RESUMO
BACKGROUND: The white-eared opossum (Didelphis albiventris) is widely distributed throughout Brazil and South America. It has been used as an animal model for studying different scientific questions ranging from the restoration of degraded green areas to medical aspects of Chagas disease, leishmaniasis and resistance against snake venom. As a marsupial, D. albiventris can also contribute to the understanding of the molecular mechanisms that govern the different stages of organogenesis. Opossum joeys are born after only 13 days, and the final stages of organogenesis occur when the neonates are inside the pouch, depending on lactation. As neither the genome of this opossum species nor its transcriptome has been completely sequenced, the use of D. albiventris as an animal model is limited. In this work, we sequenced the D. albiventris transcriptome by RNA-seq to obtain the first catalogue of differentially expressed (DE) genes and gene ontology (GO) annotations during the neonatal stages of marsupial development. RESULTS: The D. albiventris transcriptome was obtained from whole neonates harvested at birth (P0), at 5 days of age (P5) and at 10 days of age (P10). The de novo assembly of these transcripts generated 85,338 transcripts. Approximately 30% of these transcripts could be mapped against the amino acid sequences of M. domestica, the evolutionarily closest relative of D. albiventris to be sequenced thus far. Among the expressed transcripts, 2077 were found to be DE between P0 and P5, 13,780 between P0 and P10, and 1453 between P5 and P10. The enriched GO terms were mainly related to the immune system, blood tissue development and differentiation, vision, hearing, digestion, the CNS and limb development. CONCLUSIONS: The elucidation of opossum transcriptomes provides an out-group for better understanding the distinct characteristics associated with the evolution of mammalian species. This study provides the first transcriptome sequences and catalogue of genes for a marsupial species at different neonatal stages, allowing the study of the mechanisms involved in organogenesis.
Assuntos
Sequenciamento do Exoma/estatística & dados numéricos , Regulação da Expressão Gênica no Desenvolvimento , Gambás/genética , Proteínas/genética , Transcriptoma , Animais , Animais Recém-Nascidos , Brasil , Ontologia Genética , Anotação de Sequência Molecular , Gambás/crescimento & desenvolvimento , Gambás/metabolismo , Proteínas/classificação , Proteínas/metabolismo , Análise de Sequência de RNARESUMO
Torpor is a phenotype characterized by a controlled decline of metabolic rate and body temperature. During arousal from torpor, organs undergo rapid metabolic reactivation and rewarming to near normal levels. As torpor progress, animals show a preference for fatty acids over glucose as primary source of energy. Here, we analyzed for first time the changes in the maximal activity of key enzymes related to fatty acid (Carnitine palmitoyltransferase and ß-Hydroxyacyl CoA dehydrogenase) and carbohydrate (Pyruvate kinase, Phosphofructokinase and Lactate dehydrogenase) catabolism, as well as mitochondrial oxidative capacity (Citrate synthase), in six organs of torpid, arousing and euthermic Chilean mouse-opossums (Thylamys elegans). Our results showed that activity of enzymes related to fatty acid and carbohydrate catabolism were different among torpor phases and the pattern of variation differs among tissues. In terms of lipid utilization, maximal enzymatic activities differ in tissues with high oxidative capacity such as heart, kidney, and liver. In terms of carbohydrate use, lower enzymatic activities were observed during torpor in brain and liver. Interestingly, citrate synthase activity did not differ thought torpor-arousal cycle in any tissues analyzed, suggesting no modulation of mitochondrial content in T. elegans. Overall results provide an indication that modulation of enzymes associated with carbohydrate and fatty-acid pathways is mainly oriented to limit energy expensive processes and sustain energy metabolism during transition from torpor to euthermy. Future studies are required to elucidate if physiological events observed for T. elegans are unique from other marsupials, or represents a general response in marsupials. J. Exp. Zool. 325A:41-51, 2016. © 2015 Wiley Periodicals, Inc.
