RESUMEN
Two-dimensional gel electrophoresis (2-DE), matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry, and database searching were used to analyze the effects of triploidization heat shock treatment on protein expression in rainbow trout eyed embryo and fry. After fertilization, the eggs were incubated at 10 °C for 10 min. Half of the eggs were then subjected to heat shock for 10 min submerged in a 28 °C water bath to induce triploidy. The remainder was incubated normally and used as diploid controls. Specimens of eyed embryos and fry were taken on 18 and 76 days post-fertilization, respectively. In the eyed embryo extracts, seven protein spots were significantly changed in abundance between the control and heat-shocked groups and one of these was decreased while the others were increased in the heat shock-treated group. Of the spots that were shown to change in abundance in the eyed embryos with heat shock treatment, two were identified as vitellogenin, while the others were creatine kinase and angiotensin I. In the 2-DE from the fry muscle extraction, 23 spots were significantly changed in abundance between the diploid and triploid groups. Nineteen of these showed a decreased abundance in diploids, while the remaining four spots had an increased abundance. Triploidization caused differential expression of muscle metabolic proteins including triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, and beta-enolase. Myosin heavy chain as a structural protein was also found to change in abundance in triploids. The altered expression of both structural and metabolic proteins in triploids was consistent with their increased cell size and lower growth performance.
Asunto(s)
Proteínas de Peces/metabolismo , Oncorhynchus mykiss , Proteoma , Triploidía , Animales , Electroforesis en Gel Bidimensional , Oncorhynchus mykiss/embriología , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/metabolismoRESUMEN
The aim of the present study was to examine the effects of tetraploidy induction on proteome of rainbow trout during the early stages of development. After insemination, the eggs were incubated at 10°C for 350min. Thereafter, half of the eggs were exposed to a heat-shock of 28°C for 10min. The remainder were incubated normally and used as diploid controls. Fertilized egg specimens were selected 390min post-fertilization. Samples corresponding respectively to eyed embryos and fry stages were also taken on days 18 and 76 post-fertilization. Based on two-dimensional electrophoresis, all spots that were found to differ significantly in abundance between the untreated and heat-shock treated groups were selected for identification using MALDI-TOF/TOF mass spectrometry. Out of 19 protein spots showing altered abundance in the present study, 13 spots were successfully identified. Of the spots that were shown to change in abundance in the fertilized eggs with heat-shock treatment, three were identified as vitellogenin (spots 1, 2 and 3); while the others were creatine kinase (spot 5) and nucleoside diphosphate kinase (spot 6). All of the proteins identified in the embryos were related to vitellogenin (spots 8, 12 and 13). Among the identified spots from the fry muscle extracts, two were identified as beta-globin (spots 14 and 17); while the others were parvalbumin (spots 15 and 16) and creatine kinase (spot 19). The results obtained in our study may now set the ground for investigations on gene regulation and proteome modifications in polyploid fish.
Asunto(s)
Embrión no Mamífero/metabolismo , Respuesta al Choque Térmico/fisiología , Larva/metabolismo , Oncorhynchus mykiss/crecimiento & desarrollo , Oncorhynchus mykiss/metabolismo , Proteoma/análisis , Tetraploidía , Animales , Electroforesis en Gel Bidimensional , Embrión no Mamífero/citología , Fertilización , Larva/crecimiento & desarrollo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The objective of the present study was to examine the antioxidant status of rainbow trout (Oncorhynchus mykiss) during the early stages of development (fertilized egg, eyed egg, alevin and fry) as an effect of triploidy induction. Eggs and milt were taken from eight females and six males. After insemination, the eggs were incubated at 10°C for 10min. Half of the fertilized eggs were then subjected to heat-shock for 10min submerged in a 28°C water bath to induce triploidy. The remainder were incubated normally and used as diploid controls. Three batches of eggs were randomly selected from each group (control and heat-shocked) and were incubated at 10-11°C under the same environmental conditions in hatchery troughs until the fry stage. Triplicate samples of fertilized eggs from each experimental group were randomly selected 1.5h post-fertilization and at the eyed egg stage of development (18 days post-fertilization, dpf). At 27 dpf, triplicate samples of alevins were chosen from each group. Based on ploidy determination experiment performed on both groups, nine diploid and nine triploid fry (76 dpf) were also selected. The triploidy induction success rate was 87.1%. Vitamin C was in lesser concentrations in fertilized eggs and eyed eggs of the heat-shock treatment group as compared with eggs of the diploid group. Alevins of the heat-shock treatment group had a lower superoxide dismutase (SOD) activity than alevins of the diploid group. Glutathione peroxidase (GPx) level was greater in fertilized eggs and alevins of the heat-shock treatment group as compared to diploids. Catalse (CAT) activity was greater in fertilized eggs, alevins and fry of the heat-shock treatment group than those of the diploid group. Malondialdehyde (MDA), as an index of lipid peroxidation, was in greater concentration in fertilized eggs of the group that was heat-shocked, but it was lesser in alevins and fry of the group in which the eggs were heat-shocked as compared to diploid counterparts. The results demonstrate that heat-shock treatment leads to changes in the values of antioxidant enzymes such as SOD, CAT and GPx, and low molecular weight free-radical scavengers such as vitamin C, as well as level of lipid peroxidation.
