RESUMO
Transcriptional analysis of the network of transcription regulators and target pathways in exposed organisms may be a hard task when their genome remains unknown. The development of hundreds of qPCR assays, including primer design and normalization of the results with the appropriate housekeeping genes, seems an unreachable task. Alternatively, we took advantage of a whole transcriptome study on Rhinella arenarum larvae exposed to the organophosphorus pesticides azinphos-methyl and chlorpyrifos to evaluate the transcriptional effects on a priori selected groups of genes. This approach allowed us to evaluate the effects on hypothesis-selected pathways such as target esterases, detoxifying enzymes, polyamine metabolism and signaling, and regulatory pathways modulating them. We could then compare the responses at the transcriptional level with previously described effects at the enzymatic or metabolic levels to obtain global insight into toxicity-response mechanisms. The effects of both pesticides on the transcript levels of these pathways could be considered moderate, while chlorpyrifos-induced responses were more potent and earlier than those elicited by azinphos-methyl. Finally, we inferred a prevailing downregulation effect of pesticides on signaling pathways and transcription factor transcripts encoding products that modulate/control the polyamine and antioxidant response pathways. We also tested and selected potential housekeeping genes based on those reported for other species. These results allow us to conduct future confirmatory studies on pesticide modulation of gene expression in toad larvae.
Assuntos
Clorpirifos , Praguicidas , Animais , Azinfos-Metil , Clorpirifos/metabolismo , Praguicidas/farmacologia , Larva , Transcriptoma , Compostos Organofosforados/farmacologia , Antioxidantes/metabolismo , Bufo arenarum/metabolismo , Esterases/metabolismo , Poliaminas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
In the present study, the effect of nerve stimulation on the secretory activity of the ovary of adult females was analyzed for the first time in amphibians. Results revealed that in Rhinella arenarum the stimulation of nerves that supply the gonad induced an increase in estradiol and progesterone secretion, this response showing differences during the reproductive cycle of the species. During the postreproductive period, an increase in estradiol secretion was observed while, in the reproductive period, progesterone secretion increased. Our results suggest that the sympathetic division of the autonomic nervous system would be responsible for this increase, taking into account that, under our experimental conditions, acetylcholine did not affect the endocrine activity of the gonad, while adrenaline (epinephrine) was effective in inducing steroid secretion an effect that could be due to interaction with ß receptors. On the other hand, our data show that the association of adrenaline with follicle-stimulating hormone increased estradiol secretion during the postreproductive period, while the association of catecholamine with LH or hCG increased progesterone secretion during the reproductive period. Our results would suggest that nerve stimulation, mediated by the release of adrenaline, would act synergistically with gonadotrophins to stimulate steroid secretion.
Assuntos
Bufo arenarum/fisiologia , Ovário/metabolismo , Acetilcolina/farmacologia , Animais , Secreções Corporais/efeitos dos fármacos , Bufo arenarum/metabolismo , Catecolaminas/farmacologia , Estimulação Elétrica , Epinefrina/farmacologia , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/farmacologia , Técnicas In Vitro , Ovário/efeitos dos fármacos , Ovário/inervação , Progesterona/metabolismoRESUMO
Little attention has been paid to the impact of wastewater generated by mining activities on fluoride. In this study, we evaluated the hematology responses of common South American toad Rhinella arenarum inhabiting natural and artificial environments associated with a fluorite mine from central Argentina. We analyzed three sampling stations associated with the fluorite mine: (I) Los Cerros Negros stream (CN), which runs on granitic rock with a high fluorite content; (II) Los Vallecitos stream (LV), which runs on metamorphic rock with low fluorite content; and (III) artificial decantation ponds (DP) containing sediments produced by fluorite flotation process. We calculated frequencies of micronuclei, erythrocyte nuclear abnormalities, mitosis, and immature erythrocytes. In addition, we performed a differential leukocyte count and determined neutrophils/lymphocyte ratio as a stress response estimator. We found high micronucleus (MN) and erythrocyte nuclear abnormality (ENA) frequencies in DP and CN but low frequencies in LV. The neutrophil/lymphocyte ratio was different among sites, with a significant increase in individuals from DP. Values registered in DP could be caused by exposure to mixture of compounds registered in dams that hold wastewater, while high values registered in CN stream might be due to natural concentrations of fluoride. Our results suggest that blood is an effective and non-destructive sensitive indicator for monitoring genotoxic agents in freshwater ecosystems.
