of the germline is critical to ensure fertility and, ultimately, survival of the species. In humans, aberrations in germ cell development are linked to gonadal dysgenesis, infertility, and germ cell tumors (GCTs). GCTs are malignant neoplasms of germ cells that occur in neonates, infants, children, adolescents, and adults. In children the tumors are relatively rare, occurring at a rate of about 2 per million.1
The incidence in adults is 70–80 per million, and it has been rising steadily for unknown reasons.2
Testicular GCT is in fact the most common cancer in young men aged 15–40. GCTs are a heterogeneous group of neoplasms that includes yolk sac tumors (also called endodermal sinus tumors), immature and mature teratomas, choriocarcinoma, embryonal carcinoma, and germinomas (referred to as dysgerminomas in females and seminomas in males).3
Therapy for GCTs has been essentially static since the discovery that these tumors are exquisitely sensitive to cisplatin-based regimens.1,4
Although effective, these chemotherapy regimens cause hearing loss, infertility, lung damage, and kidney failure. Moreover, emerging data on long-term survivors of testicular cancer show a doubling in risk of second malignancy and of early onset of cardiovascular disease.1
Prospects for improved, targeted therapy of GCTs are poor, however, because of the lack of molecular detail about the causes of these tumors. No somatic or inherited mutations causing GCTs have been definitively identified.
Although there is overlap in tumor spectrum, it is generally helpful to consider GCTs of children and young adults as clinicopathologically distinct entities4
(). Type I tumors occur in neonates, infants, and prepubertal children, and consist of teratomas (benign) and yolk sac tumors (malignant). Characteristic chromosomal aberrations include losses of chromosome 1p and 6q, and gain of 1q and 20q. In postpubertal adolescents and adults (type II tumors), a different spectrum is seen, with the appearance of germinomas (called seminomas in males and dysgerminomas in females), which are tumors of primitive, undifferentiated germ cells. The other malignant GCTs in this age group are collectively called nongerminomas, and consist of embryonal carcinoma, yolk sac tumor, teratoma, and choriocarcinoma. Germinomas and nongerminomas appear to arise from a common precursor, the carcinoma in situ
share common chromosomal aberrations including isochromosome 12p, and are frequently found together in mixed tumors. Finally, type III GCTs are the spermatocytic seminomas occurring in older males, which are thought to arise from spermatogonia or spermatocytes and characteristically exhibit gain of chromosome 9.7,8
These tumors are confined to the testis and show a mixture of small, intermediate, and large cells.
FIG. 1. Classification and developmental origins of human germ cell tumors (GCTs). Normal gonadal development begins with the specification of primordial germ cells, which migrate to the gonadal ridges to become gonocytes. The gonad is then patterned via interactions (more ...)
Despite the differences noted above, there is striking evidence that both childhood and adolescent/adult GCTs arise due to defects in the totipotent early germline progenitors known as primordial germ cells (PGCs). First, the heterogeneous phenotype of GCTs indicates a totipotent cell of origin; GCTs can be germinomas (which retain primitive germ cell characteristics); tumors of extraembryonic tissues (yolk sac, trophoblast); and tumors differentiated to endoderm, mesoderm, and ectoderm (teratomas). Second, GCTs show persistent or ectopic expression of genes normally enriched in PGCs, such as Oct-4, NANOG, and STELLA.9,10
Third, disorders of male genitourinary development such as cryptorchidism and spermatogenic or testicular dysgenesis are strong risk factors for later developing testicular GCT,11
again supporting the idea that GCTs have their origin during embryonic and fetal development. Finally, the high frequency of extragonadal GCTs in the midline likely reflects the migratory path taken by PGCs during development, after they arise on the yolk sac.12
The development of the germline in zebrafish includes many features that are conserved from fish to mammals.13–16
Zebrafish PGCs are specified beginning with localization of specific transcripts such as vasa to the germ plasm at the four-cell stage. An initial population of four to five germ cells undergoes several rounds of division as the cells migrate to their eventual position in the presomitic mesoderm. The factors controlling PGC specification and migration are broadly conserved from flies to fish to mammals (reviewed in Refs.15,17,18
). In both sexes, the mature gonad develops between 14 and 28 days postfertilization through a primitive ovary-like stage. In presumptive males, this tissue undergoes apoptosis as the male germline emerges. Zebrafish do not have identified sex chromosomes, thus sexual development is somewhat plastic and subject to environmental influences.19–23
Previous studies have described the spontaneous development of gonadal neoplasms in male zebrafish >2 years of age. Moore et al.
described the tumor spectrum at 30–34 months of age in wild-type zebrafish and in the carriers of the gin
genomic instability phenotype.24
Testicular hyperplasias (enlarged testes containing all stages of spermatogenesis) were found in 48% of wild types and 25% of gin
heterozygotes. Benign seminomas, which they defined as tumors of predominantly one cell type derived from an early stage of spermatogenesis, were seen in 17% of wild-type fish at 30–34 months of age; the incidence was 53% in gin
carriers. Amsterdam et al.
documented the tumor spectrum in a survey of nearly 10,000 two-year-old zebrafish. Overall, 473 tumors were found, of which approximately 40% were described as seminomas of the testis.25
The testis has also been identified as a target of carcinogens in multiple fish species, including rainbow trout, medaka, and zebrafish.26,27
Spitsbergen et al
. reported testicular neoplasms of 5 of the 68 juvenile fish treated with the carcinogens N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and in 1 of the 99 juveniles treated with 7,12-dimethylbenz[a]anthracene (DMBA).28,29
Bauer and Goetz29a
carried out a histology-based genetic screen in zebrafish and identified 11 mutations that produced visible gonadal phenotypes in only one sex per family. To date, however, no line of zebrafish that consistently gives rise to GCTs of the ovary or testis has been isolated and propagated. Here we describe the identification, through an ethylnitrosourea (ENU)-based forward genetic approach, of a mutant zebrafish line that spontaneously develops highly penetrant testicular GCTs at an average age of 7 months. The GCT susceptibility trait was identified in the carriers of the shortstop
cell proliferation mutation, but we show that the trait is not linked to shortstop
. The trait is dominantly inherited, and the GCTs display disordered testicular tissue architecture and the accumulation of primitive spermatogonial-like cells, with impaired germ cell differentiation. Similar to human seminomas, the tumors are sensitive to radiation therapy. This novel model can serve as a basis for understanding the development of GCTs in humans.