Biotechnology applied to fish reproduction: tools for conservation.

This review discusses the new biotechnological tools that are arising and promising for conservation and enhancement of fish production, mainly regarding the endangered and the most economically important species. Two main techniques, in particular, are available to avoid extinction of endangered fish species and to improve the production of commercial species. Germ cell transplantation technology includes a number of approaches that have been studied, such as the transplantation of embryo-to-embryo blastomere, embryo-to-embryo differentiated PGC, larvae to larvae and embryo differentiated PGC, transplantation of spermatogonia from adult to larvae or between adults, and oogonia transplantation. However, the success of germ cell transplantation relies on the prior sterilization of fish, which can be performed at different stages of fish species development by means of several protocols that have been tested in order to achieve the best approach to produce a sterile fish. Among them, fish hybridization and triploidization, germline gene knockdown, hyperthermia, and chemical treatment deserve attention based on important results achieved thus far. This review currently used technologies and knowledge about surrogate technology and fish sterilization, discussing the stronger and the weaker points of each approach.

Triploid or hybrid tetra: Which is the ideal sterile host for surrogate technology?

This work was aimed at developing an effective procedure to obtain sterile ideal host fish in mass scale with no endogenous germ cells in the germinal epithelium, owning permanent stem-cell niches able to be colonized by transplanted germ cells in surrogate technology experiments. Thus, triploids, diploid hybrids, and triploid hybrids were produced. To obtain hybrid offspring, oocytes from a single Astyanax altiparanae female were inseminated by sperm from five males (A. altiparanae, A. fasciatus, A. schubarti, Hyphessobrycon anisitsi, and Oligosarcus pintoi). Triploidization was conducted by inhibition of the second polar body release using heat shock treatment at 40 °C for 2 min. At 9-months of age, the offspring from each crossing was histologically evaluated to access the gonadal status of the fish. Variable morphological characteristics of the gonads were found in the different hybrids offspring: normal gametogenesis, gametogenesis without production of gametes, sterile specimens holding germ cells, and sterile specimens without germ cells, which were considered "ideal hosts". However, only in the hybrid derived from crossing between A. altiparanae and A. fasciatus, 100% of the individuals were completely sterile. Among them 83.3% of the male did not present germ cells inside germinal epithelium, having only somatic cells in the gonad. The other 16.7% also presented spermatogonia inside the niches. Such a methodology allows the production of sterile host in mass scale, opening new insights for application of surrogate technologies.

Sexual plasticity of rainbow trout germ cells

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)

Sexual plasticity of rainbow trout germ cells

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.

Biomanipulation of bovine spermatogonial stem cells

The field of spermatogonial stem cell (SSC) technologies provides tools for genetic improvement of cattle herds and multiple opportunities for research. Spermatogonial stems cells belong to the male germ line and as such have high developmental potential, which offers many possibilities for transfer of relevant genetic traits across herds in a timely manner. Type A spermatogonia include a very small number of SSCs and their more numerous differentiating daughter cells. Initial attempts to isolate SSCs started with the isolation of type A spermatogonia and SSC purification. Type A spermatogonia can be obtained in large numbers from young prepubertal bulls, and it is important to note that there are breed differences. Type A spermatogonia isolation can be achieved through mechanical dissociation and enzymatic digestion of the testicular tissue followed by two purification steps, with a final typical bovine type A spermatogonia suspension of 70%. An evaluation for SSC activity using a transplantation assay adapted for bovine SSCs is described. Bovine Type A spermatogonia can be maintained in vitro for short periods (7 to 15 days) with simple culture conditions. However, expansion of SSC can only be achieved under certain conditions such as a specially supplemented medium, specific growth factors, and serial sub-culturing for longer periods of time. After expansion, bovine spermatogonia can be cryopreserved while retaining the ability to proliferate and survive. Despite all the challenges with development of SSC technologies, many questions arise focusing on how bovine SSCs work in a biotechnological setting. Progress in this field will probably result in new applications not only for bulls but also for other species with economical or ecological impact.(AU)

Biomanipulation of bovine spermatogonial stem cells

The field of spermatogonial stem cell (SSC) technologies provides tools for genetic improvement of cattle herds and multiple opportunities for research. Spermatogonial stems cells belong to the male germ line and as such have high developmental potential, which offers many possibilities for transfer of relevant genetic traits across herds in a timely manner. Type A spermatogonia include a very small number of SSCs and their more numerous differentiating daughter cells. Initial attempts to isolate SSCs started with the isolation of type A spermatogonia and SSC purification. Type A spermatogonia can be obtained in large numbers from young prepubertal bulls, and it is important to note that there are breed differences. Type A spermatogonia isolation can be achieved through mechanical dissociation and enzymatic digestion of the testicular tissue followed by two purification steps, with a final typical bovine type A spermatogonia suspension of 70%. An evaluation for SSC activity using a transplantation assay adapted for bovine SSCs is described. Bovine Type A spermatogonia can be maintained in vitro for short periods (7 to 15 days) with simple culture conditions. However, expansion of SSC can only be achieved under certain conditions such as a specially supplemented medium, specific growth factors, and serial sub-culturing for longer periods of time. After expansion, bovine spermatogonia can be cryopreserved while retaining the ability to proliferate and survive. Despite all the challenges with development of SSC technologies, many questions arise focusing on how bovine SSCs work in a biotechnological setting. Progress in this field will probably result in new applications not only for bulls but also for other species with economical or ecological impact.