Assuntos
Bufo arenarum/metabolismo , Dano ao DNA , Fluoretos/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Argentina , Feminino , Masculino , Mineração , Testes de MutagenicidadeRESUMO
In this work, we describe the validation of an electrochemiluminescence immunoassay (ECLIA) that allowed us for the first time to determine the levels of progesterone (P4 ) and testosterone (T) secreted by Rhinella arenarum follicles during the preovulatory (POP) and reproductive (RP) periods. We also verified the relation between P4 and T levels and oocyte maturation. Moreover, we demonstrated that the extraction protocol developed for the determinations of P4 and T by ECLIA proved to be efficient and reproducible since the efficacy of the extraction was above 95% in all assays conducted. The results indicate that in the validation process the variation coefficient (CV) between assays is compatible with the analytical procedures based on automated immunoassays (CV < 8%) and that the adaptation proposed for the samples allows the determination of T and P4 with the Cobas e-411 analyzer. Our results indicate that in basal conditions the levels of T released by R. arenarum follicles were higher than those of P4 during POP and RP. In these conditions, steroid secretion failed to induce germinal vesicle break down (GVBD) in the follicles. Under gonadotropin stimulation, steroidogenesis showed a remarkable increase in both periods, especially during POP. This increase was correlated with a high maturation percentage in the follicles incubated in vitro (GVBD = 72 ± 16%) during POP. During RP, human Chorionic Gonadotropin (hCG) induced 81.75 ± 9.1% GVBD. This study is the first report of the seasonal steroidogenic activity in the ovary of R. arenarum in situ using an ECLIA-modified protocol developed in our laboratory.
Assuntos
Bufo arenarum/metabolismo , Imunoensaio/métodos , Ovário/metabolismo , Progesterona/análise , Testosterona/análise , Animais , Bufo arenarum/fisiologia , Feminino , Luminescência , Oogênese , Ovário/fisiologia , Progesterona/isolamento & purificação , Testosterona/isolamento & purificaçãoRESUMO
The combined effects of two widely used pesticides, endosulfan and cypermethrin, on survival of embryo-larval development of the South American toad (Rhinella arenarum) were examined. The toxicity bioassays were performed according to the AMPHITOX test. Embryos and larvae were exposed to mixtures of these pesticides at equitoxic ratios from acute or chronic exposure to evaluate interaction effects. The results were analyzed using both Marking's additive index and combination index (CI)-isobologram methods. Acute (96-h) and intermediate (168-h) toxicity of endosulfan-cypermethrin mixtures remained almost constant for larvae and embryos, but when exposure duration was increased, there was a significant elevation in toxicity, obtaining chronic (240-h) no-observed-effect concentrations (NOEC) values of 0.045 and 0.16 mg/L for embryos and larvae, respectively. These are environmentally relevant concentrations that reflect a realistic risk of this pesticide mixture to this native amphibian species. The toxicity increment with the exposure duration was coincident with the central nervous system development on embryos reaching the larval period, the main target organ of these pesticides. The interactions of the pesticide mixtures at acute and chronic exposure were antagonistic for embryo development (CI > 1), and additive (CI = 1) for larvae, while chronic exposure interactions were synergistic (CI < 1) for both developmental periods. Data indicated that endosulfan-cypermethrin mixtures resulted in different interaction types depending on duration and developmental stage exposed. As a general pattern and considering conditions of overall developmental period and chronic exposure, this pesticide mixture usually applied in Argentine crop fields is synergistic with respect to toxicity for this native amphibian species.
Assuntos
Bufo arenarum/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Endossulfano/toxicidade , Inseticidas/toxicidade , Piretrinas/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Bufo arenarum/crescimento & desenvolvimento , Embrião não Mamífero/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimentoRESUMO
Arsenic (As), a natural element of ecological relevance, is found in natural water sources throughout Argentina in concentrations between 0.01 mg/L and 15 mg/L. The autochthonous toad Rhinella arenarum was selected to study the acute toxicity of As and the biochemical responses elicited by the exposure to As in water during its embryonic development. The median lethal concentration (LC50) value averaged 24.3 mg/L As and remained constant along the embryonic development. However, As toxicity drastically decreased when embryos were exposed from heartbeat-stage on day 4 of development, suggesting the onset of detoxification mechanisms. Given the environmental concentrations of As in Argentina, there is a probability of exceeding lethal levels at 1% of sites. Arsenic at sublethal concentrations caused a significant decrease in the total antioxidant potential but generated an increase in endogenous glutathione (GSH) content and glutathione S-transferase (GST) activity. This protective response might prevent a deeper decline in the antioxidant system and further oxidative damage. Alternatively, it might be linked to As conjugation with GSH for its excretion. The authors conclude that toad embryos are more sensitive to As during early developmental stages and that relatively high concentrations of this toxic element are required to elicit mortality, but oxidative stress may be an adverse effect at sublethal concentrations.
