Transplante interespecífico de espermatogônia tronco de piracanjuba Brycon orbignyanus
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Data
2022-05-02
Autores
López, Lucia Suárez
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Universidade Estadual Paulista (Unesp)
Resumo
A piracanjuba (Brycon orbignyanus) é uma espécie em categoria de perigo de extinção devido a ações antrópicas, como o desmatamento ciliar e a fragmentação dos rios. A associação das biotécnicas de geração de bancos géneticos in vitro, utilizando células germinativas primordiais ou células germinativas (espermatogônia ou oogônia-tronco), e do transplante destas células em receptores estéreis é uma estratégia plausível a ser utilizada em ações de conservação e reconstituição de espécies ameaçadas de extinção. O objetivo do presente trabalho foi de estabelecer procedimentos de isolamento e criopreservação de espermatogônias-tronco de piracanjuba, bem como o transplante de espermatogônias-tronco em recepitores estéreis, visando a geração de quimera germinativa. As espermatogônias-tronco foram isoladas utilizando a dissociação enzimática, gradiente de desindade de Percoll e o plaqueamento diferencial, sendo obtido neste processo 66 ± 0,01% de pureza. Espermatogônias-tronco oriundas das etapas de purificação foram caracterizadas quanto a expressão relativa de ddx4, sendo obtida maior expressão relativa, cerca de 3,5 vezes, em células oriundas do gradiente de percoll, em relação as células após plaqueamento diferencial e do tecido gonadal. Portanto, células oriundas do gradiente de percoll foram indicadas para as etapas de transplante e criopreservação in vitro. Experimentos de criopreservação de espermatogônias-tronco foram realizados e a melhor solução crioprotetora obtida foi o propanodiol a 1 M (P = 1,000). Machos adultos e juvenis diploides de Astyanax altiparanae foram utilizados nos experimentos de esterilização química para obtenção de receptores estéreis. Outro experimento de esterilização foi realizado utilizando juvenis triploides de A. altiparanae utilizando a associação da manipulação cromossômica e esterilização química. A associação das técnicas de hibridação entre espécies e manipulação cromossômica foi realizada para a obtenção dos receptores estéries de adultos híbridos triploides (A. altiparanae × A. fasciatus). Pelos resultados obtidos nos experimentos de esterilização, os juvenis triploides e os híbridos adultos triploides foram indicados como os melhores receptores estéreis para a etapa de transplante de espermatogônias-tronco. Os machos “juvenis” triploides, tratados previamente com busulfan, foram transplantados com espermatogônias-tronco recém isoladas, enquanto que os machos híbridos adultos triploides foram transplantados com células recém isoladas e células criopreservadas. No transplante realizado em “juvenis” triploides foram visualizadas células PKH26 positivas até os 80 dias após transplante. Nos receptores híbridos adultos triploides, transplantados com células recém isoladas e criopreservadas, foram visualizadas espermatogônias se proliferando nos testículos dos receptores aos 20 dias após transplante. Os resultados obtidos de isolamento e criopreservação de espermatogônias-tronco e o posterior transplante via papila urogenital, utilizando os receptores “juvenis” triploides tratados com busulfan, e híbridos adultos triploides são inéditos, inovadores e promissores para obtenção de quimera germinativa funcional. Consequentemente, os procedimentos estabelecidos neste estudo poderão ser aplicados futuramente, em ações de conservação e reconstituição da piracanjuba, em caso de extinção.
