SummaryAs complete absence of germ cells leads to sterile males in zebrafish, we explored the relationship between primordial germ cell (PGC) number and sexual development. Our results revealed dimorphic proliferation of PGCs in the early zebrafish larvae, marking the beginning of sexual differentiation. We applied morpholino-based gene knockdown and cell transplantation strategies to demonstrate that a threshold number of PGCs is required for the stability of ovarian fate. Using histology and transcriptomic analyses, we determined that zebrafish gonads are in a meiotic ovarian stage at 14 days postfertilization and identified signaling pathways supporting meiotic oocyte differentiation and eventual female fate. The development of PGC-depleted gonads appears to be restrained and delayed, suggesting that PGC number may directly regulate the variability and length of gonadal transformation and testicular differentiation in zebrafish. We propose that gonadal transformation may function as a developmental buffering mechanism to ensure the reproductive outcome.

Primordial germ cells (PGCs) arise elsewhere in the embryo and migrate into developing gonadal ridges during embryonic development. In several model animals, formation and migration patterns of PGCs have been studied, and it is known that these patterns vary. Sturgeons (genus Acipenser) have great potential for comparative and evolutionary studies of development. Sturgeons belong to the super class Actinoptergii, and their developmental pattern is similar to that of amphibians, although their phylogenetic position is an out-group to teleost fishes. Here, we reveal an injection technique for sturgeon eggs allowing visualization of germplasm and PGCs. Using this technique, we demonstrate that the PGCs are generated at the vegetal pole of the egg and they migrate on the yolky cell mass toward the gonadal ridge. We also provide evidence showing that PGCs are specified by inheritance of maternally supplied germplasm. Furthermore, we demonstrate that the migratory mechanism is well-conserved between sturgeon and other remotely related teleosts, such as goldfish, by a single PGCs transplantation (SPT) assay. The mode of PGCs specification in sturgeon is similar to that of anurans, but the migration pattern resembles that of teleosts.

The presence of germ cells in the early gonad is important for sexual fate determination and gonadal development in vertebrates. Recent studies in zebrafish and medaka have shown that a lack of germ cells in the early gonad induces sex reversal in favor of a male phenotype. However, it is uncertain whether the gonadal somatic cells or the germ cells are predominant in determining gonadal fate in other vertebrate. Here, we investigated the role of germ cells in gonadal differentiation in goldfish, a gonochoristic species that possesses an XX-XY genetic sex determination system. The primordial germ cells (PGCs) of the fish were eliminated during embryogenesis by injection of a morpholino oligonucleotide against the dead end gene. Fish without germ cells showed two types of gonadal morphology: one with an ovarian cavity; the other with seminiferous tubules. Next, we tested whether function could be restored to these empty gonads by transplantation of a single PGC into each embryo, and also determined the gonadal sex of the resulting germline chimeras. Transplantation of a single GFP-labeled PGC successfully produced a germline chimera in 42.7% of the embryos. Some of the adult germline chimeras had a developed gonad on one side that contained donor derived germ cells, while the contralateral gonad lacked any early germ cell stages. Female germline chimeras possessed a normal ovary and a germ-cell free ovary-like structure on the contralateral side; this structure was similar to those seen in female morphants. Male germline chimeras possessed a testis and a contralateral empty testis that contained some sperm in the tubular lumens. Analysis of aromatase, foxl2 and amh expression in gonads of morphants and germline chimeras suggested that somatic transdifferentiation did not occur. The offspring of fertile germline chimeras all had the donor-derived phenotype, indicating that germline replacement had occurred and that the transplanted PGC had rescued both female and male gonadal function. These findings suggest that the absence of germ cells did not affect the pathway for ovary or testis development and that phenotypic sex in goldfish is determined by somatic cells under genetic sex control rather than an interaction between the germ cells and somatic cells.

The concentrations of 14 trace elements (Li, V, Mn, Co, Cu, Zn, Se, Rb, Sr, Ag, Cd, Cs, Pb, and Hg) were determined in tissues and organs of three species and in the liver of 11 species of seabirds. Comparatively high concentrations of Li, Co, Sr, and V were found in the femur. Cd, Se, Cu, and Mn concentrations were relatively higher in the kidney than in other tissues and organs. Rb, Cs, and Pb concentrations were rather uniform among tissues. Concentrations of essential elements such as Mn, Cu, and Co were comparable among seabird species, except high Cu concentrations in northern giant petrel. Among nonessential elements, concentrations of Cd and Hg were variable according to seabird species. Pb levels were low in all the species. High Se levels (100 microg/g dry weight) were found in the liver of black-footed albatross and grey petrel. There were significant positive correlations between Se and Cd concentrations in three species and between Se and Hg in black-footed albatross, suggesting that Se has an antagonistic action on the toxic effects of Cd and Hg. Concentrations of Li, V, Ag, and Cs were usually low (less than 1 microg/g dry weight).

This time corresponds to the end of the temperature-sensitive period.These results suggest that gonadal sex was determined by temperature prior to the onset of gonadal sex differentiation in this fish.Key words: barfin flounder, sex determination, temperatureIt has been documented that sex in fish is controlled ge netically. However, many fish do not have a clear chro mosomal sex determining system,1) and therefore, genetic sex and phenotypic sex should be considered as separate en tities. Studies over the past decade have shown that exter nal factors, such as temperature and pH, influence sexual determination in some fish. Thus, temperature-dependent sex determination has been demonstrated in Menidia menidia,2) Oreochromis niloticus3) and Odontesthes bonariensis,4) while pH-dependent sex determination has been demonstrated in Xiphophorus spp.5) and Apistogram ma spp.6) The hirame flounder Paralichthys olivaceus7) in particular, has been well-studied in terms of genetic sex de termination and manipulation of physiological sex by con trolling temperature. It has been demonstrated in this spe cies, in which the male is the heterogametic sex (XX female-XY male), that genetic females reverse their sex to male if the water temperature is manipulated. Not only high but also low temperatures produced male-biased sex ratios, while intermediate temperatures yielded a 1: 1 sex ra tio.Thermolability of sex determination in another flatfish species, the barfin flounder Verasper moseri, a teleost that belongs to the Pleuronectidae family and inhabits cold sea water basins, has not been addressed. It is a commercially important species in Hokkaido and requires establishment of breeding techniques for aquaculture, like the hirame flounder. As in many other teleosts,8-10) gonadal sex in the barfin flounder can be manipulated by treatment with ex ogenous steroids during the period of gonadal sex differen tiation. l'1 However, it became evident that the population frequently had a biased sex ratio under artificial breeding conditions. A supply of seedlings with stable sex ratio for stock enhancement has not yet been realized for this spe cies. As in the hirame flounder, the biased sex ratio in barfin flounder could be caused by temperature influencing sex determination. To test this hypothesis, we evaluated sex ratios following manipulation of the temperature dur ing the larval and juvenile stage. Manipulations were done periodically in order to identify temperature sensitive periods and to relate these periods to gonadal morpholo gy. Materials and Methods AnimalsFertilized eggs of barfin flounder were acquired from a few captive-bred males and females held together in a 1 m3 tank at the Hokkaido Institute of Mariculture, Shikabe, Japan. After egg incubation and hatching at 8•Ž, tempera tures were gradually increased up to 14•Ž in 34 days. Animals were reared in flow-through seawater, larvae being fed Brachionus and Artemia nauplii, while juveniles were fed artificial diets. Experimental DesignThe experimental water temperatures ...