like growth factor-I (IGF-I) in the tissue remodeling associated with the transition of a symmetrical larva to an asymmetrical juvenile during flatfish metamorphosis is unknown. In order to investigate the potential role of these hormones in the remodeling of cranial bone and soft tissue that accompanies eye migration during metamorphosis of Atlantic halibut (Hippoglossus hippoglossus) larvae, tissue-specific gene expression was monitored by in situ hybridization for Atlantic halibut type I growth hormone receptor (hhGHR), type II hhGHR, and insulin-like growth factor-I receptor (hhIGF-IR). Polyclonal antibody generated against the extracellular domain of type I hhGHR was used for the immunohistochemical localization of type I GHR protein. Type I hhGHR, type II hhGHR, and hhIGF-IR mRNA were expressed in fibroblasts, frontal bone osteocytes, and dorsal chondrocytes at the onset of metamorphosis (stage 8), during metamorphic climax (stage 9), and in fully metamorphosed juveniles (stage 10). Type I GHR protein showed similar expression patterns to those of type I hhGHR mRNA, except in chondrocytes in which little GHR protein was detected. The localization of GHR and IGF-IR transcripts and GHR protein in cranial structures that undergo remodeling is intriguing and suggests that, in addition to thyroid hormones, the GH-IGF-I system is involved in morphological transformations during metamorphosis in Atlantic halibut.
Asunto(s)
Lenguado/crecimiento & desarrollo , Lenguado/metabolismo , Metamorfosis Biológica , Receptor IGF Tipo 1/genética , Receptores de Somatotropina/genética , Cráneo/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Condrocitos/metabolismo , Ojo/crecimiento & desarrollo , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Osteocitos/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptores de Somatotropina/química , Receptores de Somatotropina/metabolismo , Alineación de Secuencia , Cráneo/metabolismoRESUMEN
Insulin-like growth factor-I (IGF-I) is an important regulator of growth and development in vertebrates. Both the endocrine and paracrine actions of IGF-I are mediated through ligand-binding to a membrane-bound IGF-I receptor (IGF-IR). The characterization of this receptor and subsequent expression studies thus help elucidate the endocrine regulation of developmental processes. As other flatfish species, the Atlantic halibut (Hippoglossus hippoglossus) undergoes a dramatic larval metamorphosis. This process is largely under endocrine control, and data indicate that IGF-I could be a key regulator. IGF-I content increases up to late pre-metamorphosis and decreases during metamorphosis. The IGF-IR has, however, not been studied during flatfish metamorphosis. To examine IGF-IR gene expression, two IGF-IR mRNA were cloned and sequenced. These partial sequences share high identity (>or=95%) and similarity (>or=97%) with other fish IGF-IR and lower identity (>or=77%) and similarity (>or=83.5%) with Japanese flounder insulin receptors. The expression of mRNA for both IGF-IR was analyzed by quantitative real-time RT-PCR during six larval developmental stages from pre- to post-metamorphosis. IGF-IR1 and IGF-IR2 mRNA are differentially expressed during metamorphosis, but if this indicates an isoform-specific regulation of developmental processes by circulating and/or locally-secreted IGF-I is unclear. Both IGF-IR genes are down-regulated in halibut larvae experiencing arrested metamorphosis, suggesting the IGF-I system is critical for metamorphic success in halibut.
