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BOR - Papers in Press, published online ahead of print July 13, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.041871
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BIOLOGY OF REPRODUCTION 73, 1017–1024 (2005)
DOI: 10.1095/biolreprod.105.041871
© 2005 by the Society for the Study of Reproduction, Inc.

Multiple Alternative Splicing and Differential Expression of dmrt1 During Gonad Transformation of the Rice Field Eel1

Xiao Huang , Yiqing Guo , Yi Shui , Shang Gao , Hongshi Yu , Hanhua Cheng 3 , and Rongjia Zhou 2 

Department of Genetics and Center for Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, People's Republic of China


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 3' region, which generates diverse isoforms in gonads by alternative use of 3' 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 3' untranslated regions (UTRs), although these 3' 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.

alternative splicing, developmental biology, dsx, fish, ovary, sexual development; testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Morphologically distinct males and females are observed in the great majority of animal species. Why and how sex evolved and is maintained in most living organisms remain key questions in developmental and evolutionary biology. A surprising variety of sex-determining mechanisms are found in animals [1], including X- and Y-chromosome heterogamy in male mammals, Z- and W-chromosome heterogamy in female birds, temperature-dependent sex determination in some reptiles and fishes, and the ratio mechanism of X chromosomes to autosomes in Drosophila and Caenorhabditis elegans.

The established vertebrate sex-determining genes show varying degrees of evolutionary conservation. First, in mammals, male sex determination is under the control of Sry (sex-determining region on Y chromosome), which encodes a DNA-binding protein that acts dominantly to trigger differentiation of the testes from indifferent gonads that otherwise develop as ovaries [2, 3]. Sry and its functions in sex determination are conserved only in mammals. In birds and reptiles, SRY is absent, and SOX9 (an SRY-related gene that contains a similar HMG box domain) may dominantly control male differentiation [4–6]. Evidence is accumulating that SOX9 is a pivotal vertebrate (including mammals) testis-determining gene through which all male-determining switch mechanisms operate [7, 8]. However, in invertebrates, the SOX9 gene is not conserved, suggesting that diverse molecular mechanisms have evolved to control sexual development. Last, the only molecular similarity in sexual development of metazoans from flies and worms to mammals found, so far, is among doublesex (dsx) (Drosophila), mab-3 (C. elegans), and Dmrt1 (mammals) [9– 19]. These genes encode putative transcription factors that have a common DNA-binding domain, termed the DM domain. Both dsx and mab-3 control aspects of sex-specific differentiation and are functionally related [9].

The Dmrt1 also has a role in sexual development. Human DMRT1 is located on the short arm of chromosome 9 [11, 20]. Deletions of this region (9p24) are associated with XY sex reversal [21, 22], and male-specific expression for the gene in early gonadogenesis is consistent with its role in testis development [23]. In mice, high Dmrt1 expression is necessary for testicular differentiation, whereas low expression is compatible with ovarian differentiation [12, 24, 25]. A Dmrt1 knockout mouse further demonstrated that the gene is required for testis differentiation after determination, but dispensable for ovary development [11]. Furthermore, the dmrt1 gene is transposed to the Y chromosome in some fish (medaka) to become a master regulator gene (dmrt1y/dmy) in male determination [26–28]. In the chicken, DMRT1 is sex-linked [10] on the Z chromosome, and there is a higher dosage of the gene in males (ZZ) than in females (ZW) [12, 24, 29, 30].

Homologs of DMRT1 have also been cloned in reptile species. The expression of DMRT1 in turtle and alligator were found to be sexually dimorphic, and were higher in developing male gonads than in female gonads [12–14]. These data show that the expression of these DM genes from invertebrates to vertebrates is sex-specific (or sex differential), and (with the exception of Drosophila) are associated with male-specific development.

However, we know remarkably little about the evolution and roles of dmrt1 in a variety of vertebrates, especially in those with special reproduction mechanisms. Here we report that multiple alternative splicing and differential expression of dmrt1 occur during gonad differentiation from ovary via ovotestis to testis in the rice field eel, a vertebrate that undergoes natural sex reversal.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Rice field eels (Monopterus albus), a freshwater teleost fish, were obtained from markets in the Wuhan area in China. Their sex was confirmed by microscopic analysis of gonadal sections. The experiments were carried out in accordance with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction.

