ICB - Instituto de Ciências Biológicas
URI permanente desta comunidadehttps://rihomolog.furg.br/handle/1/51
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- ItemEvaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus(2009) Lanes, Carlos Frederico Ceccon; Sampaio, Luís André Nassr de; Marins, Luis Fernando FernandesSperm mediated gene transfer (SMGT) has been successfully used in mammals, amphibians, birds, and some invertebrates. In fish, this methodology has failed or had poor efficiency for the production of transgenic specimens, presumably because the processes regulating the interaction between spermatozoa and exogenous DNA are not well understood. Therefore, the objective was to develop a SMGT protocol for the Brazilian flounder Paralichthys orbignyanus, with an emphasis on the role of seminal plasma DNase on exogenous DNA uptake by fish spermatozoa. In this study, there was strong DNase activity in the seminal plasma of P. orbignyanus; however, this DNase activity was decreased or eliminated by washing the spermatozoa with solutions containing EDTA (DNase activity was completely inhibited by 40 mM EDTA). Three washing solutions were tested, all of which maintained sperm quality. Moreover, it was determined that the no more than 50 ng of exogenous DNA/106 cells should be used for SMGT in fish. Finally, it was demonstrated that fish spermatozoa were capable of spontaneous uptake of exogenous DNA after elimination of DNase activity; this was confirmed by exogenous DNA amplification (PCR using sperm genomic DNA as a template) after DNase I treatment. We concluded that whereas DNase activity was an important obstacle for exogenous DNA uptake by fish spermatozoa;controlling this activity improved the efficiency of SMGT in fish.
- ItemGH overexpression causes muscle hypertrophy independent from local IGF-I in a zebrafish transgenic model(2011) Kuradomi, Rafael Yutaka; Figueiredo, Márcio de Azevedo; Lanes, Carlos Frederico Ceccon; Rosa, Carlos Eduardo da; Almeida, Daniela Volcan; Maggioni, Rodrigo; Silva, Maeli Dal Pai; Marins, Luis Fernando FernandesThe aim of the present study was to analyse the morphology of white skeletal muscle in males and females from the GH-transgenic zebrafish(Danio rerio) lineage F0104, comparing the expression of genes related to the somatotrophic axis and myogenesis. Histological analysis demonstrated that transgenic fish presented enhanced muscle hypertrophy when compared to non-transgenic fish, with transgenic females being more hypertrophic than transgenic males. The expression of genes related to muscle growth revealed that transgenic hypertrophy is independent from local induction of insulin-like growth factor 1 gene (igf1). In addition, transgenic males exhibited significant induction of myogenin gene (myog) expression, indicating that myog may mediate hypertrophic growth in zebrafish males overexpressing GH. Induction of the a-actin gene (acta1) in males, independently from transgenesis, also was observed. There were no significant differences in total protein content from the muscle. Our results show that muscle hypertrophy is independent from muscle igf1, and is likely to be a direct effect of excess circulating GH and/or IGF1 in this transgenic zebrafish lineage.
- ItemGreen turtle (Chelonia mydas) mixed stocks in the western South Atlantic, as revealed by mtDNA haplotypes and drifter trajectories(2012) Proietti, Maíra Carneiro; Reisser, Júlia Wiener; Kinas, Paul Gerhard; Pereira, Rodrigo Kerr Duarte; Monteiro, Danielle da Silveira; Marins, Luis Fernando Fernandes; Secchi, Eduardo ResendeGenetic analyses have the potential to elucidate many aspects of juvenile green turtle (Chelonia mydas) biology and ecology, such as foraging ground composition, hatchling dispersal and migrations. To evaluate genetic structure and assess natal origins of mixed stocks in Southern Brazil, we analyzed mitochondrial DNA control region sequences from Arvoredo Island (n = 115) and Cassino Beach (n = 101), comparing them to other mixed stocks and examining their composition in terms of Atlantic Ocean stocks (nesting areas). In order to compare natal origin estimates (obtained through Bayesian Mixed Stock Analysis) with oceanographic data and develop novel informative priors for this analysis, surface drifter trajectories in the Atlantic Ocean were analyzed. Each study area presented twelve haplotypes, of which ten were shared at extremely similarfrequencies. Haplotypes CM-A8 and CM-A5 were most frequent, representing respectively around 60% and 20% of samples from both areas, and remaining haplotypes were present in less than 5% of samples. Genetic structuring was not observed between the study areas. Arvoredo Island and Cassino Beach also did not present structuring in relation to Ubatuba and Rocas/Noronha, in the southwestern Atlantic, but were structured when compared to farther feeding areas in Brazil, the Caribbean, and North America. Analysis of drifter trajectories revealed that drifters from Ascension and Trindade Islands are dominant at the eastern coast of Brazil. Informative priors developed for Mixed Stock Analysis did not greatly alter stock estimates; we do, however, consider them to be ecologically more realistic. According to the Bayesian mixed stock analyses applied here, Ascension, Aves and Trindade Islands, as well as Gulf of Guinea, were the main contributors to the Southern Brazil mixed stock. This analysis has important implications for the conservation of this species, since impacts on mixed stocks along the coast may affect some reproductive stocks which are frequently thousands of kilometers away.