Asunto(s)
Proteínas de Peces/biosíntesis , Proteínas de Peces/genética , Lenguado/embriología , Lenguado/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Receptor IGF Tipo 1/biosíntesis , Receptor IGF Tipo 1/genética , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Especificidad de Órganos/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Continuous-elution electrophoresis (CEE) has been applied to separate putative hormones from adult Atlantic halibut pituitaries. Soluble proteins were separated by size and charge on Model 491 Prep Cell (Bio-Rad), where the homogenate runs through a cylindrical gel, and protein fractions are collected as they elute from the matrix. Protein fractions were assessed by SDS-PAGE and found to contain purified proteins of molecular size from 10 to 33 kDa. Fractions containing proteins with molecular weights of approximately 21, 24, 28 and 32 kDa, were identified as putative growth hormone (GH), prolactin, somatolactin and gonadotropins, respectively. These were analyzed further by mass spectrometry and identified with peptide mass protein fingerprinting. The CEE technique was used successfully for purification of halibut GH with a 5% yield, and appears generally well suited to purify species-specific proteins often needed for research in comparative endocrinology, including immunoassay work. Thus, the GH obtained was subsequently used as standards and iodination label in a homologous radioimmunoassay, applied to analyze GH content through larval development in normally and abnormally metamorphosing larvae. As GH is mainly found in the pituitary, GH contents were analyzed in tissue extracts from the heads only. The pituitary GH content increases proportionally to increased larval weight from first feeding to metamorphic climax. No difference in relative GH content was found between normal and abnormal larvae and it still remains to be established if GH has a direct role in metamorphosis.
Asunto(s)
Electroforesis/métodos , Lenguado/embriología , Lenguado/metabolismo , Hormona del Crecimiento/aislamiento & purificación , Hipófisis/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Animales , Proteínas de Peces/química , Proteínas de Peces/aislamiento & purificación , Proteínas de Peces/metabolismo , Glicoproteínas/química , Glicoproteínas/aislamiento & purificación , Glicoproteínas/metabolismo , Gonadotropinas/química , Gonadotropinas/aislamiento & purificación , Gonadotropinas/metabolismo , Hormona del Crecimiento/química , Hormona del Crecimiento/metabolismo , Metamorfosis Biológica , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Hipófisis/química , Hormonas Hipofisarias/química , Hormonas Hipofisarias/aislamiento & purificación , Hormonas Hipofisarias/metabolismo , Prolactina/química , Prolactina/aislamiento & purificación , Prolactina/metabolismo , Radioinmunoensayo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/veterinaria , Espectrometría de Masas en TándemRESUMEN
To gain insight into the possible regulatory role of the growth hormone (GH)-insulin-like growth factor I (IGF-I) system in flatfish metamorphosis, body GHR gene expression as well as IGF-I protein content was quantified in larval Atlantic halibut throughout metamorphosis (developmental stages 5-10). The cDNA of the full-length GH receptor (hhGHR) was cloned from adult liver and characterized. The hhGHR shows common features of a GHR, including a (Y/F)GEFS motif in the extracellular domain, a single transmembrane region, and an intracellular domain containing a Box 1 and Box 2. Additionally, a truncated GHR (hhGHRtr), similar to turbot and Japanese flounder GHRtr, was cloned and sequenced. These sequences are highly similar to the full-length and truncated GHRs in turbot (89%/86%) and Japanese flounder (93%/91%) with lower identity with other fish type I GHR (81%) and type II GHRs (58%). A quantitative real-time RT-PCR assay was used to measure hhGHR and hhGHRtr mRNA content in normally and abnormally metamorphosed individuals at six developmental stages, from early pre-metamorphosis to post-metamorphosis, when the fish is considered a juvenile. The level of hhGHR gene expression was highest at pre-metamorphic stage 6 and at stage 8 at the onset of metamorphosis, and then decreased during metamorphic climax and post-metamorphosis. Expression of hhGHRtr reached highest levels at stage 6 and then decreased to post-metamorphosis. The ratio of expression between the full-length and the truncated GHR (hhGHR:hhGHRtr) varied among stages and was highest at the onset of metamorphosis and at metamorphic climax. A radioimmunoassay was used to measure halibut IGF-I body content throughout metamorphosis. IGF-I increases from early metamorphosis to the onset of metamorphosis and then decreases towards post-metamorphosis. In comparison between normally and abnormally metamorphosing larvae, IGF-I content, hhGHR and hhGHRtr mRNA levels were reduced in the abnormal fish. These data indicate that the GH-IGF-I system either has a regulatory role in metamorphosis, or is being affected as a consequence of the abnormal metamorphosis.