RESUMEN
Although the use of germ cell transplantation has been relatively well established in mammals, the technique has only been adapted for use in fish after entering the 2000s. During the last decade, several different approaches have been developed for germ cell transplantation in fish using recipients of various ages and life stages, such as blastula-stage embryos, newly hatched larvae and sexually mature specimens. As germ cells can develop into live organisms through maturation and fertilization processes, germ cell transplantation in fish has opened up new avenues of research in reproductive biotechnology and aquaculture. For instance, the use of xenotransplantation in fish has lead to advances in the conservation of endangered species and the production of commercially valuable fish using surrogated recipients. Further, this could also facilitate the engineering of transgenic fish. However, as is the case with mammals, knowledge regarding the basic biology and physiology of germline stem cells in fish remains incomplete, imposing a considerable limitation on the application of germ cell transplantation in fish. Furthering our understanding of germline stem cells would contribute significantly to advances regarding germ cell transplantation in fish.
Asunto(s)
Acuicultura/métodos , Biotecnología/tendencias , Peces/fisiología , Células Germinativas/trasplante , Reproducción/fisiología , Técnicas Reproductivas Asistidas/veterinaria , Animales , Biotecnología/métodos , Embrión no Mamífero/fisiologíaRESUMEN
The sexual plasticity of fish gonads declines after the sex-differentiation period; however, the plasticity of the germ cells themselves after this stage remains poorly understood. We characterized the sexual plasticity of gonial germ cells by transplanting them into sexually undifferentiated embryonic gonads in rainbow trout (Oncorhynchus mykiss). Spermatogonia or oogonia isolated from the meiotic gonads of vasa-green fluorescent protein (Gfp) gene transgenic trout were transplanted into the peritoneal cavity of newly hatched embryos of both sexes, and the behavior of the GFPlabeled donor cells was observed. The transplanted spermatogonia and oogonia migrated towards the recipient gonadal anlagen, and were subsequently incorporated into them. We also confirmed that the donor-derived gonial germ cells resumed gametogenesis in the recipient somatic microenvironment synchronously with the endogenous germ cells. Surprisingly, the donor-derived spermatogonia started to proliferate and differentiate into oocytes in female recipients. At 2 years post-transplantation, the eggs from mature female recipients were artificially inseminated with sperm from intact male rainbow trout. Normal, live offspring with the donor-derived haplotype were obtained. In addition, oogonia-derived sperm were produced in the male recipients. These donor-derived sperm were shown to be fully functional, as live offspring carrying GFP-labeled germ cells with the donor haplotype were obtained in the first filial (F1) generation. These findings indicate that rainbow trout pre-meiotic germ cells, which are likely to be spermatogonial or oogonial stem cells, possess a high level of sexual plasticity, and that the sexual differentiation of germ cells is controlled solely by the somatic microenvironment, rather than being cell autonomous.
Asunto(s)
Animales , Diferenciación Sexual/fisiología , Espermatogonias/crecimiento & desarrollo , Oogonios/crecimiento & desarrollo , Trasplante de Células/métodos , Trasplante de Células/veterinaria , Oncorhynchus mykiss/crecimiento & desarrollo , Trasplante Heterólogo/efectos adversosRESUMEN
The sexual plasticity of fish gonads declines after the sex-differentiation period; however, the plasticity of the germ cells themselves after this stage remains poorly understood. We characterized the sexual plasticity of gonial germ cells by transplanting them into sexually undifferentiated embryonic gonads in rainbow trout (Oncorhynchus mykiss). Spermatogonia or oogonia isolated from the meiotic gonads of vasa-green fluorescent protein (Gfp) gene transgenic trout were transplanted into the peritoneal cavity of newly hatched embryos of both sexes, and the behavior of the GFPlabeled donor cells was observed. The transplanted spermatogonia and oogonia migrated towards the recipient gonadal anlagen, and were subsequently incorporated into them. We also confirmed that the donor-derived gonial germ cells resumed gametogenesis in the recipient somatic microenvironment synchronously with the endogenous germ cells. Surprisingly, the donor-derived spermatogonia started to proliferate and differentiate into oocytes in female recipients. At 2 years post-transplantation, the eggs from mature female recipients were artificially inseminated with sperm from intact male rainbow trout. Normal, live offspring with the donor-derived haplotype were obtained. In addition, oogonia-derived sperm were produced in the male recipients. These donor-derived sperm were shown to be fully functional, as live offspring carrying GFP-labeled germ cells with the donor haplotype were obtained in the first filial (F1) generation. These findings indicate that rainbow trout pre-meiotic germ cells, which are likely to be spermatogonial or oogonial stem cells, possess a high level of sexual plasticity, and that the sexual differentiation of germ cells is controlled solely by the somatic microenvironment, rather than being cell autonomous.(AU)
