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Primordial germ cells (PGCs) generate gametes, the only cells that can transmit genetic information to the next generation. A previous report demonstrated that a fusion construct of green fluorescent protein (gfp) and zebrafish nos 1 3UTR mRNA could be used to label PGCs in a number of fish species. Here, we sought to exploit this labeling strategy to isolate teleost PGCs by flow cytometry (FCM), and to use these isolated PGCs to examine germ cell migration to the gonadal region. In zebrafish, medaka and goldfish, the PGCs were labeled by injecting the gfp-nos1 3UTR mRNA into 1- 4 cell embryos. When the embryos had developed to the somitogenesis or later stages, they were enzymatically disaggregated and GFP positive cells isolated using FCM. PGCs in the different species clustered in the same segments of the FCM scatter diagrams for total embryonic cells produced by plotting the forward scatter intensity against GFP intensity. In situ hybridization showed that the sorted zebrafish cells expressed vasa RNA in their cytoplasm, suggesting that they were PGCs. When the migration ability of the sorted cells from zebrafish was examined in an in vivo transplantation experiment, approximately 30% moved to the gonadal region of host embryos. These observations demonstrate that PGCs can be isolated without use of transgenic fishes and that the isolated PGCs retain the ability to migrate. Our data indicate that this technique will be of value for isolating PGCs from a range of fish species.

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. PGCs therefore have the potential to be of value for gene banking and cryopreservation, particularly via the production of donor gametes with germ-line chimeras. Currently, it is not clear how many PGCs are required for germ-line differentiation and formation of gonadal structures. In the present study, we achieved complete germ-line replacement between two related teleost species, the pearl danio (Danio albolineatus) and the zebrafish (Danio rerio), with transplantation of a single PGC into each host embryo. We isolated and transplanted a single PGC into each blastula-stage, zebrafish embryo. Development of host germ-line cells was prevented by an antisense dead end morpholino oligonucleotide. In many host embryos, the transplanted donor PGC successfully migrated toward the gonadal anlage without undergoing cell division. At the gonadal anlage, the PGC differentiated to form one normally sized gonad rather than the pair of gonads usually present. Offspring were obtained from natural spawning of these chimeras. Analyses of morphology and DNA showed that the offspring were of donor origin. We extended our study to confirm that transplanted single PGCs of goldfish (Carassius auratus) and loach (Misgurnus anguillicaudatus) can similarly differentiate into sperm in zebrafish host embryos. Our results show that xenogenesis is realistic and practical across species, genus, and family barriers and can be achieved by the transplantation of a single PGC from a donor species.

Abstract: The present study analyzes the temperature-dependent sex differentiation in goldfish, which have an XX–XY sex determining system. Hatchlings from an XX male–XX female cross were reared at 23±0.5 °C until Day 12 (days after fertilization), and divided into experimental groups reared at 15, 17, 20, 23, 25, 28 or 30±0.5 °C. The proportions of females were 94.6% at 15 °C, 100% at 17 °C, 94.6% at 20 °C, 90.5% at 23 °C, 46.6% at 25 °C, 15.4% at 28 °C, and 7.7% at 30±0.5 °C. This result confirms that genotypic females can be changed to phenotypic males by high temperature treatment. To determine the temperature sensitive period (TSP) of sex differentiation, the sex ratio was measured in groups of larvae shifted reciprocally between 23 and 30 °C as development progressed. The percentage of females increased in test groups shifted from 23 to 30 °C at Days 17, 22 and 27 and decreased in groups shifted from 30 to 23 °C at Day 17 and 22. The end point of the TSP occurs on Day 17 at 30 °C and on Day 22 at 23 °C, implying that the TSP is shortened at higher temperatures. To investigate the effect of fluctuating temperatures on sex differentiation, several one-day temperature pulses from 23 to 30 °C or 30 to 23 °C were applied, during the TSP. The pulse to 23 °C, from the basal temperature of 30 °C, resulted in a decreased percentage of males, however, the pulse to 30 °C, from the basal temperature of 23 °C, had no effect on the sex ratio. These results show that high temperature must be maintained during the TSP in order to produce a high proportion of masculinized genetic female goldfish. In conclusion, water temperature can play an important role in the process of sex differentiation in goldfish, as has been seen in other teleosts and reptiles, and be applied to aquaculture as a tool for control of sex. [Copyright &y& Elsevier]