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.

Viability of homologous and heterologous subcutaneous transplantation of fresh germ cells in rabbits.

OBJECTIVE: This study aimed to compare heterologous to homologous transplantation of fresh ovarian germ cells in rabbits. METHODS: Twelve female white New Zealand rabbits (Oryctolagus cuniculus) were randomly numbered and submitted to bilateral oophorectomies. The ovaries from the six odd-numbered rabbits were dissected and cortical germinal tissue was digested in collagenase type 1 to obtain six solutions containing stromal and germ cells, which were injected in the abdominal region of the odd-numbered rabbits themselves (homologous transplantation) and of the even-numbered rabbits (heterologous transplantation) off immunosuppression. Sixty days after transplantation, the tissue around the transplanted region was excised, processed and sent to histological analysis with hematoxylin-eosin staining and Bcl-2 immunohistochemistry to verify the presence and viability of the transplanted cells. RESULTS: The analyzed specimens contained ovarian stroma, while follicular cells were found in 66.6% of the homologous and in 60% of the heterologous transplant specimens. Mild inflammatory reaction was observed in all heterologous specimens, and in only one (16.7%) of the homologous specimens. However, this inflammatory reaction was not so intense as to cause the death of the implanted cells. Except for the specimens from rabbits 7 and 8, all specimens were stained for Bcl-2, indicating that most of them were viable. CONCLUSIONS: The results of this study supported the viability of heterologous transplantation of fresh ovarian germ cells. However, more studies are required to further our understanding and improve the germ cell separation technique.

Germ cell transplantation as a potential biotechnological approach to fish reproduction.

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.

An overview of functional and stereological evaluation of spermatogenesis and germ cell transplantation in fish.

Although there are almost thirty-thousand species of fish living in a great variety of habitats and utilizing vast reproductive strategies, our knowledge of morphofunctional and quantitative aspects of testis structure and spermatogenesis is still incipient for this group of vertebrates. In this review, we discuss aspects that are important to better understanding of testis structure and function, and of the development of germ cells (GC) during spermatogenesis. To achieve this, we have recently completed a number of studies presenting morphometric and functional data related to the numbers of GC and Sertoli cells (SC) per each type of spermatogenic cyst, the number of spermatogonial generations, the SC efficiency, and the magnitude of GC loss that normally occurs during spermatogenesis. We also investigated SC proliferation and the relationship of this important event to early spermatogenic cysts. The available data strongly suggest that SC proliferation in sexually mature tilapia is the primary factor responsible for the increase in testis size and for determination of the magnitude of sperm production. The influence of temperature on the duration of spermatogenesis in tilapia was also evaluated and we have used this knowledge to deplete endogenous spermatogenesis in this teleost, in order to develop an experimental system for GC transplantation. This exciting technique results in new possibilities for investigation of spermatogenesis and spermatogonial stem cell biology, creating also an entirely new and promising scenario in biotechnology-transgenic animal production and the preservation of the genetic stocks of valuable animals or endangered species.

Germinative testicular cells and bone marrow mononuclear cells transplanted to a rat model of testicular degeneration.

The aim of this study was to evaluate the ability of rat mononuclear bone marrow cells to recover testis cell associations and multiplication in busulfan-treated rats, and to compare these data to germinative testicular cell transplant. The germinative testicular cells were obtained by the trypsin digestion method, and bone marrow cells were harvested from femurs and tibias, and purified using by Ficoll gradient. Cell transplantation was performed by the injection of cells through the efferent ducts into the rete testis in busulfan-treated animals. Fifteen days after transplantation, the recipient rats were sacrificed and the testes were collected and analyzed by histology (hematoxilin-eosin and DAPI staining). Results demonstrated that germ cells transplantation promoted cellular reorganization of seminiferous epithelium 15 days later. On the other hand, no improvement in spermatogenesis regeneration was found after heterologous mononuclear bone marrow cell transplantation.

Spermatogonial Transplantation in Mammals

Spermatogonial transplantation from mouse-to-mouse was first reported by Brinster and colleagues in 1994. Since then, many important developments in this fascinating methodology such as interspecies transplants, transplants from cryopreserved and cultured spermatogonial stem cells have been made. This technique has been shown a valuable tool to study the biology of spermatogonial stem cells. Also, important functional questions regarding Sertoli-germ cell interactions and the role of the Sertoli cell and germ cells during spermatogenesis have now been answered. Transplantation of cultured spermatogonial stem cells is now opening exciting possibilities for in vitro multiplication and manipulation of male germ line cells. Spermatogonial stem cells can be considered "immortal". By freezing and storing testicular tissue, it should be possible to preserve indefinitely the genetic stocks of valuable farm animals, endangered species and uniqueexperimental animals, until a suitable recipient can be found that will maintain the germ line. Transplantation of spermatogonia has also potential clinical application to address human infertility. Overall, spermatogonial transplantation has been proved to be an extraordinary and powerful technique to investigate reproductive biology.