Germ cells are not the primary factor for sexual fate determination in goldfish

Abstract

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.

Efficient and Heritable Gene Targeting in Tilapia by CRISPR/Cas9

“…In contrast, Cyp19a1a, an ovarian-specific gene, was not detected in nanos3 mutant XX gonads. On the other hand, germ-cell deficiency in XY tilapia testis did not affect the sex differentiation in somatic cells, which is consistent with the results from the four fishes mentioned above (Slanchev et al 2005;Kurokawa et al 2007;Fujimoto et al 2010;Goto et al 2012). Together, these results demonstrate that the effects of germ-cell ablation gonadal fate are species-specific.…”

“…Germ cells were lost in the gonads after nanos2 and nanos3 mutation, as demonstrated by GFP labeling and Vasa staining. In line with the results obtained from medaka and zebrafish (Slanchev et al 2005;Kurokawa et al 2007), but contrary to those from goldfish and loach (Fujimoto et al 2010;Goto et al 2012), our study showed that germ-cell-deficient XX tilapia displayed femaleto-male sex reversal after nanos3 mutation. In contrast, Cyp19a1a, an ovarian-specific gene, was not detected in nanos3 mutant XX gonads.…”

Molecular characterization and expression pattern of a germ cell marker gene dnd in gibel carp (Carassius gibelio)

“…Dnd plays an important role in germ plasm RNAs stabilization through binding their 3′UTR and inhibiting miRNAs targeting (Kedde et al, 2007). Depletion of Dnd abolishes PGC in teleost including zebrafish (Weidinger et al, 2003), loach (Fujimoto et al, 2010), goldfish (Goto et al, 2012), Atlantic salmon (Wargelius et al, 2016), (Hong et al, 2016), and gibel carp (Liu et al, 2015a). Recent study in medaka has revealed that it is dnd but not dazl or vasa, functions as a key specifier of PGCs.…”

“…Dnd has been cloned and identified in many vertebrates, including mouse (Youngren et al, 2005), zebrafish (Weidinger et al, 2003), chicken (Aramaki et al, 2009), Xenopus (Horvay et al, 2006), medaka (Liu et al, 2009;Hong et al, 2016), loach (Fujimoto et al, 2010), goldfish (Goto et al, 2012), sterlet sturgeon (Linhartova et al, 2015), and Atlantic salmon (Wargelius et al, 2016). In most of examined teleost fishes, such as zebrafish, medaka, loach, goldfish, sterlet sturgeon, and Atlantic salmon, dnd is specifically expressed in PGCs, and the dnd translation block or depletion completely abolishes PGCs during early embryogenesis (Weidinger et al, 2003;Fujimoto et al, 2010;Goto et al, 2012;Hong et al, 2016;Wargelius et al, 2016). In some species, however, the dnd expression in adults appears to be different with sex.…”

Learning to Fish with Genetics: A Primer on the Vertebrate ModelDanio rerio

“…Females can also be mated to WT males to produce clutches of embryos without maternal gene products. Germ-line replacement has now been used on a wide variety of fish species and even in cross-species transplants, opening up additional opportunities for research on gamete development (Saito et al 2008;Shimada and Takeda 2008;Goto et al 2012).…”