Assuntos
Arsênio/toxicidade , Bufo arenarum/metabolismo , Embrião não Mamífero/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/análise , Argentina , Arsênio/análise , Arsenitos/toxicidade , Desenvolvimento Embrionário/efeitos dos fármacos , Glutationa/análise , Glutationa Transferase/análise , Medição de Risco , Compostos de Sódio/toxicidadeRESUMO
In the principal route of phosphatidylcholine (PC) synthesis the regulatory steps are catalysed by CTP:phosphocholine cytidylyltransferase (CCT) and choline kinase (CK). Knock-out mice in Pcyt1a (CCT gene) and Chka1 (CK gene) resulted in preimplantation embryonic lethality, demonstrating the essential role of this pathway. However, there is still a lack of detailed CCT and CK expression analysis during development. The aim of the current work was to study the expression during early development of both enzymes in the external-fertilization vertebrate Bufo arenarum. Reverse transcription polymerase chain reaction (RT-PCR) and western blot confirmed their presence in unfertilized eggs. Analysis performed in total extracts from staged embryos showed constant protein levels of both enzymes until the 32-cell stage: then they decreased, reaching a minimum in the gastrula before starting to recover. CTP:phosphocholine cytidylyltransferase is an amphitropic enzyme that inter-converts between cytosolic inactive and membrane-bound active forms. Immunoblot analysis demonstrated that the cytosolic:total CCT protein ratio does not change throughout embryogenesis, suggesting a progressive decline of CCT activity in early development. However, PC (and phosphatidylethanolamine) content per egg/embryo remained constant throughout the stages analysed. In conclusion, the current data for B. arenarum suggest that net synthesis of PC mediated by CCT and CK is not required in early development and that supplies for membrane biosynthesis are fulfilled by lipids already present in the egg/embryo reservoirs.
Assuntos
Bufo arenarum/embriologia , Colina Quinase/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Embrião não Mamífero/enzimologia , Fosfatidilcolinas/metabolismo , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Animais , Bufo arenarum/metabolismo , Colina Quinase/genética , Colina-Fosfato Citidililtransferase/genética , Citosol/enzimologia , Feminino , Masculino , Óvulo/enzimologia , Fosfatidilcolinas/biossíntese , Fosfatidiletanolaminas/metabolismoRESUMO
Animals with external fertilization, as amphibians, store their sperm in a quiescent state in the testis. When spermatozoa are released into natural fertilization media, the hypotonic shock triggers activation of sperm motility. Rhinella (Bufo) arenarum sperm are immotile in artificial seminal plasma (ASP, resembling testicular plasma tonicity) but acquire in situ flagellar beating upon dilution. However, if components from the egg shelly coat are added to this medium, motility shifts to a progressive pattern. Recently, we have shown that the signal transduction pathway required for in situ motility activation involves a rise in intracellular cAMP through a transmembrane adenylyl cyclase and activation of PKA, mostly in the midpiece and in the sperm head. In this report, we demonstrate that activation of calcineurin (aka PP2B and PPP3) is required for the shift from in situ to progressive sperm motility. The effect of calcineurin is manifested by dephosphorylation of PKC substrates, and can be promoted by intracellular calcium rise by Ca(2+) ionophore. Both phosphorylated PKC substrates and calcineurin localized to the flagella, indicating a clear differentiation between compartmentalization of PKA and calcineurin pathways. Moreover, no crosstalk is observed between these signaling events, even though both pathways are required for progressive motility acquisition as discussed.
