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      Clonagem e avaliação da expressão gênica do sbGnRH em machos juvenis e adultos de linguado, Paralichthys orbignyanus Translated title: Cloning and evaluation of sbGnRH gene expression in juvenile and adult males of Brazilian flounder Paralichthys orbignyanus

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          Abstract

          Este 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.

          Translated abstract

          The objectives of this study were to clone sbGnRH cDNA, evaluate the mRNA levels in different tissues of flounder, and also evaluate brain sbGnRH expression in juvenile and adult males. Using RT-PCR the cloning of a 297 nucleotides coding region of sbGnRH from Brazilian flounder brain was demonstrated for the first time. Expression of sbGnRH was detected in several peripheral tissues. Brain gene expression in the adult flounder was higher than those found in juvenile. These results suggest that sbGnRH is involved on the Brazilian flounder puberty.

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          Evaluation of DNase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of the Brazilian flounder Paralichthys orbignyanus.

          Sperm 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/10(6) 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.
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            Developmental expression of three different prepro-GnRH (gonadotrophin-releasing hormone) messengers in the brain of the European sea bass (Dicentrarchus labrax).

            In this study, we have analyzed the ontogenic expression of three gonadotrophin-releasing hormones (GnRH) systems expressed in the brain of a perciform fish, the European sea bass, using in situ hybridization. The riboprobes used correspond to the GnRH-associated peptide (GAP) coding regions of the three prepro-GnRH cDNAs cloned from the same species: prepro-salmon GnRH, prepro-seabream GnRH and prepro-chicken GnRH II. On day 4 after hatching, the first prepro-chicken GnRH-II mRNA-expressing cells appeared in the germinal zone of the third ventricle. They increased in number and size from 10 to 21 days, reaching at day 30 their adult final position, within the synencephalic area, at the transitional zone between the diencephalon and the mesencephalon. First prepro-salmon GnRH mRNA-expressing cells became evident on day 7 arising from the olfactory placode and migrating towards the olfactory nerve. On day 10, this cell group reached the olfactory bulb, being evident in the ventral telencephalon and preoptic area from days 15 and 45, respectively. Weakly labeled prepro-seabream GnRH mRNA-expressing cells were first detected at 30 days in the olfactory area and ventral telencephalon. On day 45, prepro-seabream GnRH mRNA-expressing cells were also present in the preoptic region reaching the ventrolateral hypothalamus on day 60. The results obtained in sea bass indicate that sGnRH and sbGnRH cells have a common origin in an olfactory primordium suggesting that both forms might arise from a duplication of a single ancestral gene, while cGnRH-II cells develop from a synencephalic primordium.
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              Phylogeny Inference Package (PHYLIP). Version 3.5

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                Author and article information

                Contributors
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Role: ND
                Journal
                abmvz
                Arquivo Brasileiro de Medicina Veterinária e Zootecnia
                Arq. Bras. Med. Vet. Zootec.
                Universidade Federal de Minas Gerais, Escola de Veterinária (Belo Horizonte )
                1678-4162
                February 2011
                : 63
                : 1
                : 239-246
                Affiliations
                [1 ] Universidade Federal de Pelotas Brazil
                [2 ] Universidade Federal do Rio Grande Brazil
                Article
                S0102-09352011000100034
                10.1590/S0102-09352011000100034

                http://creativecommons.org/licenses/by/4.0/

                Product
                Product Information: SciELO Brazil
                Categories
                VETERINARY SCIENCES

                General veterinary medicine

                fish, sbGnRH, mRNA, puberty, peixe, RNAm, puberdade

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