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...
Sox9 plays an essential role in mammalian testis formation. It has been reported that gene expression in the testes is regulated by enhancers. Among them, mXYSRa/Enh13—which is located at far upstream of the transcription start site—plays a critical role, wherein its deletion causes complete male-to-female sex reversal in mice. It has been proposed that the binding sites (BSs) of SOX9 and SRY, the latter of which is the sex determining gene on the Y chromosome, are associated with mXYSRa/Enh13. They function as an enhancer, whereby the sequences are evolutionarily conserved and in vivo binding of SOX9 and SRY to mXYSRa/Enh13 has been demonstrated previously. However, their precise in vivo functions have not been examined to date. To this end, this study generated mice with substitutions on the SOX9 and SRY BSs to reveal their in vivo functions. Homozygous mutants of SOX9 and SRY BS were indistinguishable from XY males, while double mutants had small testes, suggesting that these functions are redundant and that there is another functional sequence on mXYSRa/Enh13, since mXYSRa/Enh13 deletion mice are XY females. In addition, the majority of hemizygous mice with substitutions in SOX9 BS and SRY BS were female and male, respectively, suggesting that SOX9 BS contributes more to SRY BS for mXYSRa/Enh13 to function. The additive effect of SOX9 and SRY via these BSs was verified using an in vitro assay. In conclusion, SOX9 BS and SRY BS function redundantly in vivo, and at least one more functional sequence should exist in mXYSRa/Enh13.
The sex-determining region of the Y chromosome, Sry/SRY, is an initiation factor for testis development in both humans and mice. Although the functional compatibility between murine SRY and human SRY was previously examined in transgenic mice, their equivalency remains inconclusive. As molecular interaction and timeline of mammalian sex determination were mostly described in murine experiments, we generated a mouse model in which Sry was substituted with human SRY to verify the compatibility. The mouse model had the human SRY open reading frame at the locus of murine Sry exon 1 (Sry (SRY) mice) and was generated using the CRISPR/Cas9 system. The reproductive system of the mice was analyzed. The expression of human SRY in the fetal gonadal ridge of Sry (SRY) mice was detected. The external and internal genitalia of adult Sry (SRY) mice were similar to those of wild-type females, without any significant difference in anogenital distance. Sry (SRY) mice obtained gonads, which were morphologically considered as ovaries. Histological analysis revealed that the cortical regions of gonads from adult Sry (SRY) mice contained few follicles. We successfully replaced genes on the Y chromosome with targeted genome editing using the CRISPR/Cas9 system. Since the Sry (SRY) XY mice did not develop testis, we concluded that human SRY was insufficient to drive testis development in mouse embryos. The difference in response elements and lack of glutamine-rich domains may have invalidated human SRY function in mice. Signal transduction between Sry/SRY expression and Sox9/SOX9 activation is possibly organized in a species-specific manner.
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