Assuntos
Proteínas de Anfíbios/metabolismo , Bufo arenarum/metabolismo , Calcineurina/metabolismo , Proteína Quinase C/metabolismo , Transdução de Sinais , Motilidade dos Espermatozoides , Espermatozoides/enzimologia , Animais , Inibidores de Calcineurina , Ionóforos de Cálcio/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Flagelos/enzimologia , Masculino , Pressão Osmótica , Fosforilação , Transdução de Sinais/efeitos dos fármacos , Peça Intermédia do Espermatozoide/enzimologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Cauda do Espermatozoide/enzimologia , Espermatozoides/efeitos dos fármacos , Especificidade por SubstratoRESUMO
The authors evaluated biomarker responses in caged larvae of the amphibian Rhinella arenarum in water channels during fruit production season and compared them with those elicited by a transient exposure to azinphos methyl (AzM) (0.02-2 mg/L; 4 h), the main pesticide applied in the Alto Valle region, Patagonia, Argentina, taking into account the maximum environmental concentration detected in superficial water (22.5 µg/L). The traditional biomarkers of organophosphate exposure, acetylcholinesterase (AChE) and carboxylesterase, were inhibited in tadpoles after one week of exposure in channels potentially receiving pesticide drift, whereas the antioxidant glutathione (GSH) and the detoxifying activity of GSH S-transferase (GST) were induced. In a two-week monitoring study, AChE activity was induced in larvae exposed at the agricultural site, and carboxylesterase showed an inhibition followed by return to control values, suggesting an exposure-recovery episode. Antioxidant glutathione levels were first depleted and then surpassed control levels, whereas GST activity was continuously induced. These responses were mimicked in the laboratory by 2 mg/L AzM-pulse exposure, which notably exceeds the expected environmental concentrations. The results draw attention to the complexity of responses after pesticide exposure, strongly depending on exposure time-concentration and recovery periods, among other possible factors, and support the necessity of the integrated use of biomarkers to assess exposure episodes in agricultural areas.
Assuntos
Agricultura , Azinfos-Metil/farmacologia , Biomarcadores/análise , Bufo arenarum/metabolismo , Praguicidas/farmacologia , Poluentes Químicos da Água/farmacologia , Acetilcolinesterase/análise , Irrigação Agrícola , Animais , Argentina , Carboxilesterase/análise , Exposição Ambiental , Monitoramento Ambiental , Frutas , Glutationa/análise , Glutationa Transferase/análise , Larva/efeitos dos fármacos , Estações do AnoRESUMO
Organophosphorus pesticides (OPs) are widely applied in the Alto Valle of Río Negro and Neuquén, Argentina, due to intensive fruit growing. Amphibians are particularly sensitive to environmental pollution, and OPs may transiently accumulate in ponds and channels of the region during their reproductive season. Organophosphorus pesticide exposure may alter amphibian embryonic development and the reproductive success of autochthonous species. In the present study, embryos of the common toad Rhinella arenarum were employed to assess developmental alterations and to study polyamine metabolism, which is essential to normal growth, as a possible target underlying the effects of the OP chlorpyrifos. As the duration of chlorpyrifos exposure increased and embryonic development progressed, the median lethal concentration (LC50) values decreased, and the percentage of malformed embryos increased. Developmental arrest was also observed and several morphological alterations were recorded, such as incomplete and abnormal closure of the neural tube, dorsal curvature of the caudal fin, reduction of body size and caudal fin length, atrophy, and edema. An early decrease in ornithine decarboxylase (ODC) activity and polyamine levels was also observed in embryos exposed to chlorpyrifos. The decrease in polyamine contents in tail bud embryos might be a consequence of the reduction in ODC activity. The alteration of polyamine metabolism occurred before embryonic growth was interrupted and embryonic malformations were observed and may be useful as a biomarker in environmental studies.
Assuntos
Bufo arenarum/anormalidades , Bufo arenarum/metabolismo , Clorpirifos/toxicidade , Embrião não Mamífero/anormalidades , Embrião não Mamífero/metabolismo , Poluentes Ambientais/toxicidade , Praguicidas/toxicidade , Poliaminas/metabolismo , Animais , Argentina , Bufo arenarum/embriologia , Desenvolvimento Embrionário , Feminino , Dose Letal Mediana , Ornitina Descarboxilase/metabolismoRESUMO
The reduction of A-ring of glucocorticoids to produce 5α-dihydro-derivatives by 5α-reductases has been considered as a pathway of irreversible inactivation. However, 5α-reduced metabolites of corticosterone and testosterone have significant biological activity. In this paper, we investigated whether toad testicular 5α-reductase (5α-Red) is able to transform corticosterone into 5α-dihydrocorticosterone. Furthermore, we studied the role of 5α-reduced metabolite of corticosterone as a glucocorticoid receptor (GR) agonist. The activity of 5α-Red was assayed in subcellular fractions with [(3)H]corticosterone or [(3)H]testosterone as substrate. The enzyme localizes in microsomes and its optimal pH is between 7 and 8. The activity is not inhibited by finasteride. These results support the conclusion that toad 5α-Red resembles mammalian type 1 isoenzyme. Kinetic studies indicate that neither K(m) nor V(max) for both corticosterone and testosterone were significantly different among reproductive periods. The K(m) value for testosterone was significantly higher than that for corticosterone, indicating that the C-21 steroid is the preferred substrate for the enzyme. Studies of the binding capacity of 5α-dihydrocorticosterone (5α-DHB) to the testicular GR show that 5α-DHB is able to displace the binding of [(3)H]dexamethasone to testicular cytosol with a similar potency than corticosterone. The inhibition constant (Ki) values for corticosterone and 5α-DHB were similar, 31.33±2.9 nM and 35.24±2.3 nM, respectively. In vitro experiments suggest that 5α-DHB is an agonist of toad testicular GR, decreasing the activity of the key enzyme for androgen synthesis, the cytochrome P450 17-hydroxylase, C17,20-lyase.
