RESUMEN
Arsenic, a natural element of ecological relevance, is one of the most toxic elements present in various regions of the world. It can be found in natural water sources throughout Argentina in concentrations between 0.01 and 15mgL-1. The Argentinean autochthonous toad Rhinella arenarum was selected to study the molecular mechanisms involved in the effects and response to the chronic As exposure along its embryonic and larval development. We evaluated the effects on MAPK signal transduction pathway and transcription factors c-FOS and c-JUN, and the regulation of the expression at protein levels of different antioxidant enzymes. Our results indicated that As is modulating the MAPK pathway, increasing MEK and ERK levels both in the nuclear and post-nuclear fraction along the embryonic development and mainly at the beginning of the larval stage. Through this pathway, As can upregulate transcription factors like c-FOS and c-JUN, impacting the antioxidant response of the exposed embryos and larvae through antioxidant enzymes and recycling of GSH. Arsenic triggered specifically the synthesis of antioxidant enzymes in exposed R. arenarum embryo and larvae. In particular, the expression levels of SOD, CAT and GST enzymes analyzed by Western blot showed a similar behavior to their enzymatic activities in our previous work. This fact suggests that not only the synthesis of these antioxidant enzymes but also their rapid degradation after inactivation would be regulated in response to ROS levels. Antioxidant enzymes may show dual responses of induction and inactivation followed by degradation depending on the levels of oxidative stress and impact on ROS targets when the exposure is sustained in time and intensity. We also performed a probability of exceedence analysis including our previous results to visualize a progression of the response in time and also established the best early-responding biomarkers at the lowest As concentrations. As a conclusion, the molecular biomarkers such as the MAPKs MEK and ERK and transcription factors c-FOS and c-JUN are early induced in the response of developing toad embryos exposed to very low As concentrations in water. The advantage of counting with molecular biomarkers early responding to low concentrations of As in a chronic exposure is that they may anticipate the irreversible damage at later developmental stages due to the constant oxidative challenge.
Asunto(s)
Arsénico/toxicidad , Biomarcadores/metabolismo , Bufo arenarum/embriología , Bufo arenarum/genética , Desarrollo Embrionario/efectos de los fármacos , Exposición a Riesgos Ambientales/análisis , Animales , Antioxidantes/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/enzimología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glutatión Transferasa/metabolismo , Larva/efectos de los fármacos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Análisis de Componente Principal , Probabilidad , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Contaminantes Químicos del Agua/toxicidadRESUMEN
Perturbations of water bodies near agricultural and livestock systems can affect embryonic and larval stages of anurans and negatively impact adult populations and structure of amphibian communities. This study is focused on early development of Rhinella arenarum, for which body growth, abnormalities in the oral disc and genetic damage on erythrocytes were analyzed to establish the impact of agroecosystems on local populations of amphibians. Tadpoles and metamorphs of R. arenarum were collected in three agroecosystems (namely, C1, C2, and C3) and in a site without agricultural and livestock activities (SM) from central Argentina. Egg masses of C1 were extracted for breeding tadpoles under laboratory conditions (Lab). Tadpoles were in small size and lighter in weight in C1 and C2. Metamorphs were shorter and lighter in weight in C1 and C3. In SM and Lab samples, no tadpoles with abnormal LTRF (labial tooth row formula) or without labial teeth were observed. In C1, the highest frequency of abnormal LTRF was recorded and was the only site in which tadpoles without labial teeth were found. In C1 and C2 the tadpoles had highest micronucleus frequencies and nuclear abnormalities. C1 can be considered as the site with the highest anthropogenic perturbation and with less healthy tadpoles. Livestock practices such as alternating cattle between parcel and keeping a buffer between crops and water bodies, would allow a better development of the first aquatic stages that are essential for the conservation of the anuran populations.
