Explore the vast range of titles available.

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.

Understanding the mechanisms that regulate germ-cell development is crucial to reproductive medicine and animal production. Animal gametes originally derive from sexually undifferentiated primordial germ cells (PGCs), which develop into mitotic germ cells (oogonia or spermatogonia) before proceeding to meiosis [Wylie, C. (1999) Cell 96, 165-174]. Spermatogonia are thought to include a population of cells with stem cell activity, which proliferate throughout the lifespan of male animals and produce spermatozoa [Zhao, G. Q. & Garbers, D. L. (2002) Dev. Cell 2, 537-547]. However, the functional differences between PGCs and spermatogonial stem cells are poorly understood. Here we show that transplanted adult testicular germ cells can colonize sexually undifferentiated embryonic gonads and resume gametogenesis. Testicular germ cells containing spermatogonial stem cells isolated from adult male rainbow trout (Oncorhynchus mykiss) were transplanted into the peritoneal cavity of newly hatched embryos of both sexes, and the behavior of the donor cells was observed. The testicular germ cells differentiated into spermatozoa in male recipients and fully functional eggs in female recipients. Furthermore, the donor-derived spermatozoa and eggs obtained from the recipient fish were able to produce normal offspring. These findings indicate that fish testicular germ cells, probably spermatogonial stem cells, possess a high level of developmental plasticity and sexual bipotency, even after the animal reaches maturity. Furthermore, our results suggest that spermatogonial stem cells are at least partly functionally similar to PGCs.

A worldwide decline in the number of wild salmonids calls for strategies to restore endangered populations. Here we show that germ cells can be transplanted between two different salmonid species, with the subsequent production of xenogenic, donor-derived offspring. This pioneering xenotransplantation technology may eventually find applications in facilitating the production of commercially valuable fish, as well as in species conservation.

If in vitro-cultured cells could be converted into individual fish, they would have numerous applications in the field of genome biology and biotechnology. In spite of the benefits, however, no such system is presently available. Because primordial germ cells (PGCs) have the potential to be converted into individual fish via maturation and fertilization processes, we chose them to be the starting material for our system. As the first step, we visualized live PGCs in rainbow trout. Because the vasa transcript is restricted to the germ cell lineage, its regulatory regions are activated only in PGCs. Therefore, we produced transgenic strains carrying the green fluorescent protein (GFP) gene driven by the vasa gene regulatory regions. The resulting transgenic embryos showed green fluorescence specifically in PGCs. As the second step, the GFP-labeled PGCs were purified by flow cytometry. The genital ridges isolated from the transgenic embryos were dissociated by trypsin and sorted into GFP-positive and GFP-negative cells. The GFP-positive cells possessed morphological characteristics typical of PGCs. In addition, the vasa gene was expressed only in the GFP-positive cells, confirming that they were PGCs. To obtain functional gametes derived from isolated PGCs, a technique for converting PGCs into eggs and sperm is necessary. For this purpose, we developed a method for transplanting PGCs into developing embryos in order to incorporate them into the germ cell lineage of the recipient embryos. Approximately 10 PGCs isolated from newly hatched embryos were transplanted into the peritoneal cavity of recipient hatchlings. The transplanted PGCs actively migrated towards, and were finally incorporated into, the genital ridge. The PGCs that settled in the genital ridges of the recipient embryos proliferated, started meiosis, and differentiated into eggs and sperm in synchrony with the germ cells of allogenic recipient embryos. Further, the donor-derived gametes produced normal progenies through fertilization. These techniques, in combination with in vitro culture, genetic modification, and cryopreservation of PGCs, are expected to have numerous applications in the field of fish bioengineering.

A method for cryopreserving trout primordial germ cells (PGCs) was established. Trout PGCs were successfully cryopreserved with high survival by freezing with medium containing 1.8 M ethylene glycol. Transplantation experiment revealed that frozen/thawed PGCs colonized and proliferated in the recipients' gonads.

Albinism in animals is generally a recessive trait, but in Japan a dominant oculocutaneous albino (OCA) mutant strain has been isolated in rainbow trout (Oncorhyncus mykiss). After confirming that this trait is not due to a tyrosinase gene mutation that causes OCA1 (tyrosinase-negative OCA), we combined the amplified fragment length polymorphism (AFLP) technique with bulked segregant analysis (BSA) to map the gene involved in dominant oculocutaneous albinism. Four AFLP markers tightly linked to the dominant albino locus were identified. One of these markers was codominant and we have it converted into a GGAGT-repeat microsatellite marker, OmyD-AlbnTUF. Using this pentanucleotide-repeat DNA marker, the dominant albino locus has been mapped on linkage group G of a reference linkage map of rainbow trout. The markers identified here will facilitate cloning of the dominant albino gene in rainbow trout and contribute to a better understanding of tyrosinase-negative OCA in animals.