Assuntos
Bufo arenarum/metabolismo , Colestenona 5 alfa-Redutase/metabolismo , Glucocorticoides/metabolismo , Receptores de Glucocorticoides/metabolismo , Testículo/metabolismo , Inibidores de 5-alfa Redutase/farmacologia , Animais , Western Blotting , Colestenona 5 alfa-Redutase/antagonistas & inibidores , Finasterida/farmacologia , Cinética , Masculino , Testículo/enzimologiaRESUMO
The glycoprotein envelope surrounding the Bufo arenarum egg exists in different functional forms. Conversion between types involves proteolysis of specific envelope glycoproteins. When the egg is released from the ovary, the envelope cannot be penetrated by sperm. Conversion to a penetrable state occurs during passage through the pars recta portion of the oviduct, where oviductin, a serine protease with trypsin-like substrate specificity, hydrolyzes two kinds of envelope glycoproteins: gp84 and gp55. The nucleotide sequence of a 3203 bp B. arenarum oviductin cDNA was obtained. Deduced amino acid sequence showed a complete open reading frame encoding 980 amino acids. B. arenarum oviductin is a multi-domain protein with a protease domain at the N-terminal region followed by two CUB domains and toward the C-terminal region another protease domain, which lacked an active histidine site, and one CUB domain. Expression of ovochymase 2, the mammalian orthologous of amphibian oviductin, was assayed in mouse female reproductive tract. Ovochymase 2 mRNA was unnoticeable in the mouse oviduct but expression was remarkable in the uterus. Phylogenetic relationship between oviductin and ovochymase 2 opens the possibility to understand the role of this enzyme in mammalian reproduction.
Assuntos
Proteínas de Anfíbios/genética , Bufo arenarum/genética , Regulação Enzimológica da Expressão Gênica , Serina Endopeptidases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bufo arenarum/metabolismo , Clonagem Molecular , DNA Complementar/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Tubas Uterinas/citologia , Tubas Uterinas/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Fases de Leitura Aberta , Filogenia , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade por Substrato , Útero/citologia , Útero/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
Integrins are cell adhesion molecules that are thought to be involved in sperm-oocyte interaction. Nevertheless, their function in mammalian fertilization is still controversial, as different species behave differently. In amphibians, their role is mainly supported by Xenopus laevis studies, where RGDS peptide induces oocyte activation. We recently provided evidence suggesting the presence and involvement of integrins in the interaction of the oocyte plasma membrane (PM) with sperm in the amphibian Bufo arenarum. In order to understand the role of integrin homologs in oocytes and their possible contribution to egg activation mechanisms, we examined the presence of integrin subunits and the effect of RGDS peptide on oocytes and during fertilization. Western blot studies detected integrin subunits α5, αV and ß1 in oocytes. In sperm, we could detect only the αV integrin subunit. We found that RGDS peptide was unable to elicit egg activation or MAPK dephosphorylation, but can induce reversible inhibition of fertilization. A similar partial inhibition was produced by an anti-ß1 integrin antibody. Using an anti-phosphotyrosine antibody we found major changes in phosphotyrosine-containing proteins in egg extracts minutes after fertilization. Cytosol and PMs isolated from oocytes and fertilized eggs showed additional fertilization-induced phosphorylated proteins. Some of these were also present in cytosol and PMs from RGDS-treated oocytes (partially mimicking fertilization). These findings suggest that B. arenarum fertilization involves integrins (e.g. ß1 subunit) as adhesion proteins. Our data support the view that RGDS-binding receptors may function as signaling receptors in B. arenarum oocytes, but integrin engagement by RGDS is not sufficient for oocyte activation.