Asunto(s)
Antioxidantes/metabolismo , Oncorhynchus mykiss/genética , Animales , Ácido Ascórbico/metabolismo , Embrión no Mamífero/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Calor , Peroxidación de Lípido , Masculino , Oncorhynchus mykiss/fisiología , TriploidíaRESUMEN
A proteomic screening approach was employed to achieve a better understanding of the changes that occur in protein expression patterns associated with skeletal deformities in both diploid and triploid rainbow trout larvae. Triploidy was induced through the application of heat shock of 28°C for 10min to eggs 10-min post fertilization in an aquarium equipped with a heater. Percentage of skeletal deformity in heat-shocked larvae (2.88±0.30, mean±S.E.) was significantly (P<0.05) greater than that of the diploids (0.55±0.24). At five days after hatching, proteins of normal and deformed specimens of deyolked larvae were subjected to proteomic analysis using two-dimensional electrophoresis and mass spectrometry. Among the identified protein spots from diploids, creatine kinase was found to be increased in larvae with skeletal deformities, while apolipoprotein A-I-2, apolipoprotein A-II and calmodulin were found to be decreased in deformed fish. Among the five protein spots that were identified in heat-shocked fish, apolipoprotein A-I-2, apolipoprotein A-II, parvalbumin, myosin light chain 1-1 and nucleoside diphosphate kinase were found to be decreased in deformed larvae. The identification of nine protein spots showing altered expression in deformed fish allows us to reach a preliminary view of the molecular mechanisms that are involved in the development of skeletal malformations in diploid and triploid fish.
Asunto(s)
Huesos/metabolismo , Diploidia , Proteínas de Peces/metabolismo , Larva/metabolismo , Oncorhynchus mykiss/metabolismo , Proteoma/análisis , Triploidía , Animales , Huesos/anomalías , Electroforesis en Gel Bidimensional , Larva/crecimiento & desarrollo , Oncorhynchus mykiss/anomalías , Oncorhynchus mykiss/genética , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The aim of this study was to compare effects of triploidy induction on basal physiological and immunological characteristics in rainbow trout at three developmental stages including fertilized eggs, eyed eggs and fry. Eggs and milt were taken from eight females and six males. The gametes were pooled to minimize the individual differences. After insemination, the eggs were incubated at 10°C for 10min. Half of the fertilized eggs were then subjected to heat shock for 10min submerged in a 28°C water bath to induce triploidy. The remainder were incubated normally and used as diploid controls. Three batches of eggs were randomly selected from each group and were incubated at 10-11°C under the same environmental conditions in hatchery troughs until the fry stage. The first-feeding offspring were also reared under the same environmental and nutritional conditions for 38 days. Triplicate samples of 30 eggs (10 eggs per trough) from each group were selected 1.5h post-fertilization and at the eyed stage. Based on red blood cell analysis, nine diploid and nine triploid fish were also selected for study. The triploidy induction success rate was 87.1%. While diploid fish had greater body weights than those in the heat-shock treatment group, weight gain (WG%) was not different between the fry of the diploid and heat-shock treatment groups. Of thyroid hormones measured, 3,5,3'-triiodo-l-thyronine (T3) was less (P<0.05) in eyed eggs of the heat-shock treatment group, but thyroxine (T4) was greater in fry of the heat-shock treatment group as compared to those that were diploid. Cortisol concentration was greater in fry of the heat-shock treatment group as compared to those that were diploid suggesting that fry in the triploid state may be more susceptible to stressors. Concentrations of immune variables (lysozyme, ACH50, albumin, IgM, total protein, globulin and complement) were either comparable or greater in fry of the heat-shock treatment group suggesting that the immune system is not impaired in fish as a result of triploidy induction.
Asunto(s)
Embrión no Mamífero/fisiología , Oncorhynchus mykiss/genética , Óvulo/fisiología , Triploidía , Albúminas/metabolismo , Animales , Diploidia , Embrión no Mamífero/inmunología , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Calor , Larva/inmunología , Larva/fisiología , Masculino , Oncorhynchus mykiss/embriología , Oncorhynchus mykiss/inmunología , Oncorhynchus mykiss/fisiología , Óvulo/inmunología , Hormonas Tiroideas/metabolismoRESUMEN
The aim of the present study was to explore proteome changes in rainbow trout (Oncorhynchus mykiss) fertilized eggs as an effect of triploidization heat-shock treatment. Eggs and milt were taken from eight females and six males. The gametes were pooled to minimize the individual differences. After insemination, the eggs were incubated at 10°C for 10min. Half of the fertilized eggs were then subjected to heat shock for 10min submerged in a 28°C water bath to induce triploidy. The remainder were incubated normally and used as diploid controls. Three batches of eggs were randomly selected from each group and were incubated at 10-11°C under the same environmental conditions in hatchery troughs until the fry stage. Triplicate samples of 30 eggs (10 eggs per trough) from each group were randomly selected 1.5h post-fertilization for proteome extraction. Egg proteins were analyzed using two-dimensional electrophoresis (2-DE) and MALDI-TOF/TOF mass spectrometry. Based on the results from the statistical analyses, 15 protein spots were found to decrease significantly in abundance in heat-shock treated group and were selected for identification. Out of 15 protein spots showing altered abundance, 14 spots were successfully identified. All of the egg proteins identified in our study were related to vitellogenin (vtg). Decreased abundance of vitellogenin in heat-shock treated eggs in our study may either be explained by (i) higher utilization of vtg as an effect of increased cell size in triploids or (ii) changed metabolism in response to heat-shock stress and (iii) diffusion of vtg through chorion due to incidence of egg shell damage. Decreased abundance of vitellogenin in heat-shock treated eggs was associated with reduced early survival rates and lowered growth performance of triploid fish.