Asunto(s)
Animales , Diferenciación Sexual/fisiología , Trasplante de Células/métodos , Trasplante de Células/veterinaria , Oogonios/crecimiento & desarrollo , Espermatogonias/crecimiento & desarrollo , Oncorhynchus mykiss/crecimiento & desarrollo , Trasplante Heterólogo/efectos adversosRESUMEN
The Nile tilapia (Oreochromis niloticus) stands out as one of the most important fresh water edible fish, possessing remarkable characteristics that make it desirable for both commercial culture and as a laboratory model. For the utilization of tilapia in germ cell transplantation experiments, appropriate cell markers are required to evaluate the colonization behavior of donor-derived germ cells in recipient gonads. Here we report the production of a medaka β-actin/EGFP transgenic tilapia strain expressing the green fluorescent protein (GFP) reporter gene in several tissues including germ cells in testis and ovary. Fluorescent observations in F2 generation transgenic individuals showed GFP positive cells along the body axis in pre-hatched embryos, while in hatching embryos the GFP gene was strongly expressed in the area surrounding the gills, operculum and in the cephalic region. In early larvae, fluorescent cells were scattered throughout the body, forming aggregations around the dorsal-cephalic and mouth areas. At 38 days post-fertilization, juvenile fish expressed the GFP homogeneously in the whole body. GFP fluorescence was also observed in caudal fins, muscle, and in several internal organs (gills, heart, testes, and ovaries) in 140 and 240 day F2 and F3 individuals. Immunohistochemistry using a monoclonal anti-GFP antibody in juvenile and adult gonads showed that both mitotic and meiotic germ cells were labeled with GFP. The utilization of this transgenic line in a germ cell transplantation system could offer a fast and reliable screening of donor-derived transgenic offspring, as well as accurate tracing of donor-derived cell colonization in the recipient gonad by means of immunohistochemistry using GFP antibodies. In the future, germ cell transplantation using Nile tilapia also could help to preserve the genetic resources of threatened cichlids, through cryopreservation and interspecies transplantation of germ cells from endangered cichlids into O. niloticus recipients.
Asunto(s)
Animales , Células Germinativas/citología , Proteínas/análisis , Trasplante/métodos , Peces/clasificación , TilapiaRESUMEN
The Nile tilapia (Oreochromis niloticus) stands out as one of the most important fresh water edible fish, possessing remarkable characteristics that make it desirable for both commercial culture and as a laboratory model. For the utilization of tilapia in germ cell transplantation experiments, appropriate cell markers are required to evaluate the colonization behavior of donor-derived germ cells in recipient gonads. Here we report the production of a medaka β-actin/EGFP transgenic tilapia strain expressing the green fluorescent protein (GFP) reporter gene in several tissues including germ cells in testis and ovary. Fluorescent observations in F2 generation transgenic individuals showed GFP positive cells along the body axis in pre-hatched embryos, while in hatching embryos the GFP gene was strongly expressed in the area surrounding the gills, operculum and in the cephalic region. In early larvae, fluorescent cells were scattered throughout the body, forming aggregations around the dorsal-cephalic and mouth areas. At 38 days post-fertilization, juvenile fish expressed the GFP homogeneously in the whole body. GFP fluorescence was also observed in caudal fins, muscle, and in several internal organs (gills, heart, testes, and ovaries) in 140 and 240 day F2 and F3 individuals. Immunohistochemistry using a monoclonal anti-GFP antibody in juvenile and adult gonads showed that both mitotic and meiotic germ cells were labeled with GFP. The utilization of this transgenic line in a germ cell transplantation system could offer a fast and reliable screening of donor-derived transgenic offspring, as well as accurate tracing of donor-derived cell colonization in the recipient gonad by means of immunohistochemistry using GFP antibodies. In the future, germ cell transplantation using Nile tilapia also could help to preserve the genetic resources of threatened cichlids, through cryopreservation and interspecies transplantation of germ cells from endangered cichlids into O. niloticus recipients.(AU)