Assuntos
Proteínas de Anfíbios/metabolismo , Bufo arenarum/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fertilização/efeitos dos fármacos , Integrinas/metabolismo , Oligopeptídeos/farmacologia , Oócitos/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Interações Espermatozoide-Óvulo/efeitos dos fármacos , Animais , Western Blotting , Ativação Enzimática , Feminino , Masculino , Microscopia de Fluorescência , Oócitos/enzimologia , Fosforilação , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Fatores de Tempo , TirosinaRESUMO
Nickel, a widely distributed heavy metal in the biosphere, produces systemic, carcinogenic, and teratogenic effects. The objectives of the present study are to report the acute, short-term chronic, and chronic toxicity of Ni in Rhinella arenarum embryos as well as the stage-dependent susceptibility to this heavy metal, including oxygen consumption, teratogenesis, and adverse effects on cell differentiation processes. The stages evaluated were blastula (S.7), gastrula (S.11), tail bud (S.17), fin circulation (S.22), and complete operculum (S.25), in this last case by means of toxicity profile curves. Nickel increases its adverse effects gradually, with a maximum value after 96 h. The 50% lethal concentrations (LC50s) for 96, 168, and 240 h at S.25 were 1.14, 0.60, and 0.48 mg Ni²(+) /L, respectively; S.11 and S.22 were the least and most susceptible to Ni with, LC50s 96 h of 6.12 and 0.19 mg Ni²(+) /L, respectively. A reduction of approximately 25% in oxygen consumption anticipates lethal effects from S.17 onward. The main teratogenic effects were retarded growth and development, extremely severe axis incurvations, persistent yolk plug, asymmetry, microcephaly and mouth and gill agenesia, and limited neuromuscular activity. Ciliated cells were not functional. The possibility of associating the remarkable stage-dependent susceptibility to Ni with environmental changes during the evolutionary process is also considered.
Assuntos
Bufo arenarum/fisiologia , Diferenciação Celular/efeitos dos fármacos , Morfogênese/efeitos dos fármacos , Níquel/toxicidade , Consumo de Oxigênio/efeitos dos fármacos , Oligoelementos/toxicidade , Animais , Bufo arenarum/embriologia , Bufo arenarum/metabolismo , Carcinógenos/toxicidade , Relação Dose-Resposta a Droga , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/patologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , América do Sul , Teratogênicos/toxicidadeRESUMO
Vitellogenin (Vtg), a large lipoglycophosphoprotein, is the most important precursor of the yolk proteins, and the major source of nutrients for the developing embryo in oviparous species. After its uptake by the oocytes, Vtg is converted into lipovitellins (high and light) and phosvitin, which are deposited into crystalline yolk platelets. We describe here the presence of two high molecular mass lipovitellin isoforms in Bufo arenarum mature oocytes with masses of 113 and 100 kDa, respectively. The amino acid sequence analysis of p113 and p100 peptides showed a high sequence homology between both polypeptides and the complete reported sequences of Xenopus laevis vitellogenin. Using specific antibodies, we determined that the Vtg uptake begins early during oogenesis, at the previtellogenic stage, and continues until oocytes have reached their mature status. In addition, we found that large endocytic vesicles mediate Vtg uptake in stage I oocytes, and that the size of the endocytic vesicles declines with oogenesis progression. In terms of the Vtg protein trafficking, we detected the Vtg precursor (190 kDa) in the liver of estradiol-injected females. Finally, we propose a subclassification of B. arenarum stage II oocytes into three physiologically and morphologically distinct periods (early, mid and late).
