SRY-related cDNA encoding a protein with a high-mobility-group (HMG) box and a leucine zipper motif, which was designated SOX-LZ, was isolated from a rainbow trout testis cDNA library. Comparison of this cDNA with the mouse homologous cDNA isolated from a testis cDNA library exhibits an overall amino acid sequence identity of 77%, which is in striking contrast to the abrupt loss of amino acid sequence homology outside the HMG box found among mammalian SRY genes. In both rainbow trout and mice, Northern (RNA) blot analyses have revealed the presence of a testis-specific 3-kb-long SOX-LZ mRNA, and this transcript appeared coincidentally with the protamine mRNA, suggesting its expression in the germ line. A recombinant HMG box region protein encoded by SOX-LZ could bind strongly with an oligonucleotide containing an AACAAT sequence, which is also recognized by mouse Sry and Sox-5. Upon cotransfection into CHO cells, In fish, sex determination and differentiation are attributed to both genetic and environmental factors (4,7,45). With regard to salmonids, male heterogamety has been demonstrated cytologically for several species (30,39,40), and a Y chromosome-specific DNA fragment has been isolated (11), although their sex chromosomes show little morphological differentiation, suggesting an early stage of heteromorphic evolution. Genetic evidence has also indicated that an XY system operates to control sex determination (6,21,24). However, in contrast to mammals, sex determination does not appear to be so strictly bound to the sex chromosomes, and functional sex inversion can be induced in both directions by treatment with exogenous sex steroids (12,20). Thus, fish provide a particularly intriguing biological system in which to study sex determination and differentiation. SOX-LZSRY, a candidate gene responsible for testis determination, has been isolated from the sex determining regions of human and mouse Y chromosomes (16,36). Analyses of XY sexreversed females (2,18,19,23) and testis formation of mice transgenic for Sry (27) have equated SRY and the testis-determining factor. SRY encodes a protein with a DNA-binding motif known as the high-mobility-group (HMG) box (16,36). Surprisingly, comparison of mammalian SRY sequences reveals that sequence conservation is largely confined to the HMG box, suggesting its rapid evolution (13,16,36,41,43). In addition to SRY, genes encoding an SRY type HMG box (SOX genes) have been identified by PCR with primers based on the conserved amino acid sequences of the HMG boxes of mammalian SRY (5,9,10,15,29,42,44). To investigate sex determination and differentiation in fish, we have isolated cDNA clones for a rainbow trout SOX gene (SOX-LZ) expressed in testes. From a phylogenetic interest, we have also isolated mouse homologous cDNA clones. SOX-LZ genes of both species show not only an overall structural conservation but also similar mRNA expression patterns. MATERIALS AND METHODSAmplification of rainbow trout SRY-related HMG box sequence. Total RNA was extracted from rainbow trout t...

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The doublesex and mab-3-related transcription factor 1 (DMRT1) is involved in testis formation in a variety of vertebrates. In the teleost fish, Medaka, DMY/DMRT1Y on the Y chromosome, a duplicate of the autosomal DMRT1 gene, is characterized as a sex-determining gene. We report here the characterization of the Xenopus DMRT1 genes. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that X. laevis DMRT1 was expressed throughout the embryo during early development and was restricted to the primordial gonads after embryogenesis. Whole-mount in situ hybridization analysis of the gene confirmed its specific expression in the primordial gonads. To study the transcriptional control of DMRT1 gene expression, we isolated the predicted promoter region of X. tropicalis DMRT1 using databases for this species. Analysis of transgenic tadpoles with a green fluorescence protein (GFP) reporter showed that approximately 3 kb of the 5′-flanking sequence of the DMRT1 gene was implicated in DMRT1 expression in the primordial gonads. We also showed that the C-terminal region of DMRT1 functioned as a transactivation domain in cultured cells, by a luciferase reporter assay using fusion proteins with the DNA-binding domain of GAL4. These findings suggest that DMRT1 functions as an activator of one or more genes involved in sex determination or gonadal differentiation.

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The molecular mechanisms of vertebrate ZZ/ZW-type sex-determining systems remain unclear. We recently indicated that a W-linked gene, DM-W is a likely ovary-determining gene in Xenopus laevis. We first examined whether Cyp19 for estrogen-synthesizing enzyme P450 aromatase and Foxl2 showed female-specific expression in developing gonads. Both genes showed much higher expression in ZW than in ZZ gonads during and after sex determination. Importantly, transgenic ZZ gonads expressing exogenous DM-W at the sex-determining stage showed a ZW-type pattern of Cyp19 and Foxl2 expression. These results suggest that DM-W up-regulates Cyp19 and Foxl2 expression to guide primary ovary development in X. laevis.