In mammals, testis determination is under the control of the sex-determining gene SRY. This Y-linked gene encodes a protein with a DNA binding domain similar to those found in high-mobility-group proteins. Here we report the cloning and sequences of the SRY genes of yak and Chinese native cattle. Our data show that SRY genes in Bovidae are less divergent, especially in the coding and 30 regions.

Several mechanisms were used in determination of the development of the male or female of vertebrates. The genes for determination of sequential hermaphrodite sex are unknown. Here, we reported cloning, alternative splicing, and expression patterns of the CYP17 gene of the rice field eel, a teleost fish with a characteristic of nature sex reversal. The CYP17 gene of the rice field eel was clustered into the CYP17 gene group of all the other vertebrates, especially into the fish subgroup. Four isoforms of the CYP17 were generated in gonads by alternative splicing and polyadenylation. Alternative splicing events of all these isoforms occurred in 30 regions, which encoded three different sizes (517, 512, and 159 aa) of proteins. RT-PCR results indicate specific expression in gonads of these isoforms. Northern blot analysis shows that expression patterns of the CYP17 (dominantly expressed in testis, less in ovary, and the least in ovotestis) are consistent with the sex reversal process of the rice field eel. In situ hybridization further shows its specific expression in germinal lamellae, the gonadal epithelium of the gonads. These findings indicate that CYP17 is differentially regulated in a sex-and developmentally specific manner, suggesting that the CYP17 potentially has important roles in gonad differentiation during sex reversal of the rice field eel.

The mammalian sex determining gene, SRY, is the founding member of the new growing family of Sox (SRY-like HMG-box gene) genes. Sox genes encode transcription factors with diverse roles in development, and a few of them are involved in sex determination and differentiation. We report here the existence of Sox genes in the rice feld eel, Monopterus albus, and DNA sequence information of the HMG box region of ve Sox genes. The Sox1, Sox4 and Sox14 genes do not have introns in the HMG box region. The Sox9 gene and Sox17 gene, which each have an intron in the conserved region, show strong identity at the amino acid level with the corresponding genes of mammals and chickens. Similar structure and identity of the Sox9 and Sox17 genes among mammals, chickens and sh suggest that these genes have evolutionarily conserved roles, potentially including sex determination and differentiation.

The DM domain gene family has at least eight members with conserved DNA-binding DM domain, which encodes putative transcription factors related to the sexual regulator Dsx of Drosophila and Mab-3 of Caenorhabditis elegans. Although some of the DM genes are involved in sexual development, the function of most of these genes remains unclear. There is also few structural and functional analysis concerning DM domain genes of the model fish, zebrafish. We report here molecular cloning, sequence, and expression of zebrafish Dmrt5, which consists of two exons, and encodes a 440-amino acid protein with conserved DMA and DMB domains in addition to DM domain. Phylogenetic analysis shows that zebrafish Dmrt5 fits within the Dmrt5 clade of fish and mammals. Zebrafish Dmrt5 was expressed in early gastrula period, subsequently increased to a high level in late stage of gastrula period (bud stage) and lower until the hatch period. In situ hybridization analysis showed its expression in developing central nervous system of embryos, especially in mid-brain and mid-hind brain boundary. In adult, its expression was restricted in brain and developing germ cells, especially in spermatogonia, spermatocytes, spermatids, and sperm cells, and in developing oocytes, including early perinucleolus stage oocyte, late yolk vesicle stage oocyte, and oil drop stage oocyte. These data suggest that zebrafish Dmrt5 have potentially important roles in gonadal development and may have contributed to the functional endocrine axis.

Many basic cellular processes are shared across vast phylogenetic distances, whereas sex-determining mechanisms are highly variable between phyla although the existence of two sexes is nearly universal in the animal kingdom. The only molecular similarity in sex determination found so far between phyla is among the fly doublesex, worm mab-3, and vertebrate Dmrt1/DMY, which contain a zinc-finger-like DNA-binding motif, DM domain. Here we report that three isoforms of the zebrafish Dmrt1 were generated in gonads by multiple alternative splicing, which encoded predicted proteins with 267, 246, and 132 amino acids, respectively. By cDNA cloning and genomic structure analysis, we found that there were seven exons of Dmrt1, which were alternatively spliced to generate the Dmrt1 isoforms. Northern blotting analysis revealed that expression of zebrafish Dmrt1 was higher in testis than ovary. Real time fluorescent quantitative RT-PCR indicated that expression of isoform a of Dmrt1 was dominantly higher than those of Dmrt1 b and c. Furthermore, in situ hybridization to gonads sections showed that Dmrt1 was expressed in developing germ cells of both testis and ovary, suggesting that the Dmrt1 gene is not only associated with testis development, but also, may be important in ovary differentiation of zebrafish.

Morphologically distinct males and females are observed throughout the animal kingdom. Why and how sex evolved and is maintained in most living organisms remains a key question in cellular and evolutionary biology. Here we report that four isoforms of dmrt1 (dsx-and mab3-related transcription factor 1) are generated in testis, ovotestis, and ovary by alternative splicing in the rice field eel, a fresh water fish that undergoes natural sex reversal from female to male during its life cycle. These transcripts encode four different size proteins with 301, 196, 300, and 205 amino acids. Like fly doublesex splicing, the dmrt1 of the rice field eel is also alternatively spliced at the 39 region, which generates diverse isoforms in gonads by alternative use of 39 sequences. Not only is dmrt1 expressed specifically in gonads, but its multiple isoforms are differentially coexpressed in gonadal epithelium during gonad transformation. Expression levels of a and b isoforms of dmrt1 ranged from low to high (ovary,ovotestis I, ovotestis II, ovotestis III,testis), based on comparisons of mean values from real-time fluorescent quantitative reverse transcription-polymerase chain reaction analysis. The overall expression level of dmrt1 b was much lower than that of dmrt1 a. Expression of dmrt1 d was not only low, but it also did not change significantly during sex transformation. The differential expression of dmrt1 isoforms may also be regulated by their 39 untranslated regions (UTRs), although these 39 UTRs do not contribute to intracellular localization of the Dmrt1 protein. These results provide new insight into roles of regulation at the level of splicing of dmrt1 in governing the sex differentiation cascade.