- ItemCharacterization of growth-related genes in the south-western Atlantic pink shrimp Farfantepenaeus paulensis (Perez-Farfante 1967) through a modified DDRT-PCR protocol(2008) Toth, Michel Kamimura; Meier, Karina Maria; Cavalli, Ronaldo Olivera; Laurino, Jomar Pereira; Maggioni, Rodrigo; Marins, Luis Fernando FernandesAn exploratory methodology has been used to identify genes related to growth of the shrimp Farfantepenaeus paulensis (Pe¤rez-Farfante, 1967). Six hundred postlarvae, with an average ( SD) initial weight of 0.022 g ( 0.008), were reared for 60 days in salinity 30 g L 1. In the end, the 15 heaviest (41.2 g) and the15 lightest (o0.6 g) animalswere frozen in liquid nitrogen. A modi¢ed di¡erential display reverse transcription-polymerase chain reaction technique was used to generate expression pro¢les for all individuals. The resulting cDNA from reverse transcription were ampli¢ed by pairing 31 arbitrary primers with a reverse primer anchored to the cDNA tails. From the comparison between the expression pro¢les of the organisms in the two size classes, differences could be pinpointed. Bands of interest were collected, puri¢ed, cloned and sequenced. Despite the relatively small number of primers used, the methodology allowed the identi¢cation of three genes, not described previously for the investigated species and possibly related to growth. These partial sequences are likely to belong to genes that code formyosin heavy chain, cyclophilin and haemocyanin as revealed through an amino acid similarity search conducted using the BLASTP on-line tool.
- ItemSOCS1 and SOCS3 are the main negative modulators of the somatotrophic axis in liver of homozygous gh-transgenic zebrafish (danio rerio)(2009) Studzinski, Ana Lupe Motta; Almeida, Daniela Volcan; Lanes, Carlos Frederico Ceccon; Figueiredo, Marcio de Azevedo; Marins, Luis Fernando FernandesHomozygote individuals (HO) of the GH-transgenic zebrafish lineage (F0104), despite expressing double the amount of growth hormone (GH) in relation to the hemizygote (HE) individuals, presented smaller growth in relation to the last, and similar to the non-transgenic (NT) group. Through the analysis of the expression of genes of the somatotrophic axis in the livers of HO and NT individuals, it was verified that GHR, JAK2 and STAT5.1 did not present significant differences among the analyzed genotypes (NTand HO). However, in the IGF-I gene expression, an accentuated decrease was observed in group HO(p < 0.01), suggesting a resistance effect to excess GH. This resistance could be related to the insufficient amount of energy for supporting the accelerated metabolic demand caused by excess circulating GH. Analysis of the genes involved in the regulation of GH signalization by dephosphorylation (PTP-H1 and PTP-1B) did not show any significant alteration when comparing groups HO and NT. However, the analysis of the SOCS1 and SOCS3 genes showed an induction in homozygotes of 2.5 times (p < 0.01) and 4.3 times (p < 0.05), respectively, in relation to non-transgenics. The results of the present work demonstrate that, in homozygotes, GH signaling is reduced by the action of the SOCS1 and SOCS3 proteins.
- ItemClonagem e avaliação da expressão gênica do sbGnRH em machos juvenis e adultos de linguado, Paralichthys orbignyanus(2011) Campos, Vinicius Farias; Collares, Tiago Veiras; Deschamps, João Carlos; Seixas, Fabiana Kömmling; Okamoto, Marcelo Hideo; Sampaio, Luís André Nassr de; Marins, Luis Fernando Fernandes; Robaldo, Ricardo BerteauxEste estudo buscou clonar o cDNA do sbGnRH, identificar sua expressão em diferentes tecidos do linguado, bem como avaliar possíveis diferenças no RNA mensageiro (RNAm) desse gene no cérebro de linguados machos juvenis e adultos. Por meio da RT-PCR, demonstrou-se pela primeira vez, a clonagem da região codificadora do sbGnRH contendo 297 nucleotídeos do cérebro do linguado. A expressão do sbGnRH foi detectada em vários tecidos periféricos. Foram detectados níveis mais elevados de RNAm do sbGnRH no hipotálamo dos animais adultos. Estes resultados sugerem que o sbGnRH está envolvido na puberdade do linguado.
