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The tiger puffer Takifugu rubripes is one of the most popular aquacultural fish; however, there are two major obstacles to selective breeding. First, they have a long generation time of 2 or 3 years until maturation. Second, the parental tiger puffer has a body size (2-5 kg) much larger than average market size (0.6-1.0 kg). The grass puffer Takifugu niphobles is closely related to the tiger puffer and matures in half the time. Furthermore, grass puffer can be reared in small areas since their maturation weight is about 1/150 that of mature tiger puffer. Therefore, to overcome the obstacles of maturation size and generation time of tiger puffer, we generated surrogate grass puffer that can produce tiger puffer gametes through germ cell transplantation. Approximately 5000 tiger puffer testicular cells were transplanted into the peritoneal cavity of triploid grass puffer larvae at 1 day post hatching. When the recipient fish matured, both males and females produced donor-derived gametes. Through their insemination, we successfully produced donor-derived tiger puffer offspring presenting the same body surface dot pattern, number of dorsal fin rays, and DNA fingerprint as those of the donor tiger puffer, suggesting that the recipient grass puffer produced functional eggs and sperm derived from the donor tiger puffer. Although fine tunings are still needed to improve efficiencies, surrogate grass puffer are expected to accelerate the breeding process of tiger puffer because of their short generation time and small body size.

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.

Exogenous factors such as temperature, social behavior, and salinity play a crucial role during the critical sensitive period of sex differentiation in many vertebrates. In fishes, amphibians, and reptiles temperature treatment is known to induce all-male (or female) individuals, and genes related to sex differentiation have been studied. The Japanese pufferfish, Takifugu rubripes, possesses the most compact genome among vertebrates and has immense potential for studies focusing on comparative genome analysis. In this study, we describe gonadal morphology and vasa (germ cell marker) and dmrt1 (Sertoli cell marker) expression on a molecular level in relation to the development of temperature-treated pufferfish. To investigate the relationship between temperature and gonadal development, pufferfish were exposed to high-temperature conditions (32°C) during early gonadal development. Morphological observations showed that this high-temperature treatment did not influence sexual differentiation as determined by ovarian cavity characteristics; however, high-temperature treatment induces gonadal degeneration that is devoid of germ cells. RT-PCR results revealed no vasa expression within germ cell-degenerated gonads. In situ hybridization results showed that dmrt1 was expressed in somatic cells of germ cell-degenerated ovaries. These results suggest that high-temperature treatment during early gonadal development induces germ cell degeneration and masculinization of ovarian somatic cells in pufferfish.

Estrogens are responsible for most characteristics of the female sex of a species, such as metabolic, behavioral, and morphological changes during reproduction. Artificial estradiol-17beta (E2) treatment Induces sex reversal in some fish. The Japanese pufferfish (Takifugu rubripes) has the most compact genome among vertebrates and great pottial for comparative genome analysis. In this paper, we describe the Influence of E2 treatment during gonadal development in the pufferfish. After hatching, fry were treated with no (control) or a 0.1, 1, 10, or 100 microg/g diet from 21 to 80 days after hatching (dah). Doublesex-mab3-related transcription factor (DMRT1) is Involved in testicular development. VASA is responsible for germ cell development, and CYP19A plays a role in E2 biosynthesis during ovarian development across animal phyla as well as in gonadal morphology after E2 treatment. DMRT1, VASA, and CYP19A were Investigated in the gonads of E2-treated pufferfish. Fish fed with the highest dose (E2 100 microg/g diet) developed Intersexual gonads in the testis; the majority of germ cells were oocytes, but some spermatocytes were detected. RT-PCR results showed the expression of VASA and CYP19A in all intersexual gonads and DMRT1 in some. Furthermore, abnormalities in the epithelium-tunica layer were detected, and gonadal somatic cells (e.g., granulosa cells, theca cells, or germinal epithelium) proliferated extensively in the intersexual gonad. These results suggest that E2 treatment Induces ovarian development in the bipotential gonads of genetic males by modification of gonadal somatic cells and E2 production, mediated by CYP19A.