Assuntos
Bufo arenarum/metabolismo , Proteínas do Ovo/química , Proteínas do Ovo/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Vitelogênese , Sequência de Aminoácidos , Animais , Bufo arenarum/crescimento & desenvolvimento , Bufo arenarum/fisiologia , Proteínas do Ovo/isolamento & purificação , Feminino , Imuno-Histoquímica , Dados de Sequência Molecular , Peso Molecular , Oócitos/ultraestrutura , Transporte Proteico , Análise de Sequência de DNA , Fatores de TempoRESUMO
SummaryBufo arenarum oocytes are oviposited surrounded by jelly coats, one component of the extracellular matrix required for fertilization. The secretion, released to the oviductal lumen, was analysed by SDS-PAGE. The coomassie blue staining evidenced an electrophoretic pattern with molecules ranging between 300 and 19 kDa that showed variations in their secretion profiles during the sexual cycle. In the preovulatory period the densitometric analysis showed the presence of nine peaks with marked predominance of the 74 kDa molecule. Once ovulation has occurred, the jelly coats become arranged around the oocytes during their transit throughout the oviductal pars convoluta (PC), revealing the addition of three proteins only observed during this period, which suggests a differential secretion. Some of these proteins could not diffuse under any extraction treatment, indicating for them a structural or in situ function. Proteins of low molecular mass diffused totally while others showed a partial diffusing capacity. After ovulation a marked decrease in the relative amount of all the proteins released to the lumen, especially the 74 kDa protein, could be detected. During this period, unlike the other stages of the sexual cycle, a differential secretion pattern was observed along the PC. The histochemical analysis performed during the ovulatory period showed the presence of glycoconjugates including both acidic and neutral groups. The present results are in agreement with previous ultrastructural and histochemical studies that describe the role of Bufo arenarum jelly coats in fertilization.
Assuntos
Proteínas de Anfíbios/análise , Bufo arenarum/metabolismo , Matriz Extracelular/química , Oócitos/química , Oviductos/metabolismo , Animais , Eletroforese em Gel de Poliacrilamida , FemininoRESUMO
Amphibian embryos are naturally exposed to prooxidant conditions throughout their development. Environmental exposure to contaminants may affect their capacity to respond to challenging conditions, to progress in a normal ontogenesis, and finally to survive and succeed in completing metamorphosis. We studied the effects of the exposure to two anticholinesterase agents, the carbamate carbaryl and the organophosphate azinphos methyl, on the antioxidant defenses of developing embryos of the toad Rhinella (Bufo) arenarum. Reduced glutathione (GSH) levels were increased early by carbaryl, but were decreased by both pesticides at the end of embryonic development. The GSH-dependent enzymes glutathione reductase and glutathione peroxidases showed oscillating activity patterns that could be attributed to an induction of activity in response to oxidative stress and inactivation by excess of reactive oxygen species. Glutathione-S-transferases, which may participate in the conjugation of lipid peroxide products in addition to pesticide detoxification, showed an increase of activity at the beginning and at the end of development. Catalase also showed variations in the activity suggesting, successively, induction and inactivation in response to pesticide exposure-induced oxidative stress. Superoxide dismutase activity was increased by carbaryl and transiently decreased by azinphos methyl exposure. Judging from the depletion in GSH levels and glutathione reductase inhibition at the end of embryonic development, the oxidative stress caused by azinphos methyl seemed to be greater than that caused by carbaryl, which might be in turn related with a higher number of developmental alterations caused by the organophosphate. GSH content is a good biomarker of oxidative stress in the developing embryos exposed to pesticides. The antioxidant enzymes are in turn revealing the balance between their protective capacity and the oxidative damage to the enzyme molecules, decreasing their activity.
Assuntos
Antioxidantes/metabolismo , Azinfos-Metil/toxicidade , Bufo arenarum/metabolismo , Carbaril/toxicidade , Inseticidas/toxicidade , Poluentes Químicos da Água/toxicidade , Anormalidades Induzidas por Medicamentos/etiologia , Anormalidades Induzidas por Medicamentos/metabolismo , Animais , Bufo arenarum/embriologia , Catalase/metabolismo , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Feminino , Glutationa/metabolismo , Glutationa Peroxidase/metabolismo , Glutationa Redutase/metabolismo , Glutationa Transferase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
The localization of calcium and Ca-ATPase activity in Bufo arenarum oocytes was investigated by ultracytochemical techniques during progesterone-induced nuclear maturation, under in vitro conditions. No Ca2+ deposits were detected in either control oocytes or progesterone-treated ones for 1-2 h. At the time when nuclear migration started, electron dense deposits of Ca2+ were visible in vesicles, endoplasmic reticulum cisternae and in the space between the annulate lamellae membranes. Furthermore, Ca-ATPase activity was also detected in these membrane structures. As maturation progressed, the cation deposits were observed in the cytomembrane structures, which underwent an important reorganization and redistribution. Thus, they moved from the subcortex and became located predominantly in the oocyte cortex area when nuclear maturation ended. Ca2+ stores were observed in vesicles surrounding or between the cortical granules, which are aligned close to the plasma membrane. The positive Ca-ATPase reaction in these membrane structures could indicate that the calcium deposit is an ATP-dependent process. Our results suggest that during oocyte maturation calcium would be stored in membrane structures where it remains available for release at the time of fertilization. Data obtained under our experimental conditions indicate that calcium from the extracellular medium would be important for the oocyte maturation process.