Asunto(s)
Agricultura , Bufo arenarum/fisiología , Ecosistema , Animales , Argentina , Bufo arenarum/anomalías , Bufo arenarum/genética , Bufo arenarum/crecimiento & desarrollo , Contaminantes Ambientales/toxicidad , Larva/efectos de los fármacos , Larva/genética , Larva/crecimiento & desarrollo , Larva/fisiología , Metamorfosis Biológica/efectos de los fármacos , Metamorfosis Biológica/genética , Metamorfosis Biológica/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas Anfibias/genética , Bufo arenarum/genética , Regulación Enzimológica de la Expresión Génica , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bufo arenarum/metabolismo , Clonación Molecular , ADN Complementario/genética , Endopeptidasas/genética , Endopeptidasas/metabolismo , Trompas Uterinas/citología , Trompas Uterinas/metabolismo , Femenino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Filogenia , Señales de Clasificación de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de ADN , Especificidad por Sustrato , Útero/citología , Útero/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
BACKGROUND INFORMATION: The egg envelope is an extracellular matrix that surrounds oocytes. In frogs and mammals, a prominent feature of envelope modification following fertilization is the N-terminal proteolysis of the envelope glycoproteins, ZPA [ZP (zona pellucida) A]. It was proposed that ZPA N-terminal proteolysis leads to a conformational change in egg envelope glycoproteins, resulting in the prevention of polyspermy. Bufo arenarum VE (vitelline envelope) is made up of at least four glycoproteins: gp120 (glycoprotein 120), gp75, gp41 and gp38. The aim of the present study was to identify and characterize the baZPA (B. arenarum ZPA homologue). Also, our aim was to evaluate its integrity and functional significance during fertilization. RESULTS: VE components were labelled with FITC in order to study their sperm-binding capacity. The assay showed that gp75, gp41 and gp38 possess sperm-binding activity. We obtained a full-length cDNA of 2062 bp containing one ORF (open reading frame) with a sequence for 687 amino acids. The predicted amino acid sequence had close similarity to that of mammalian ZPA. This result indicates that gp75 is the baZPA. Antibodies raised against an N-terminal sequence recognized baZPA and inhibited sperm-baZPA extracted from VE binding. This protein does not induce the acrosome reaction in homologue sperm. Northern-blot studies indicated that the transcript is exclusively expressed in the ovary. In situ hybridization studies confirmed this and pointed to previtellogenic oocytes and follicle cells surrounding the oocyte as the source of the transcript. baZPA was cleaved during fertilization and the N-terminal peptide fragment remained disulfide bonded to the glycoprotein moiety following proteolysis. CONCLUSION: From the sequence analysis, it was possible to consider that gp75 is the baZPA. It is expressed by previtellogenic oocytes and follicle cells. Also, it can be considered as a sperm receptor that undergoes N-terminal proteolysis during fertilization. The N-terminal peptide could be necessary for sperm binding.
Asunto(s)
Bufo arenarum/metabolismo , Proteínas del Huevo/metabolismo , Fertilización , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , Reacción Acrosómica/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Anticuerpos/farmacología , Secuencia de Bases , Bufo arenarum/genética , Adhesión Celular/efectos de los fármacos , Clonación Molecular , Proteínas del Huevo/química , Proteínas del Huevo/genética , Proteínas del Huevo/aislamiento & purificación , Femenino , Técnica del Anticuerpo Fluorescente , Hibridación in Situ , Masculino , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Péptidos/inmunología , ARN Mensajero/análisis , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/aislamiento & purificación , Homología de Secuencia , Interacciones Espermatozoide-Óvulo/efectos de los fármacos , Membrana Vitelina/química , Membrana Vitelina/metabolismo , Glicoproteínas de la Zona PelúcidaRESUMEN
A 15.7 kDa fatty acid-binding protein from toad heart was purified by gel-filtration chromatography on Sephadex G-75 followed by anion-exchange chromatography on a Mono-Q column. Purity was confirmed by gel electrophoresis and isoelectric focusing. Molecular mass, isoelectric point, amino acid composition and partial internal sequence showed that the protein is related to mammalian heart fatty acid-binding proteins.