Luteinizing hormone- (LH)-dependent ovarian follicle maturation has been recently described in two stages for teleost fishes. The oocyte's ability to respond to the steroidal maturation-inducing hormone (MIH), also known as oocyte maturational competence (OMC), is acquired during the first stage+ADs- whereas the MIH-dependent resumption of meiosis occurs during the second stage. However, studies directly addressing OMC have been performed with a limited number of species and therefore the general relevance of the two-stage model and its mechanisms remain uncertain. In this study, we examined the hormonal regulation of OMC and its basic transduction mechanisms in ovarian follicles of the sciaenid teleost, Nibe (Nibea mitsukurii). Exposure to MIH +AFs-17,20+ACY-bgr+ADs--dihydroxy-4-pregnen-3-one or 17,20+ACY-bgr+ADs-,21-trihydroxy-4-pregnen-3-one] stimulated germinal vesicle breakdown (index of meiotic resumption) in full-grown follicles primed with human chorionic gonadotropin (HCG, an LH-like gonadotropin) but not in those pre-cultured in plain incubation medium. The induction of OMC by HCG was mimicked by protein kinase A (PKA) activators (forskolin and dibutyryl cyclic AMP), and blocked by specific inhibitors of PKA (H89 and H8) as well as inhibitors of RNA (actinomycin D) and protein (cycloheximide) synthesis. Forskolin-induced OMC was also inhibited by actinomycin D and cycloheximide. A strong activator of protein kinase C, PMA, inhibited HCG-dependent OMC. In conclusion, OMC in Nibe ovarian follicles is gonadotropin-dependent and requires activation of the PKA pathway followed by gene transcription and translation events. These observations are consistent with the two-stage model of ovarian follicle maturation proposed for other teleosts, and suggest that Nibe can be used as new model species for mechanistic studies of ovarian follicle differentiation and maturation in fishes.[PUBLICATION ABSTRACT]

The feasibility of using rainbow trout Oncorhynchus mykiss embryos as an expression system for proteins was investigated. For model proteins, we selected two goldfish gonadotropins (GTHs), follicle-stimulating hormone (FSH) and luteinizing hormone (LH). To produce single-chain goldfish FSH (scgfFSH) and LH (scgfLH), cDNAs encoding glycoprotein hormone (GP) alpha and FSH beta were fused in tandem, and cDNAs encoding GP alpha and LH beta were fused in tandem. The fused cDNAs were ligated with beta -actin promoter, and microinjected into fertilized rainbow trout eggs. After 4-days incubation, the embryos were subjected to western blotting and in vitro bioassays. The recombinant proteins produced by the embryos were immunoreactive to antisera against goldfish GP alpha , N-glycosylated, and biologically active. We conclude that scgfFSH and scgfLH were successfully produced in transgenic rainbow trout.

In this study, a Pacific bluefin tuna (Thunnus orientalis) homolog of the Drosophila vasa gene, BtVLG (bluefin tuna vasa-like gene), was cloned and characterized for use as a molecular marker for germ cells in this species. Analysis of the nucleotide sequence revealed that BtVLG comprises 2,394 bps with an open reading frame of 1,932 bps encoding 644 amino acids. The deduced amino acid sequence contained arginine-glycine or arginine-glycine-glycine motifs and eight conserved motifs belonging to the DEAD-box protein family. The BtVLG sequence showed high similarity to Drosophila vasa (69.1%), zebrafish vasa homolog (80.5%), and tilapia vasa homolog (91.2%). In adult tissues, the BtVLG transcripts were specifically detected in ovary and testis. In situ hybridization analysis showed that BtVLG messenger RNA (mRNA) was detected in oogonia and previtellogenic oocytes in the ovary. In the testis, while BtVLG mRNA was detected in spermatogonia, it was not detected in the primary and secondary spermatocytes, spermatids, spermatozoa or gonadal somatic cells. Consequently, consensus sequences, sequence similarity, and specific localization of BtVLG mRNA in the germ cells all suggest that BtVLG is the bluefin tuna vasa homolog of the Drosophila vasa gene. Further, BtVLG can be used as a molecular marker for bluefin tuna germ cells.

Recombinant gonadotropins (GTH), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) of goldfish Carassius auratus were produced by baculovirus in silkworm larvae. Hemolymph containing recombinant FSH (rFSH) or LH (rLH) was collected from silkworm larvae, and its biological activity was examined in vivo using male goldfish and female bitterling Rhodeus ocellatus ocellatus. Injection of hemolymph containing rFSH or rLH induced milt production in male goldfish, whereas only rLH induced ovulation in female bitterling. These results suggest, that biologically active goldfish recombinant GTH could be applied for the induction of gonadal development in aquaculture fishes as a substitute of pituitary extract, if a method of large scale production is established.