Assuntos
Metabolismo Energético/genética , Marsupiais/metabolismo , Mitocôndrias/metabolismo , Gambás/genética , 3-Hidroxiacil-CoA Desidrogenases/metabolismo , Animais , Carnitina O-Palmitoiltransferase/metabolismo , Citrato (si)-Sintase/metabolismo , L-Lactato Desidrogenase/metabolismo , Marsupiais/genética , Mitocôndrias/enzimologia , Mitocôndrias/genética , Gambás/metabolismo , Piruvato Quinase/metabolismo , Torpor/genética , Torpor/fisiologiaRESUMO
The aim of this study was to identify and quantify the argyrophil, argentaffin and insulin-immunoreactive cells (IIC) in the small intestine of the opossum Didelphis aurita. Seven adult male specimens of opossums were investigated. The animals were captured, and their blood insulin levels were determined. After euthanasia, fragments of the small intestine were processed for light microscopy and transmission electron microscopy, and submitted to histochemistry and immunohistochemistry for identification of argyrophil and argentaffin endocrine cells, and IIC. Argyrophil and argentaffin cells were identified in the intestinal villi and Liberkühn crypts, whereas IIC were present exclusively in the crypts. Ultrastructure of the IIC revealed cytoplasmic granules of different sizes and electron densities. The numbers of IIC per mm(2) in the duodenum and jejunum were higher than in the ileum (p<0.05). The animals had low levels of blood insulin (2.8 ± 0.78 µIU/ml). There was no correlation between insulin levels and the number of IIC in the small intestine. The IIC presented secretory granules, elongated and variable morphology. It is believed that insulin secretion by the IIC may influence the proliferation of cells in the Liberkühn crypts, and local glucose homeostasis, primarily in animals with low serum insulin levels, such as the opossum.
Assuntos
Didelphis/metabolismo , Células Endócrinas/metabolismo , Células Endócrinas/ultraestrutura , Células Enterocromafins/metabolismo , Insulina/metabolismo , Intestino Delgado/metabolismo , Animais , Proliferação de Células , Grânulos Citoplasmáticos/ultraestrutura , Didelphis/imunologia , Células Endócrinas/citologia , Células Enterocromafins/citologia , Células Enterocromafins/ultraestrutura , Imuno-Histoquímica , Insulina/imunologia , Mucosa Intestinal/citologia , Mucosa Intestinal/ultraestrutura , Intestino Delgado/ultraestrutura , Masculino , Microscopia Eletrônica de Transmissão , Gambás/metabolismoRESUMO
We investigated the structural and molecular organization of the extracellular matrix in Thylamys elegans, a marsupial representative of the mammalian order Didelphimorphia. Perineuronal nets (PNs) associated with distinct types of neurons were visualized by detection of chondroitin sulfate proteoglycans and hyaluronan, and by labeling with Wisteria floribunda agglutinin (WFA), a marker for PNs in the mammalian brain. In the neocortex of Thylamys, these methods revealed PNs on pyramidal cells. In contrast, parvalbumin-immunoreactive interneurons in the neocortex and hippocampal formation (displaying robust, WFA-labeled PNs in placental mammals) were ensheathed only with a delicate rim of hyaluronan and proteoglycans not detectable with WFA. The absence of WFA staining was characteristic also of some subcortical regions which contained PNs intensely labeled for chondroitin sulfate proteoglycan and hyaluronan. However, corresponding to placental mammals, numerous subcortical nuclei showed clearly WFA-stained PNs. Similar as in placental mammals, cholinergic basal forebrain neurons and tyrosine hydroxylase-immunoreactive neurons of the substantia nigra and locus coeruleus were devoid of PNs. Together with our earlier study on Monodelphis, the present results reveal that South American opossums show either a particular "marsupial" or "Didelphid" type of extracellular matrix chemoarchitecture, supporting the view that these components may vary phylogenetically as integral parts of neuronal physiology at the systems and single cell level.
Assuntos
Encéfalo/metabolismo , Matriz Extracelular/metabolismo , Neurônios/metabolismo , Gambás/metabolismo , Animais , Encéfalo/citologia , Mapeamento Encefálico , Chile , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Corantes , Matriz Extracelular/ultraestrutura , Ácido Hialurônico/metabolismo , Imuno-Histoquímica , Interneurônios/metabolismo , Interneurônios/ultraestrutura , Masculino , Neurônios/citologia , Gambás/anatomia & histologia , Parvalbuminas/metabolismo , Lectinas de Plantas , Células Piramidais/metabolismo , Células Piramidais/ultraestrutura , Receptores de N-Acetilglucosamina , Especificidade da EspécieRESUMO
Previous studies have disclosed three types of mast cell in opossums: connective tissue (CTMC), mucosal (MMC), and lymphatic sinus (LSMC). In contrast to most opossum lymph nodes, the mesenteric lymph node is virtually devoid of LSMC, displaying medullary cord CTMC. The present study aimed to describe the development of these mast cell populations. Toluidine blue staining and a histochemical method for demonstrating heparin allowed the identification of immature and mature mast cells. Immature CTMC devoid of detectable heparin were rare until postnatal day 10. Mature CTMC filled with heparin-containing granules became numerous by day 30 to day 40. In the ileum, despite the presence of mature CTMC in the submucosa and mucosa (villus base), immature mast cells first appeared in the villus core by day 65 and adult features were apparent by day 100. In LSMC-containing lymph nodes, immature mast cells were found in lymphatic sinuses by day 10. Clear signs of LSMC differentiation were observed from day 20. Compared with the 10-day value, the mean diameter of cytoplasmic granules at day 40 had doubled and that at day 110 had tripled. In the mesenteric lymph nodes, immature mast cells differentiated into lymphatic sinus CTMC-like cells. After day 80, most of them were located in medullary cords. Weaning and complete maturation of mucosa preceded the differentiation of MMC. In lymph nodes, LSMC differentiation occurred in parallel with the development of the medullary region and deep cortex units.
