[Toxicity of Takifugu exascurusCollected from the Sea of Kumano].

Marine pufferfish Takifugu exascurus is not approved for human consumption due to the lack of information on its toxicity. To clarify the toxicity of T. exascurus, ten live specimens were collected from the Sea of Kumano, Japan, and the toxicity and tetrodotoxin (TTX) concentration were determined using mouse bioassay and high performance liquid chromatography-fluorescence detection (HPLC-FLD), respectively. Toxicity was observed in the skin, liver, and ovaries, but the testes and muscle were non-toxic (<10 MU/g). On the other hand, HPLC-FLD revealed that TTX was detected in the muscle in two of the 10 specimens (1.4 and 1.5 MU/g). Based on the results, TTX is the main toxic component contributing to toxicity in T. exascurus.

Production of donor-derived eggs after ovarian germ cell transplantation into the gonads of adult, germ cell-less, triploid hybrid fish†.

In animals, spermatogonial transplantation in sterile adult males is widely developed; however, despite its utility, ovarian germ cell transplantation is not well developed. We previously showed that the interspecific hybrid offspring of sciaenid was a suitable model for germ cell transplantation studies as they have germ cell-less gonads. However, all these gonads have testis-like characteristics. Here, we tested whether triploidization in hybrid embryos could result in germ cell-less ovary development. Gonadal structure dimorphism and sex-specific gene expression patterns were examined in 6-month-old triploid hybrids (3nHybs). Thirty-one percent of 3nHybs had germ cell-less gonads with an ovarian cavity. cyp19a1a and foxl2, ovarian differentiation-related genes, were expressed in these gonads, whereas dmrt1 and vasa were not expressed, suggesting ovary-like germ cell-less gonad development. Some (26%) 3nHybs had testis-like germ cell-less gonads. Ovarian germ cells collected from homozygous green fluorescent protein (GFP) transgenic blue drum (BD) (Nibea mitsukurii) were transplanted into 6-month-old 3nHybs gonads via the urogenital papilla or oviduct. After 9 months, the recipients were crossed with wild type BD. Among the six 3nHyb recipients that survived, one female and one male produced fertile eggs and motile sperm carrying gfp-specific DNA sequences. Progeny tests revealed that all F1 offspring possessed gfp-specific DNA sequences, suggesting that these recipients produced only donor-derived eggs or sperm. Histological observation confirmed donor-derived gametogenesis in the 3nHyb recipients' gonads. Overall, triploidization reduces male-biased sex differentiation in germ cell-less gonads. We report, for the first time, donor-derived egg production in an animal via direct ovarian germ cell transplantation into a germ cell-less ovary.

Germ cell-less hybrid fish: ideal recipient for spermatogonial transplantation for the rapid production of donor-derived sperm†.

An interspecific hybrid marine fish that developed a testis-like gonad without any germ cells, i.e., a germ cell-less gonad, was produced by hybridizing a female blue drum Nibea mitsukurii with a male white croaker Pennahia argentata. In this study, we evaluated the suitability of the germ cell-less fish as a recipient by transplanting donor testicular cells directly into the gonads through the urogenital papilla. The donor testicular cells were collected from hemizygous transgenic, green fluorescent protein (gfp) (+/-) blue drum, and transplanted into the germ cell-less gonads of the 6-month-old adult hybrid croakers. Fluorescent and histological observations showed the colonization, proliferation, and differentiation of transplanted spermatogonial cells in the gonads of hybrid croakers. The earliest production of spermatozoa in a hybrid recipient was observed at 7 weeks post-transplantation (pt), and 10% of the transplanted recipients produced donor-derived gfp-positive spermatozoa by 25 weeks pt. Sperm from the hybrid recipients were used to fertilize eggs from wild-type blue drums, and approximately 50% of the resulting offspring were gfp-positive, suggesting that all offspring originated from donor-derived sperm that were produced in the transplanted gfp (+/-) germ cells. To the best of our knowledge, this is the first report of successful spermatogonial transplantation using a germ cell-less adult fish as a recipient. This transplantation system has considerable advantages, such as the use of comparatively simple equipment and procedures, and rapid generation of donor-derived spermatogenesis and offspring, and presents numerous applications in commercial aquaculture.

Hybrid Sterility in Fish Caused by Mitotic Arrest of Primordial Germ 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.