DMY gene induces male development in genetically female (XX) medaka fish

Abstract

Although the sex-determining gene SRY/Sry has been identified in mammals, homologues and genes that have a similar function have yet to be identified in nonmammalian vertebrates. Recently, DMY (the DM-domain gene on the Y chromosome) was cloned from the sex-determining region on the Y chromosome of the teleost fish medaka (Oryzias latipes). DMY has been shown to be required for the normal development of male individuals. In this study, we show that a 117-kb genomic DNA fragment that carries DMY is able to induce testis differentiation and subsequent male development in XX (genetically female) medaka. In addition, overexpression of DMY cDNA under the control of the CMV promoter also caused XX sex reversal. These results demonstrate that DMY is sufficient for male development in medaka and suggest that the functional difference between the X and Y chromosomes in medaka is a single gene. Our data indicate that DMY is an additional sex-determining gene in vertebrates.

Fig. 1.

Transgenic construct of the DMY genomic region. (A) Transgenic construct derived from the BAC clone mCON051H1, which contains DMY^HNI and two SfiI sites. The red coloration of the BAC clone represents the insert. This fragment was digested with SfiI and ligated into the pCC1 vector by using linkers. (B) Genomic PCR products digested with StuI. The DMY^d-rR allele is digested, whereas the DMY^HNI allele is not digested. M represents the DNA marker. The arrowheads show the positions of the 1.5-kb and 1.0-kb DNA fragments. (C) Sequence chromatograms of the RT-PCR products of transgenic medaka. The DNA polymorphisms between DMY^HNI and DMY^d-rR are shown by arrowheads. Transgenic XX individuals have only the DMY^HNI allele, whereas transgenic XY individuals have both the DMY^HNI and DMY^d-rR alleles.

Fig. 2.

Phenotypic analyses of DMY transgenic adult medaka. (A–C) The external secondary sexual characteristics and body coloration of the DMY transgenic fish in the adult stage are shown. The secondary sexual characteristics are manifested in the size and shape of dorsal and anal fins (white arrowhead). Both dorsal and anal fins of males are larger then those of females. The transgenic medaka include: a white (XX) female (A), a white (XX) male (B), and an orange-red (XY) male (C). Body coloration is clearly discernable at the basal parts of the fins (white asterisks). Gonadal histologies of a transgenic XX medaka, which has an ovary (D), an XX medaka, which has testes (E and E′), and an XY medaka (F and F′). ED, efferent duct. (Scale bars: D, E′, and F′, 200 μm; E and F, 50 μm.)

Fig. 3.

Analyses of DMY transgenic medaka at different developmental stages. (A–C) DMY mRNA expression in the gonads of the fry is revealed by digoxigenin-labeled in situ hybridization of larval sections. (A) DMY-negative gonad of a transgenic XX fry. Strong signals for DMY are seen in the cells surrounding the germ cells in the XX fry (B) and XY fry (C). (A–C) Germ cells are denoted by asterisks. Gonad histologies at 2 DAH (D–F) and 30 DAH (G–I). (E and H) Transgenic XX medaka with testis-like gonads. (F and I) The transgenic XY individuals have gonads with typical testicular structure. The gonadal region is outlined by dotted lines. (Scale bars: 20 μm.)

Fig. 4.

Numbers of germ cells in transgenic and control medaka fry. (A) Schematic diagram depicting the procedure. Total numbers of germ cells at 0 DAH (B) and 5 DAH (C). Open circles represent individuals that have oocytes in the zygotene or pachytene stage, whereas filled circles represent individuals that lack oocytes in the zygotene or pachytene stage. (D) The numbers of germ cells at the different developmental stages of 5 DAH XX fry are shown. The control and DMY transgenic were compared within each stage. The results are presented as the mean ± SEM of each stage.