Assuntos
Diferenciação Celular/fisiologia , Sistema Imunitário/crescimento & desenvolvimento , Mastócitos/citologia , Gambás/crescimento & desenvolvimento , Gambás/imunologia , Células-Tronco/citologia , Envelhecimento/imunologia , Animais , Tecido Conjuntivo/crescimento & desenvolvimento , Tecido Conjuntivo/imunologia , Células do Tecido Conjuntivo/citologia , Células do Tecido Conjuntivo/imunologia , Feminino , Sistema Imunitário/citologia , Mucosa Intestinal/citologia , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/imunologia , Linfonodos/citologia , Linfonodos/imunologia , Masculino , Mastócitos/imunologia , Gambás/metabolismo , Células-Tronco/imunologia , DesmameRESUMO
The potential for thermal acclimation in marsupials is controversial. Initial studies suggest that the thermoregulatory maximum metabolic rate (MMR) in metatherians cannot be changed by thermal acclimation. Nevertheless, recent studies reported conspicuous seasonality in both MMR and in basal metabolic rate (BMR). We studied the role of thermal acclimation in the Chilean mouse-opossum, Thylamys elegans, by measuring MMR and BMR before and after acclimation to cold or warm conditions. Following acclimation we also measured the mass of metabolically active organs, and the activity of a key digestive enzyme, aminopeptidase-N. No significant effect of thermal acclimation (i.e. between cold- and warm-acclimated animals) was observed for body mass, MMR, body temperature or factorial aerobic scope. However, the BMR of cold-acclimated animals was 30 % higher than for warm-acclimated individuals. For organ mass, acclimation had a significant effect on the dry mass of caecum, liver and kidneys only. Stepwise multiple regression using pooled data showed that 71 % of the variation in BMR is explained by the digestive organs. Overall, these results suggest that MMR is a rather rigid variable, while BMR shows plasticity. It seems that T. elegans cannot respond to thermal acclimation by adjusting its processes of energy expenditure (i.e. thermogenic capacity and mass of metabolically active organs). The lack of any significant difference in aminopeptidase-N specific activity between warm- and cold-acclimated animals suggests that this response is mainly quantitative (i.e. cell proliferation) rather than qualitative (i.e. differential enzyme expression). Finally, as far as we know, this study is the first to report the effects of thermal acclimation on energy metabolism, organ mass and digestive enzyme activity in a marsupial.
Assuntos
Gambás/anatomia & histologia , Gambás/metabolismo , Aclimatação/fisiologia , Animais , Metabolismo Basal , Regulação da Temperatura Corporal/fisiologia , Antígenos CD13/metabolismo , Sistema Digestório/enzimologia , Metabolismo Energético , Feminino , Masculino , Gambás/fisiologia , Tamanho do ÓrgãoRESUMO
The histochemistry for the mitochondrial enzyme cytochrome oxidase (CO) was used to evaluate the levels of metabolic activity in neurons of the nucleus of the optic tract (NOT) and dorsal terminal nucleus (DTN) in the opossum (Didelphis aurita). The observations were performed in four groups: normal juveniles (4 months old), monocularly enucleated juveniles analysed when adults, normal adults (8 to 18 months old) and monocularly enucleated adults. CO labeled cells were observed to have a similar distribution along the NOT-DTN anteroposterior axis in both juvenile and adult normal animals. Monocular enucleation performed in adults produced a significant reduction of the reactive neuropil but not of the number of CO labeled cells in the deafferented NOT-DTN: the number of labeled neurons per section in the deafferented side matched those of the ipsilateral complex. In juveniles, however, this procedure caused a systematic reduction of the number of CO labeled cells in the contralateral NOT-DTN in comparison to the spared complex. The lack of reduction in the number of neurons found on the deafferented side of the NOT-DTN of monocularly enucleated adult opossums compared with the ipsilateral side might result from the presence of compensatory inputs to maintain their metabolic equivalence. However, when the monocular enucleation was performed in juvenile opossums, a statistically significant asymmetry of CO neurons in the NOT-DTN was observed. In other words, the compensatory mechanisms proposed for the adults were either absent or insufficient to achieve symmetry in juveniles, suggesting a more heavily reliance in the retinal input.