- ItemNeuropeptide y gene expression around meal time in the brazilian flounder paralichthys orbignyanus(2012) Campos, Vinicius Farias; Robaldo, Ricardo Berteaux; Deschamps, João Carlos; Seixas, Fabiana Kömmling; Alan, John Alexander McBride; Marins, Luis Fernando Fernandes; Okamoto, Marcelo Hideo; Sampaio, Luís André Nassr de; Collares, Tiago VeirasNeuropeptide Y (NPY) is considered the major stimulant for food intake in mammals and fish. Previous results indicate that NPY is involved in the feeding behaviour of the Brazilian flounder, Paralichthys orbignyanus. In this study, we evaluated hypothalamic NPY expression before (−2 h), during (0 h) and after feeding (+2 h) in two independent experiments: (1) during a normal feeding schedule and (2) in fish fasted for 2 weeks. During normal feeding, changes in the levels of NPY mRNA were periprandial, with expression levels being significantly elevated at meal time (P<0.05) and significantly reduced 2 h later (P<0.05). Comparing the fasting and unfasted groups, NPY mRNA levels were significantly higher (P<0.05) at −2 h and +2 h in the fasting group, but there was no difference at 0 h. In addition, the higher NPY mRNA levels that were observed in the fasting group were maintained throughout the sampling period. In summary, our results show that NPY expression was associated with meal time (0 h) in food intake regulation.
- ItemMuscle-specific growth hormone receptor (GHR) overexpression induces hyperplasia but not hypertrophy in transgenic zebrafish(2012) Figueiredo, Marcio de Azevedo; Mareco, Edson Assunção; Silva, Maeli Dal Pai; Marins, Luis Fernando FernandesEven though growth hormone (GH) transgenesis has demonstrated potential for improved growth of commercially important species, the hormone excess may result in undesired collateral effects. In this context, the aim of this work was to develop a new model of transgenic zebrafish (Danio rerio characterized by a muscle-specific overexpression of the GH receptor (GHR) gene, evaluating the effect of transgenesis on growth, muscle structure and expression of growth-related genes. In on line of transgenic zebrafish overexpressing GHR in skeletal muscle, no significant difference in total weight in comparison to non-transgenics was observed. This can be explained by a significant reduction in expression of somatotrophic axis-related genes, in special insulin-like growth factor I (IGF-I). In the same sense, a significant increase in expression of the suppressors of cytokine signaling 1 and 3 (SOCS) was encountered in transgenics. Surprisingly, expression of genes coding for the main myogenic regulatory factors (MRFs) was higher in transgenic than non-transgenic zebrafish. Genes coding for muscle proteins did not follow the MRFs profile, showing a significant decrease in their expression. These results were corroborated by the histological analysis, where a hyperplasic muscle growth was observed in transgenics. In conclusion, our results demonstrated that GHR overexpression does not induce hypertrophic muscle growth in transgenic zebrafish probably because of SOCS impairment of the GHR/IGF-I pathway, culminating in IGF-I and muscle proteins decrease. Therefore, it seems that hypertrophy and hyperplasia follow two different routes for entire muscle growth, both of them triggered by GHR activation, but regulated by different mechanisms.
- ItemA comparative expression analysis of gene transcripts in brain tissue of non-transgenic and GH-transgenic zebrafish (Danio rerio) using a DDRT-PCR approach(2012) Alves-Costa, Fernanda Antunes; Figueiredo, Marcio de Azevedo; Lanes, Carlos Frederico Ceccon; Almeida, Daniela Volcan; Marins, Luis Fernando Fernandes; Wasko, Adriane PintoThe presence of higher level of exogenous growth hormone (GH) in transgenic animals could lead to several physiological alterations. A GH transgenic zebrafish (Danio rerio) line was compared to nontransgenic (NT) samples of the species through a DDRT-PCR approach, with the goal of identifying candidate differentially expressed transcripts in brain tissues that could be involved in GH overexpression. Densitometric analyses of two selected amplification products, p300 and ADCY2, pointed to a significant lower gene expression in the transgenic zebrafish (104.02 ± 57.71; 224.10 ± 91.73) when compared to NT samples (249.75 ± 30.08; 342.95 ± 65.19). The present data indicate that p300 and ADCY2 are involved in a regulation system for GH when high circulating levels of this hormone are found in zebrafishes.
- ItemElectrophoretic characterization of a hybrid between Eretmochelys imbricata and Caretta caretta (Cheloniidae)(1990) Conceição, Moisés Basilio da; Sabaj, José Alberto Levy; Marins, Luis Fernando Fernandes; Marcovaldi, Maria Angela Azevedo Guagni DeiAn intermediate morphotype between Eretmochelys imbricata and Caretta caretta was studied in Praia do Forte, Bahia, Brazil. Three enzymatic systems were successfully analyzed: SOD, lactate dehydrogenase (LDH) and esterase (EST). Isoelectfic focusing of total soluble proteins of muscle and transferrin were shown. Esterase exhibited nine phenotype patterns, seven in C, caretta and one in the others morphotypes. SOD phenotypes were identical in the three morphotypes. Lactate dehydrogenase and transferrins were characteristic for each species. Jaccard's measure of similarity was calculated and a phenogram with the three morphotypes were constructed using isoelectric focusing of total soluble proteins.