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.

Biological characteristics of fish germ cells and their application to developmental biotechnology.

We have revealed several unique characteristics of germ cell development using rainbow trout, including the fact that spermatogonia transplanted into the peritoneal cavity of newly hatched embryos migrate toward recipient gonads, that spermatogonia transplanted into female recipients start oogenesis and produce functional eggs and that diploid germ cells transplanted into triploid trout can complete gametogenesis. By combining these unique features of fish germ cells, we established allogeneic and xenogeneic transplantation systems for spermatogonia in several fish species. Spermatogonia isolated from the mature testes of vasa-green fluorescent protein (Gfp) transgenic rainbow trout were transplanted into the peritoneal cavity of triploid masu salmon newly hatched embryos. These spermatogonia migrated toward recipient salmon genital ridges with extending pseudopodia and were subsequently incorporated into them. We further confirmed that the donor-derived spermatogonia resumed gametogenesis and produced sperm and eggs in male and female salmon recipients, respectively. By inseminating the resulting eggs and sperm, we obtained only rainbow trout offspring in the F1 generation, suggesting that the triploid salmon recipients produced functional gametes derived only from donor trout. We further confirmed that this intra-peritoneal transplantation of germ cells is applicable to several marine fishes, which could be of benefit in the production of bluefin tuna that has a large broodstock (>100 kg) and is difficult to maintain in captivity. Gamete production of bluefin tuna could be more easily achieved by generating a surrogate species, such as mackerel, that can produce tuna gametes.

[Measurement of prostaglandin E2 content in lung tissue of mouse].

A radioimmunoassay (RIA) was applied for the determination of PGE2 levels in the lung from 4 groups of male ICR mice 1) fed a standard diet, 2) fed a 20% corn oil-supplemented diet (HFD) for 2 weeks, 3) given a single s.c. injection of 15 mg/kg of 4NQO, a potent lung carcinogen and 4) given a s.c. injection of 4NQO and subsequently fed HFD for 2 weeks. Animals were sacrificed at week 3, and the lung was carefully excised and frozen in liquid nitrogen to prevent the postmortem synthesis of PGE2. PGE2, extracted from the lung tissue, was purified and then measured with or without adding a known amount of PGE2. The lung levels of PGE2 were shown to be significantly higher in the groups treated with HFD and/or 4NQO than the group fed a standard diet. These results show that the modified RIA method can be used for the measurement of PGE2 contents in the tissue of animals.

[Twenty-eight day repeated dose toxicity testing of diphenylamine in F344 rats].

A twenty-eight day repeated dose toxicity test of diphenylamine (DPA) was carried out in male and female F344 rats at dose levels of 1000, 333, 111 or 0 mg/kg/day. Thirty-six animals of both sexes were divided into 6 groups of equal number, 4 groups being used for the 28 days dosing study and the remainder for investigation of recovery. Inhibition of body weight gain, increase of liver, spleen and kidney weights, and anemia were observed in the highest dose groups in both sexes. The same groups demonstrated mucosal hyperplasia in the forestomach, dilatation, degeneration or necrosis of renal tubules in the corticomedullary junction, and hyperplasia in the bone marrow histopathologically. Slight increase of spleen, liver and kidney weights as well as slight degeneration of renal tubules were evident in several animals receiving the dose level of 333 mg/kg/day. Repair of histopathological lesions and anemia occurred within 14-day resting period. Based on these findings, under the present experimental conditions, the no observable effect level of DPA was 111 mg/kg/day.