Caracterização e transplante de células germinativas primordiais em peixes neotropicais
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Data
2021-10-01
Autores
Coelho, Geovanna Carla Zacheo
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
Atividades antrópicas aumentam o número de espécies de peixes ameaçadas, e, portanto, esforços na conservação são necessários para atenuar tais efeitos. O estabelecimento de bancos genéticos e procedimentos de reconstituição são então essenciais para a manutenção da ictiofauna, incluindo o transplante de células germinativas primordiais (PGCs), para posterior reconstituição de espécies ameaçadas de extinção, por meio da geração de quimeras germinativas e propagação substituta. Apesar do potencial mencionado acima, a produção de quimeras germinativas em espécies de peixes nativas ainda não foi estabelecida. O objetivo deste trabalho foi produzir quimeras germinativas em espécies nativas por meio do transplante de PGCs em receptores estéreis em fase de blástula. A marcação e rastreabilidade da rota de migração de PGCs in vivo em Prochilodus lineatus e Piaractus mesopotamicus foi realizada com sucesso, utilizando microinjeção de mRNA sintetizado in vitro, constituído pela sequência codificante do gene repórter GFP fusionada com a região reguladora de tradução 3’ UTR do gene nanos1 de Danio rerio. As PGCs de A. altiparanae e P. lineatus cultivadas em solução salina ou meio suplementado foi obtida e transplantada em receptores triploides de (A. altiparanae) e/ou híbridos triploides (A. altiparanae X A. fasciatus). As PGCs de A. altiparanae cultivadas em meio suplementado tiveram migração direcionada para a região da crista gonadal em 4,5% dos receptores triplóides e em 19,3% dos híbridos triplóides, enquanto as PGCs cultivadas em solução salina apresentaram apenas migração ectópica. No transplante de PGCs de P. lineatus, apenas migração ectópica foi observada nos receptores híbridos triploides. A análise do nível de expressão de genes específicos de PGCs foi realizada em diferentes tecidos, além da comparação da expressão entre PGCs isoladas em diferentes meios de cultura. Os resultados indicaram que a maior parte da expressão gênica é detectada apenas em gônadas adultas. A expressão gênica em PGCs isoladas demonstra maior nível de expressão em dnd1, ddx4 e dazl em PGCs cultivadas em meio de cultura suplementado em comparação com PGCs de solução salina, indicando que o meio de cultura ajuda a manter as características dessas células. As expressões de nanos e cxcr4b foram diminuídas em PGCs do meio de cultura suplementado em relação à solução salina. Esses genes são marcadores de PGCs e estão diretamente ligados a diferenciação e migração dessas células, alterações nos níveis de expressão podem levar a perda da identidade celular e migração ectópica. O presente estudo traz novas abordagens sobre o transplante de células germinativas primordiais em espécies nativas de peixes, incluindo procedimentos de micromanipulação e transplante de PGCs em espécies da região neotropical. Essas informações são importantes para a construção de bancos genéticos e a reconstituição de espécies ameaçadas.
