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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.

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.

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.

It has been controversial whether female germline stem cells (FGSCs) are present in postnatal mammalian ovaries. Cells from a line derived from FGSCs isolated from adult mice that were transplanted into ovaries of infertile animals underwent oogenesis and generated offspring. The idea that females of most mammalian species have lost the capacity for oocyte production at birth 1 , 2 , 3 , 4 , 5 has been challenged recently by the finding that juvenile and adult mouse ovaries possess mitotically active germ cells 6 . However, the existence of female germline stem cells (FGSCs) in postnatal mammalian ovaries still remains a controversial issue among reproductive biologists and stem cell researchers 6 , 7 , 8 , 9 , 10 . We have now established a neonatal mouse FGSC line, with normal karyotype and high telomerase activity, by immunomagnetic isolation and culture for more than 15 months. FGSCs from adult mice were isolated and cultured for more than 6 months. These FGSCs were infected with GFP virus and transplanted into ovaries of infertile mice. Transplanted cells underwent oogenesis and the mice produced offspring that had the GFP transgene. These findings contribute to basic research into oogenesis and stem cell self-renewal and open up new possibilities for use of FGSCs in biotechnology and medicine.

Spermatogonial stem cells (SSCs) are the most primitive spermatogonia in the testis and have an essential role to maintain highly productive spermatogenesis by self-renewal and continuous generation of daughter spermatogonia that differentiate into spermatozoa, transmitting genetic information to the next generation. Since the 1950s, many experimentalmethods, including histology, immunostaining, whole-mount analyses, and pulse-chase labeling, had been used in attempts to identify SSCs, but without success. In 1994, a spermatogonial transplantation method was reported that established a quantitative functional assay to identify SSCs by evaluating their ability to both self-renew and differentiate to spermatozoa. The system was originally developed using mice and subsequently extended to nonrodents, including domestic animals and humans. Availability of the functional assay for SSCs has made it possible to develop culture systems for their ex vivo expansion, which dramatically advanced germ cell biology and allowed medical and agricultural applications. In coming years, SSCs will be increasingly used to understand their regulation, as well as in germline modification, including gene correction, enhancement of male fertility, and conversion of somatic cells to biologically competent male germline cells.

Sterility in hybrid animals is widely known to be due to a cytological mechanism of aberrant homologous chromosome pairing during meiosis in hybrid germ cells. In this study, the gametes of four marine fish species belonging to the Sciaenid family were artificially fertilized, and germ cell development was examined at the cellular and molecular levels. One of the intergeneric hybrids had gonads that were testis-like in structure, small in size, and lacked germ cells. Specification of primordial germ cells (PGCs) and their migration toward genital ridges occurred normally in hybrid embryos, but these PGCs did not proliferate in the hybrid gonads. By germ cell transplantation assay, we showed that the gonadal microenvironment in hybrid recipients produced functional donor-derived gametes, suggesting that the germ cell-less phenotype was caused by cell autonomous proliferative defects of hybrid PGCs. This is the first evidence of mitotic arrest of germ cells causing hybrid sterility in animals.

In germ cell transplantation experiments, the use of sterile recipients that do not produce their own gametes is an important prerequisite. Triploidization and dnd gene knockdown (KD) methods have been widely used to produce sterile fish. However, triploidization does not produce complete sterility in some fish species, and gene KD is labor and time intensive since it requires microinjection into individual fertilized eggs. To overcome these problems, in this study, we generated homozygous mutants of the dead end (dnd) gene in rainbow trout (Oncorhynchus mykiss) using the clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, analyzed their reproductive capacity, and evaluated their suitability as recipients for germ cell transplantation. By crossing F1 heterozygous mutants produced from founders subjected to genome editing, an F2 generation consisting of approximately 1/4 homozygous knockout mutants (dnd KO) was obtained. The dnd KO hatchlings retained the same number of primordial germ cells (PGCs) as the wild-type (WT) individuals, after which the number gradually decreased. At 1 year of age, germ cells were completely absent in all analyzed individuals. To evaluate the dnd KO individuals as recipients for germ cell transplantation, germ cells prepared from donor individuals were transplanted into the abdominal cavity of dnd KO hatchlings. These cells migrated to the recipient gonads, where they initiated gametogenesis. The mature recipient individuals produced only donor-derived sperm and eggs in equivalent numbers to WT rainbow trout. These results indicate that dnd KO rainbow trout are suitable recipient candidates possessing a high capacity to nurse donor-derived germ cells.