The piracanjuba (Brycon orbignyanus) is an endangered species due to anthropic actions, such as riparian deforestation and the fragmentation of rivers. The association of biotechniques for the generation of in vitro genetic banks, constituted by primordial germ cells (PGCs) and germ lineage cells (spermatogonial and oogonial stem cells) with cell transplantation in sterile recipients, can be a strategy to be applied for the conservation and reconstitution of endangered species. This study aimed to establish the procedures for isolation and cryopreservation of the spermatogonia stem cells from piracanjuba, as well for the spermatogonia stem cells transplantation in sterile recipients, for the generation of germline chimera generation. Spermatogonial stem cells were isolated by enzymatic dissociation, Percoll density gradient, and differential plating, being reached a purity of 66 ± 0.01%. Spermatogonial stem cells from purification steps were characterized by relative ddx4 gene expression. A higher expression level, approximately 3.5 times, was observed in cells from the Percoll density gradient than in cells from differential plating and gonadal tissue. Therefore, cells from the Percoll density gradient were appointed for use in transplantation and in vitro cryopreservation procedures. The experiments of spermatogonial stem cell cryopreservation were performed and the best cryoprotectant observed was the 1 M propanediol solution (P = 1.000). Adult male diploid and juvenile diploid of Astyanax altiparanae were used in the experiments of chemical sterilization to generate sterile recipients. Another experiment was performed using juvenile triploid by the association of chromosomal manipulation and chemical sterilization. The association of hybridization between species and chromosomal manipulation was applied for the generation of triploid hybrids (A. altiparanae × A. fasciatus) as sterile recipients. In the experiments of sterilization, juvenile triploids and adult triploid hybrids were suggested as the best recipients for spermatogonial stem cells transplantation. “Juvenile” triploids treated with busulfan received transplantation of newly isolated spermatogonial stem cells, and adult triploid hybrids received transplantation of newly isolated cells and cryopreserved cells. In the transplant performed for “juvenile” triploids, PKH26-positive cells were visualized up to 80 days after transplantation. In adult triploid hybrids, both from newly isolated and cryopreserved cells, spermatogonia were visualized proliferating in the recipient's gonads 20 days after transplantation. Results from the isolation and cryopreservation of spermatogonial stem cells and the subsequent transplantation via the urogenital papilla in the sterile recipients, "juvenile" triploid and adult triploid hybrids, are unprecedented, innovative, and promissory for obtaining a functional germline chimera. Consequently, the procedures established in this study can be applied in the future in actions of conservation and reconstitution of piracanjuba in case of extinction.
The piracanjuba (Brycon orbignyanus) is an endangered species due to anthropic actions, such as riparian deforestation and the fragmentation of rivers. The association of biotechniques for the generation of in vitro genetic banks, constituted by primordial germ cells (PGCs) and germ lineage cells (spermatogonial and oogonial stem cells) with cell transplantation in sterile recipients, can be a strategy to be applied for the conservation and reconstitution of endangered species. This study aimed to establish the procedures for isolation and cryopreservation of the spermatogonia stem cells from piracanjuba, as well for the spermatogonia stem cells transplantation in sterile recipients, for the generation of germline chimera generation. Spermatogonial stem cells were isolated by enzymatic dissociation, Percoll density gradient, and differential plating, being reached a purity of 66 ± 0.01%. Spermatogonial stem cells from purification steps were characterized by relative ddx4 gene expression. A higher expression level, approximately 3.5 times, was observed in cells from the Percoll density gradient than in cells from differential plating and gonadal tissue. Therefore, cells from the Percoll density gradient were appointed for use in transplantation and in vitro cryopreservation procedures. The experiments of spermatogonial stem cell cryopreservation were performed and the best cryoprotectant observed was the 1 M propanediol solution (P = 1.000). Adult male diploid and juvenile diploid of Astyanax altiparanae were used in the experiments of chemical sterilization to generate sterile recipients. Another experiment was performed using juvenile triploid by the association of chromosomal manipulation and chemical sterilization. The association of hybridization between species and chromosomal manipulation was applied for the generation of triploid hybrids (A. altiparanae × A. fasciatus) as sterile recipients. In the experiments of sterilization, juvenile triploids and adult triploid hybrids were suggested as the best recipients for spermatogonial stem cells transplantation. “Juvenile” triploids treated with busulfan received transplantation of newly isolated spermatogonial stem cells, and adult triploid hybrids received transplantation of newly isolated cells and cryopreserved cells. In the transplant performed for “juvenile” triploids, PKH26-positive cells were visualized up to 80 days after transplantation. In adult triploid hybrids, both from newly isolated and cryopreserved cells, spermatogonia were visualized proliferating in the recipient's gonads 20 days after transplantation. Results from the isolation and cryopreservation of spermatogonial stem cells and the subsequent transplantation via the urogenital papilla in the sterile recipients, "juvenile" triploid and adult triploid hybrids, are unprecedented, innovative, and promissory for obtaining a functional germline chimera. Consequently, the procedures established in this study can be applied in the future in actions of conservation and reconstitution of piracanjuba in case of extinction.
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Palavras-chave
Manipulação cromossômica, Hibridação entre espécies, Esterilização química, Espécie ameaçada, Quimera germinativa, Chromosomal manipulation, Hybridization between species, Chemical sterilization, Endangered species, Germ chimera