Anthropogenic activities increase the number of threatened fish species and, therefore, conservation efforts are necessary attenuate such effects. The establishment of gene banks and reconstitution procedures are then essential for the maintenance of the ichthyofauna, including the transplantation of primordial germ cells (PGCs), for subsequent reconstitution of endangered species, through the generation of germline chimera and surrogate propagation. Despite the potential mentioned above, the production of germline chimeras in native fish species has not yet been established. The objective of this work was to produce germline chimera within native species, by the transplantation of PGCs into sterile hosts at the blastula stage. The labelling and traceability of the migration route of PGCs in vivo in Prochilodus lineatus and Piaractus mesopotamicus was successfully performed, using microinjection of mRNA synthesized in vitro, constituted by coding sequence of the gfp reporter gene fused with the 3' UTR translation regulatory region of the gene nanos1 from Danio rerio. The fluorescence labeling of PGCs was specific for the germline of both species studied. The PGC from A. altiparanae and P. lineatus cultured into saline solution or supplemented medium was achieved and transplanted into triploid hosts of (A. altiparanae) and/or triploid hybrids (A. altiparanae X A. fasciatus). PGCs of A. altiparanae cultured in supplemented medium, had directed migration to the gonadal ridge region was in 4.5% of triploid hosts and in 19.3% of triploid hybrid hosts, while PGCs cultured in saline solution showed only ectopic migration. In the transplantation of PGCs from P. lineatus, only ectopic migration was observed in the triploid hybrid hosts. The level expression analysis of specific genes of PGCs was performed in different tissues, in addition to the comparison of expression between PGCs isolated in different culture media. The results indicated that most gene expression is detected only in adult gonads. Gene expression in isolated PGCs demonstrates greater expression of dnd1, ddx4 and dazl in PGCs grown in supplemented culture medium compared to PGCs from saline solution, indicating that the culture medium helps to maintain the characteristics of PGCs. Nanos and cxcr4b expressions were decreased in PGCs form supplemented culture medium in relation to saline solution. These genes are markers of PGCs and are directly linked to differentiation and migration of these cells, changes in expression levels can lead to loss of cell identity and ectopic migration. The present study brings new insights regarding the transplantation of primordial germ cells in native fish species, including micromanipulation procedures and PGCs transplantation in species from the neotropical region. Such information is important for genebanking and reconstitution of threatened species.
Anthropogenic activities increase the number of threatened fish species and, therefore, conservation efforts are necessary attenuate such effects. The establishment of gene banks and reconstitution procedures are then essential for the maintenance of the ichthyofauna, including the transplantation of primordial germ cells (PGCs), for subsequent reconstitution of endangered species, through the generation of germline chimera and surrogate propagation. Despite the potential mentioned above, the production of germline chimeras in native fish species has not yet been established. The objective of this work was to produce germline chimera within native species, by the transplantation of PGCs into sterile hosts at the blastula stage. The labelling and traceability of the migration route of PGCs in vivo in Prochilodus lineatus and Piaractus mesopotamicus was successfully performed, using microinjection of mRNA synthesized in vitro, constituted by coding sequence of the gfp reporter gene fused with the 3' UTR translation regulatory region of the gene nanos1 from Danio rerio. The fluorescence labeling of PGCs was specific for the germline of both species studied. The PGC from A. altiparanae and P. lineatus cultured into saline solution or supplemented medium was achieved and transplanted into triploid hosts of (A. altiparanae) and/or triploid hybrids (A. altiparanae X A. fasciatus). PGCs of A. altiparanae cultured in supplemented medium, had directed migration to the gonadal ridge region was in 4.5% of triploid hosts and in 19.3% of triploid hybrid hosts, while PGCs cultured in saline solution showed only ectopic migration. In the transplantation of PGCs from P. lineatus, only ectopic migration was observed in the triploid hybrid hosts. The level expression analysis of specific genes of PGCs was performed in different tissues, in addition to the comparison of expression between PGCs isolated in different culture media. The results indicated that most gene expression is detected only in adult gonads. Gene expression in isolated PGCs demonstrates greater expression of dnd1, ddx4 and dazl in PGCs grown in supplemented culture medium compared to PGCs from saline solution, indicating that the culture medium helps to maintain the characteristics of PGCs. Nanos and cxcr4b expressions were decreased in PGCs form supplemented culture medium in relation to saline solution. These genes are markers of PGCs and are directly linked to differentiation and migration of these cells, changes in expression levels can lead to loss of cell identity and ectopic migration. The present study brings new insights regarding the transplantation of primordial germ cells in native fish species, including micromanipulation procedures and PGCs transplantation in species from the neotropical region. Such information is important for genebanking and reconstitution of threatened species.
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Palavras-chave
Biotecnologia, Reprodução, Transplante, Células germinativas, Biotechnology, Reproduction, Transplant, Germ cells