Rapid Amplification of cDNA Ends Analysis and Cloning of Alternatively Spliced and Full-Length cDNAs of dmrt1

Primers for rapid amplification of cDNA ends (RACE) were designed based on sequence information of the DM domain of the rice field eel dmrt1 that we previously cloned [31]. SMART cDNAs were made from gonad RNA using the SMART cDNA library construction kit (Clontech, Palo Alto, CA). 5' RACE was performed using the DM domain primer, DM3, 5'-TCTCGGCTATCAGTTTACATTTGG-3' and the SMART primer, SMARTIII, 5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3'. Polymerase chain reaction (PCR) cycling conditions were as follows: 35 cycles for 30 sec each at 94°C, 40 sec at 65°C, and 40 sec at 72°C in a 20-µl reaction mix containing 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 200 µM deoxynucleotide triphosphate (dNTP), 0.2 µM each primer, 1 U Taq DNA polymerase, and 1 µl of the cDNA. After the PCR, nested PCR was performed using nest primer NDM3, 5'-TCCCTCCAGTTGCAGAAGCG-3' and the same 5' primer, SMARTIII. We performed 3' RACE using the DM domain primer DM5, 5'-TGAAGGGGCACAAGCGCTTCTGC-3'; and the CDSIII primer, 5'-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N–1N-3' (where N = A, G, C, or T; and N–1 = A, G, or C) 3'. After 3' RACE, nested PCR was used to specifically amplify the 3' end of the dmrt1 gene using the same conditions described above. The nested primer pair was NDM5, 5'-GACTGCCAGTGCTCCAAATG-3' and the same CDSIII primer. All bands amplified were gel-purified, cloned, and sequenced. Full lengths of cDNAs alternatively spliced were cloned by using PCR and sequenced. The primers were 5' end primers, 5'-TCTAACCAGCCTTGTCGTCCA-3'; nest primer, 5'-GTTTGGCAGTTGGCAGCATAGT-3'; and 3' end primers, 5'-AGTTTGATTCTGAAGAGTGCCCC-3' for dmrt1 a, 5'-GGGGCACCATTTTGGAAGATA-3' for dmrt1 b, and 5'-GGACACTGACTTACAGCTGAATAA-3' for dmrt1 d. The PCR conditions were the same as those mentioned above. The sequence for dmrt1 c was the same as that for dmrt1 a except for the deletion of a CAG coded for glutamine at the 186 amino acid, and a region of 3' UTR after the first polyadenylation signal sequence, so that it cannot be distinguished from the dmrt1 a by PCR.

Sequences and Secondary Structure Analysis

All sequences were analyzed with the Vector NTI software. Sequence alignments were performed using Clustal W and analyzed using the basic local alignment search tool supplied by GenBank online. 3' UTR sequences of both dmrt1 a and dmrt1 b were analyzed by the GeneQuest program in DNAStar software (3' UTR sequences of both dmrt1 c and d were too short to be analyzed). Minimal free energy was calculated allowing G-U everywhere at 30°C so as to increase the complexity of secondary structures.

Reverse Transcription-PCR

RNAs were isolated from gonad using the RNeasy Mini kit (Qiagen, Germany). DNase was used to exclude DNA contamination. Complementary DNAs were reverse-transcribed from gonad RNAs using Maloney murine leukemia virus reverse transcriptase (Promega, Madison, WI). Reverse transcription (RT)-PCR (nonquantification) was used to amplify individual isoforms of dmrt1 from the cDNAs of different tissues of the rice field eel. PCR cycling conditions were as follows: 35 cycles for 30 sec each at 94°C, 40 sec at 63°C or 58°C (beta-actin), and 40 sec at 72°C in a 20-µl reaction mix containing 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 200 µM dNTP, 0.2 µM each primer, 1 U Taq DNA polymerase. The designed primers flanked one or more introns, and the sequences were as follows: dmrt1 a, 5'-ATGGTGATGGACGCCTGTCT-3' and 5'-GTCGATAATGGAGTCAACGG-3'; dmrt1 b, 5'-GGAGTCGCTCGGCGTCAT-3' and 5'-GGGGCACCATTTTGGAAGATA-3'; dmrt1 d, 5'-CTCTAACCAGCCTTGTCGTCCA-3' and 5'-GGACACTGACTTACAGCTGAATAA-3'; and for beta-actin, 5'-TGGATGATGATATTGCTGC-3' and 5'-ATCTTCTCCATATCATCCC-3'.