Many freshwater fish face extinctions due to habitat destruction and overfishing. Bitterlings, which lay their eggs in the gill chambers of freshwater mussels, are in steep decline worldwide, along with mussels, owing to the concreting and siltation of riverbeds. In particular, Tokyo bitterling is listed as “endangered” on the Red List and is on the verge of extinction. Although in situ and ex situ conservation efforts are underway, their effectiveness is limited. Here, we developed a method for long-term preservation of genetic resources by combining germ cell cryopreservation and transplantation. We transplanted Tokyo bitterling germ cells stored in liquid nitrogen into oily bitterling recipients lacking endogenous germ cells due to dnd knockdown, and the recipients matured and repeatedly produced many eggs and sperm. The resulting offspring had the Tokyo bitterling genotype and morphology. This study shows that even extinct species can be revived if their germ cell bank is available.

Murine animal models from genetically modified pluripotent stem cells (PSCs) are essential for functional genomics and biomedical research, which require germline transmission for the establishment of colonies. However, the quality of PSCs, and donor-host cell competition in chimeras often present strong barriers for germline transmission. Here, we report efficient germline transmission of recalcitrant PSCs via blastocyst complementation, a method to compensate for missing tissues or organs in genetically modified animals via blastocyst injection of PSCs. We show that blastocysts from germline-deficient Prdm14 knockout rats provide a niche for the development of gametes originating entirely from the donor PSCs without any detriment to somatic development. We demonstrate the potential of this approach by creating PSC-derived Pax2/Pax8 double mutant anephric rats, and rescuing germline transmission of a PSC carrying a mouse artificial chromosome. Furthermore, we generate mouse PSC-derived functional spermatids in rats, which provides a proof-of-principle for the generation of xenogenic gametes in vivo. We believe this approach will become a useful system for generating PSC-derived germ cells in the future. The uptake of donor pluripotent stem cells (PSCs) in hosts of different species and subsequent germline transmission is very inefficient. Here, the authors show, using Prdm14 gene depleted rat host blastocysts to remove functional sperm, that germline transmission from donor rat or mouse PSCs is possible.

Zebra finch is a unique model for behavioral, neural, and genomic studies of vocal learning. Several transgenic zebra finches have been produced, although the germline transmission efficiencies are reportedly low. Recently, there have been attempts to produce germline chimeras using primordial germ cells (PGCs). However, this has been hampered by difficulties associated with the manipulation of the small eggs and the fact that the zebra finch is an altricial species that requires parental care after birth, unlike precocial chickens. Consequently, it is difficult to transplant PGCs into embryos and maintain the chimeras. Here, we developed a busulfan-mediated system for transplantation of PGCs into adult testes, to produce germline chimeras with an improved germline transmission capacity. We established microsomal glutathione-S-transferase II (MGSTII)-overexpressing PGCs that are resistant to busulfan, which induces germ cell-specific cytotoxicity, and transplanted them into testes rendered temporarily infertile by busulfan. The recipients were given a second dose of busulfan to deplete endogenous germ cells and enrich the transplanted cells, and donor cell-derived spermatogenesis was accomplished. This method requires fewer recipients due to higher survival rates, and there is no need to wait for maturation of the founders, which is required when transplanting PGCs into embryos. These results are expected to improve transgenic zebra finch production. Summary Sentence Transplantation of busulfan-resistant PGCs into the adult testes and enrichment of donor cells by eliminating endogenous germ cells with busulfan treatment of recipients improved the efficiency of germline chimera production in songbirds.