Assuntos
Bufo arenarum/fisiologia , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Membrana Celular/fisiologia , Núcleo Celular/metabolismo , Retículo Endoplasmático/fisiologia , Oócitos/fisiologia , Animais , Bufo arenarum/metabolismo , Membrana Celular/ultraestrutura , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Feminino , Microscopia Eletrônica de Transmissão , Oócitos/metabolismo , Oócitos/ultraestrutura , Progesterona/farmacologiaRESUMO
Cell adhesion molecules act as signal transducers from the extracellular environment to the cytoskeleton and the nucleus and consequently induce changes in the expression pattern of structural proteins. In this study, we showed the effect of thyroid hormone (TH) inhibition and arrest of metamorphosis on the expression of E-cadherin, beta-and alpha-catenin in the developing kidney of Bufo arenarum. Cell adhesion molecules have selective temporal and spatial expression during development suggesting a specific role in nephrogenesis. In order to study mechanisms controlling the expression of adhesion molecules during renal development, we blocked the B. arenarum metamorphosis with a goitrogenic substance that blocks TH synthesis. E-cadherin expression in the proximal tubules is independent of thyroid control. However, the blockage of TH synthesis causes up-regulation of E-cadherin in the collecting ducts, the distal tubules and the glomeruli. The expression of beta-and alpha-catenin in the collecting ducts, the distal tubules, the glomeruli and the mesonephric mesenchyme is independent of TH. TH blockage causes up-regulation of beta-and alpha-catenin in the proximal tubules. In contrast to E-cadherin, the expression of the desmosomal cadherin desmoglein 1 (Dsg-1) is absent in the control of the larvae kidney during metamorphosis and is expressed in some interstitial cells in the KClO4 treated larvae. According to this work, the Dsg-1 expression is down-regulated by TH. We demonstrated that the expression of E-cadherin, Dsg-1, beta-catenin and alpha-catenin are differentially affected by TH levels, suggesting a hormone-dependent role of these proteins in the B. arenarum renal metamorphosis.
Assuntos
Bufo arenarum/embriologia , Moléculas de Adesão Celular/metabolismo , Rim/embriologia , Percloratos/farmacologia , Compostos de Potássio/farmacologia , Tri-Iodotironina/antagonistas & inibidores , Animais , Bufo arenarum/metabolismo , Caderinas/metabolismo , Embrião não Mamífero , Feminino , Imuno-Histoquímica , Rim/metabolismo , alfa Catenina/metabolismo , beta Catenina/metabolismoRESUMO
Cellular nucleic-acid-binding protein (CNBP) plays an essential role in forebrain and craniofacial development by controlling cell proliferation and survival to mediate neural crest expansion. CNBP binds to single-stranded nucleic acids and displays nucleic acid chaperone activity in vitro. The CNBP family shows a conserved modular organization of seven Zn knuckles and an arginine-glycine-glycine (RGG) box between the first and second Zn knuckles. The participation of these structural motifs in CNBP biochemical activities has still not been addressed. Here, we describe the generation of CNBP mutants that dissect the protein into regions with structurally and functionally distinct properties. Mutagenesis approaches were followed to generate: (i) an amino acid replacement that disrupted the fifth Zn knuckle; (ii) N-terminal deletions that removed the first Zn knuckle and the RGG box, or the RGG box alone; and (iii) a C-terminal deletion that eliminated the three last Zn knuckles. Mutant proteins were overexpressed in Escherichia coli, purified, and used to analyze their biochemical features in vitro, or overexpressed in Xenopus laevis embryos to study their function in vivo during neural crest cell development. We found that the Zn knuckles are required, but not individually essential, for CNBP biochemical activities, whereas the RGG box is essential for RNA-protein binding and nucleic acid chaperone activity. Removal of the RGG box allowed CNBP to preserve a weak single-stranded-DNA-binding capability. A mutant mimicking the natural N-terminal proteolytic CNBP form behaved as the RGG-deleted mutant. By gain-of-function and loss-of-function experiments in Xenopus embryos, we confirmed the participation of CNBP in neural crest development, and we demonstrated that the CNBP mutants lacking the N-terminal region or the RGG box alone may act as dominant negatives in vivo. Based on these data, we speculate about the existence of a specific proteolytic mechanism for the regulation of CNBP biochemical activities during neural crest development.