Assuntos
Adaptação Fisiológica/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/fisiologia , Mesencéfalo/enzimologia , Plasticidade Neuronal/fisiologia , Neurônios/enzimologia , Vias Visuais/enzimologia , Envelhecimento/fisiologia , Animais , Denervação , Regulação para Baixo/fisiologia , Enucleação Ocular , Lateralidade Funcional/fisiologia , Histocitoquímica , Mesencéfalo/citologia , Mesencéfalo/crescimento & desenvolvimento , Neurônios/citologia , Nistagmo Optocinético/fisiologia , Gambás/anatomia & histologia , Gambás/crescimento & desenvolvimento , Gambás/metabolismo , Vias Visuais/citologia , Vias Visuais/crescimento & desenvolvimentoRESUMO
From Didelphis marsupialis serum, two antihemorrhagic proteins were isolated by DEAE-Sephacel, Phenyl-Sepharose and Superdex 200 and characterized. Their masses by mass spectrometry were 40318 AMU for DM40 and 42373 and 43010 AMU for DM43, indicating the presence of isoforms for the last. Molecular masses of 44.8 and 47.3 were obtained by SDS-PAGE, respectively for DM40 and DM43. Both inhibitors showed isoelectric points lower than 3.5 and glycosylation percentages varying from 20.5 to 29.0%, as estimated by chemical deglycosylation and amino acid analysis. N-terminal sequences of the first 17 residues of DM40 and DM43 were identical except for the exchange of R9 for P9. Both were homologous to oprin, a similar inhibitor from Didelphis virginiana serum. No evidence of complex formation between DM40 and DM43 was observed either by native PAGE or gel filtration chromatography. In addition to the antihemorrhagic activity, DM40 and DM43 inhibited the hydrolysis of casein, fibrinogen and fibronectin by Bothrops jararaca venom. DM43 also showed antilethal, antiedematogenic and antihyperalgesic activities. None of the inhibitors showed enzymatic activity on casein. Both proteins formed stable complexes with jararhagin and inhibited its hemorrhagic effect as well as the enzymatic activity of this toxin on fluorogenic substrate.
Assuntos
Antivenenos/isolamento & purificação , Proteínas Sanguíneas/isolamento & purificação , Gambás/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Antivenenos/química , Proteínas Sanguíneas/química , Caseínas/antagonistas & inibidores , Cromatografia em Gel , Venenos de Crotalídeos/química , Venenos de Crotalídeos/enzimologia , Eletroforese em Gel de Poliacrilamida , Glicosilação , Ponto Isoelétrico , Espectrometria de Massas , Metaloendopeptidases/química , Dados de Sequência Molecular , Peso Molecular , Gambás/sangue , Proteínas/química , Proteínas/isolamento & purificação , Homologia de Sequência de Aminoácidos , Veneno de Bothrops jararacaRESUMO
The expression of neuronal nitric oxide synthase (nNOS) in the superior colliculus (SC) of the opossum Didelphis marsupialis was studied by NADPH diaphorase (NADPH-d) histochemistry and nNOS immunohistochemistry. In addition, the activity of nNOS was quantified by measurement of [(3)H]-L-arginine conversion to [(3)H]-L-citrulline in tissue extracts from SC superficial layers in opossums and rats. Our results show that the number of NADPH-d stained cells was small and virtually identical in stratum opticum (SO) and stratum griseum superficiale (SGS) and their staining was very light, particularly in SGS. Neuropil staining was heavier in the stratum zonale (SZ) than in SGS or SO. The intermediate and deep layers contained heavily stained cells and moderate neuropil staining. Surprisingly, nNOS-immunoreactive cells were far more numerous than NADPH-d+ cells in every layer. The production of [(3)H]-L-citrulline from [(3)H]-L-arginine in tissue extracts enriched in superficial layers indicated that nNOS specific activity is as high in the opossum as in the rat. Our results suggest that the location of nNOS-expressing neurons in retino-receptive layers may be related to inter-specific differences in the processing of visual information.
Assuntos
Óxido Nítrico Sintase/biossíntese , Gambás/metabolismo , Colículos Superiores/enzimologia , Animais , Citrulina/metabolismo , Imuno-Histoquímica , NADPH Desidrogenase/análise , NADPH Desidrogenase/metabolismo , Óxido Nítrico Sintase Tipo I , Ratos , Colículos Superiores/anatomia & histologiaRESUMO
The ultra-structural development of synapses in retino-receptive layers of the opossum superior colliculus was studied by the ethanolic phosphotungstic acid (E-PTA) method. There was a tendency for a slight reduction in the diameter of synaptic disks, a rise and fall of numerical densities and, except for an ephemeral period, a general increase in the proportion of "frown" among curve synapses. The lack of strict synchrony and the occurrence of different patterns of changes suggest that multiple factors contribute to synaptic maturation.