A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes

Abstract

The genes that determine the development of the male or female sex are known in Caenorhabditis elegans, Drosophila, and most mammals. In many other organisms the existence of sex-determining factors has been shown by genetic evidence but the genes are unknown. We have found that in the fish medaka the Y chromosome-specific region spans only about 280 kb. It contains a duplicated copy of the autosomal DMRT1 gene, named DMRT1Y. This is the only functional gene in this chromosome segment and maps precisely to the male sex-determining locus. The gene is expressed during male embryonic and larval development and in the Sertoli cells of the adult testes. These features make DMRT1Y a candidate for the medaka male sex-determining gene.

Figure 1

Southern blot of EcoRI-digested male and female DNA with a human DMRT1 cDNA (a), and rehybridized with medaka genomic DMRT1 (b) and DMRT1Y (c) probes. DMRT1 and DMRT1Y show some cross-hybridization to each other even under conditions of highest stringency because of the high sequence similarity. The hybridization conditions in a were moderate stringency and high stringency in b and c. (d and e) Hybridization under even further increased stringency conditions of PstI-digested DNAs. (f) PCR from DNA of female and male medakas (strain i-3) with DMRT1Y- and DMRT1-specific primers. Cosmid 9 contains the autosomal DMRT1 gene (26) and cosmid 13 contains DMRT1Y. (g) Genetic linkage map of the region flanking the male SD, based on meiosis in sex-reversed XY females. Numbers indicate genetic distances in centimorgans (cM). Markers DMRT1Y and SDY (locus determining the male sex phenotype) showed no recombination.

Figure 2

Sequence comparison of the medaka DMRT1 and DMRT1Y genes with the DMRT1s from other teleosts. Ola, Oryzias latipes (Medaka); Tru, Takifugu rubripes; Oni, Oreochromis niloticus (Tilapia); Omy, Oncorhynchus mykiss (rainbow trout).

Figure 3

Identification of the medaka Y chromosome: metaphases from male (a) and female (b) showing the hybridization signals of two BAC probes (15H17: DMRT1Y; 98C17: SL1). Note the presence of three hybridization spots for the BAC 15H17 in males as compared with the two spots in female (red signal). The additional fluorescence in situ hybridization signal in male is on the Y chromosome. The two relatively weak signals (arrows) in both male and female metaphase spreads represent the autosomal DMRT1 locus (linkage group 9). The SL1 marker containing BAC 98C17 detects both sex chromosomes (green signal). (c) Two highly enlarged XY chromosome pairs from two metaphases of male medaka showing hybridization to both sex chromosomes of BACs, which contain sequences flanking the Y-specific region on either side (95J10 and 125L10) (red signals). The SL1 marker containing BAC98C17 (green signals) was used to identify the sex chromosomes.

Figure 4

Schematic representation of the DMRT1Y-containing region. Lines above show the analyzed BAC and cosmid clones. Genes and sequences with predicted homology to known genes are shown as boxes: striped, DMRT1Y; hatched, ϕDMRT3; light gray, KIAA0032; black, ΔKIAA0172 and ϕMHCL. The region upstream of ΔKIAA0172 contains only repetitive DNA and sequences with similarity to transposable elements of various organisms. Two genes upstream of DMRT1, a myosin heavy chain like gene (MHCL) and an ankyrin repeat containing gene (orthologous to human KIAA0172), are part of the duplicated fragment from linkage group 9 on the Y. The duplicated Y-chromosomal copy of MHCL is, however, destroyed by insertion of a poseidon element, a non-long terminal repeat retroposon (40), in Southern medaka and additionally a TX-1-related transposon in Northern medaka. The Y-chromosomal version of KIAA0172 is corrupted by a deletion that takes out two exons. In addition, the 5′ part of the gene is missing, indicating the border of the duplicated fragment. In intron 4 of DMRT1Y an insertion has occurred. This insertion contains a duplicated copy of the putative medaka homologue of the human brain and testes antigen gene MAP1 that is located on medaka linkage group 19 (M.K., H. Mitani, A.S., and M.S., unpublished work). The Y-chromosomal copy of MAP1, however, has a frameshift mutation that leads to a prematurely terminated protein. Downstream of the Y-chromosomal DMRT1 a copy of DMRT3 is found. But its coding sequence is lacking the ATG start codon and it has several frameshifts. DMRT2, which is the next gene following DMRT3 on the autosomal cluster, is not found in the Y-specific region.

Figure 5

Expression of DMRT1Y and DMRT1. (a) Reverse transcription–PCR with DMRT1Y-specific primers of total RNA from organs of adult male and female medaka. Actin expression was determined for calibration. (b) Whole-mount RNA in situ hybridization in adult testes with DMRT1Y antisense probe (Upper Left), DMRT1 sense control (Upper Right), and section of testes showing staining in Sertoli cells (arrows, Lower). (c) Reverse transcription–PCR of medaka embryos and hatchlings with the same primers as in a. (Left) Analysis from Carbio strain samples. (Right) Analysis from Quart strain samples. (d) DMRT1Y expression in 17β estradiol-treated Quart embryos and sex-reversed adult XY females.