Germ cells are essential for sexual dimorphism in the medaka gonad

Abstract

To further elucidate the roles of germ cells in the sex differentiation of gonads, we have used the medaka, a teleost fish, to generate mutants that lack germ cells from the onset of gonadogenesis by the morpholino-mediated knockdown of cxcr4. The resulting germ-cell-deficient medaka show female-to-male sex reversal of their secondary sex characteristics, accompanied by increased levels of androgen and reduced levels of estrogen. A failure to maintain granulosa cells or estrogen-producing cells also occurs at early stages of sex differentiation in the cxcr4 morphants, before the initiation of gonadal morphogenesis. In contrast, androgen-producing cells are unaffected in germ-cell-deficient medaka of either sex. In addition, a single tube-like gonad that expresses male-specific genes is formed in these mutants irrespective of the genetic sex. Significantly, each of these mutant phenotypes occurs in a somatic cell-autonomous manner, suggesting that gonadal somatic cells are predisposed toward male development in the absence of germ cells. This highlights the importance of germ cells in the sexual dimorphism of the gonads.

Fig. 1.

Schematic representation of medaka gonadal development from stage 33 (4 dpf) to adulthood. Morphological events are indicated by black characters. Red characters indicate both the timing and location of the gene expression tested in this study. The early stages of gonadal development include the period of cell differentiation, which should be discriminated from later morphogenic stages. Secondary sex characteristics in the medaka manifest after 40–50 dph in both sexes.

Fig. 2.

Sex reversal of germ-cell-deficient medaka and its associated gonadal morphology. (A--C) Representative images of the secondary sex characteristics of medaka adults that are genetically female (XX) (A), male (XY) (B), and cxcr4-morphant (germ-cell-deficient) female (XX) (C). (Top) External genitalia. White arrowheads indicate the anus. A developed urinogenital papilla is observed only in the wild-type female. (Middle and Bottom) Dorsal and anal fins, respectively. Phenotypic males (B and C) display a sharp and long anal fin, whereas a round-shaped anal fin is characteristic of a phenotypic female (A). (D) Dorsal views of an ovary (left), testis (second from left) and germ cell-deficient XX and XY gonads (second from right and right). (E–G), Cross-sections of ovary (E; scale bar, 500 μm), testis (F; scale bar, 200 μm), and germ-cell-deficient gonad (G; scale bar, 100 μm). A single empty lumen with several foldings is present in the germ-cell-deficient gonad. Arrowheads indicate blood vessels. (H and I) Electron micrographs of germ-cell-deficient medaka gonads. The lumen (L), basement membrane (arrowheads), and desmosomes (arrows) are indicated. A single layer of innermost cells is shaded with a pale blue color.

Fig. 3.

Gene expression analysis of germ-cell-deficient gonads in the adult medaka. (A) RT-PCR analysis of the indicated genes in normal ovaries (Ov), normal testes (Ts), and germ-cell-deficient gonads from sex-reversed genetically XX and XY medaka (MO). RT−, control reaction without reverse transcriptase. (B–E) Gene expression patterns of P450c17 (B), P45011β (C), DMRT1 (D), and ftz-f1 (E) analyzed by in situ hybridization. Nuclei are counterstained with neutral red. Insets show the whole section in each case. (Scale bars, 20 μm.)

Fig. 4.

Gene expression analysis of both wild-type and germ-cell-deficient developing gonads. The XX gonads of 10 dph wild-type (A–C and G–I), 10 dph morphant (D–F and J–L), and 20 dph morphant (M–O) were analyzed by in situ hybridization for the marker genes, which are indicated on the top of columns. Nuclei are counterstained with neutral red. The gonad in each image is enclosed by a black dotted line. Nephric duct (nd), gut (g). (Scale bars, 20 μm.)