Real-Time Fluorescent Quantitative RT-PCR

Real-time RT-PCR was used for to quantify dmrt1 expression using the multichannel RotorGene 3000 (Corbett Research, Australia), according to the supplied protocol. PCR cycling conditions were as follows: 5 min at 95°C, 40 cycles of 15 sec at 95°C, 30 sec at 58°C (actin) and 65°C (dmrt1), and 1 min at 72°C in a 25-µl reaction mix containing 0.5x SYBR Green I. The primers and cDNA templates used were the same as those used in RT-PCR. A serial dilution of cDNA samples was simultaneously used to amplify beta-actin to determine the quantity of cDNAs as standards. Based on the standards, dmrt1 cDNAs were then amplified using a serial dilution of the cDNAs as templates and relative expression levels of the dmrt1 to beta-actin (expression percentage) were determined. For robustness issues, determinations were performed three to six times on each sample (samples: testis, 6; ovary, 4; ovotestis, 3). Data were produced by the Rotor-gene version 4.6 software. The mean percentage of dmrt1 to beta-actin expression of each sex was obtained.

Northern Blotting Hybridization

Northern blots were performed as routine protocols, except that hybridization at 42°C was performed in ULTRAhyb solution (Ambion, Austin, TX) with an [alpha-32P]dCTP-labeled probe. A fragment including the DM domain of dmrt1 a cDNA was used as the probe in the hybridizations.

In Situ Hybridization Analysis

For in situ hybridization to gonadal sections, antisense and sense RNA probes were prepared separately from a region that included the DM domain of dmrt1 a cDNA, which can hybridize to all isoforms of dmrt1 under the same conditions, and labeled with digoxigenin-uridine triphosphate using SP6 or T7 RNA polymerase. Gonad tissues were cryosectioned, and the sections were immediately hybridized at 42°C, and hybridization signals were detected by the NBT/BCIP system according to the manufacturer's instructions (Boehringer, Mannheim, Germany).

3' UTR Analysis

The 3' UTRs of dmrt1 isoforms were cloned using PCR. The primers were as follows: 5Fa, 5'-TAAGCGGCCGCAGGAGCCTAA-3' and 3Ra, 5'-AGCCTCGAGGGTGAGCTACAA-3' for dmrt1 a; 5Fb, 5'-ATAGCGGCCGCCACTGCAACTGTGTTT-3' and 3Rb, 5'-TAACTCGAGGGGTGCGGCATTAAAAA-3' for dmrt1 b; and 5Fa and 3Rd, 5'-CGCCTCGAGATCTCATAACGC-3' for dmrt1 d. After digestion with NotI and XhoI, the PCR products were ligated with the egfp coding fragment of pEGFP-N1 digested with NotI and BamHI. The recombined fragments (egfp-3' UTR) were then cloned into Amp-col E1 ori-cmv of pIRES-egfp (predigested with BamHI and XhoI) to obtain gfp a, b, and d constructions. These recombinants were transfected into COS-7 cells. For equal transfection efficiency, the pcDNA3.0 was used to adjust the transfected DNAs to the same DNA quantity (an equal amount of DNA in each transfection). The ratio of DNA:Lipofectamine 2000 was 0.8 µg:2.5 µl. The gfp expression levels in the cells were examined using flow cytometry. Each sample was transfected three times and mean values were calculated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
cDNA Cloning of Multiple Alternative Transcripts of dmrt1 in Gonads

In an attempt to isolate the dmrt1 gene from gonads of this species, we previously searched for one or more DM domain genes transcribed in the testis of the male rice field eel using a degenerate RT-PCR strategy [31]. Based on the DNA sequence information, we designed primers for cloning full-length dmrt1. The cDNAs synthesized from the SMART primer were used as a template for both 5' RACE and 3' RACE by combined use of the primer pairs of the DM domain, the SMART primer, and nested PCR. After obtaining both 5' and 3' half regions of the dmrt1 sequence, which overlap in the DM domain region, the full-length dmrt1 sequence was amplified by PCR based on the 5' and 3' sequence information. Four bands of different sizes were observed when the 3' RACE products were run on gels. Sequence analysis revealed that these four products represented four 3' regions of dmrt1 transcripts generated by multiple alternative splicing, and dmrt1 d was also truncated by alternative polyadenylation. The dmrt1 a, b, and c isoforms used a common splicing site at amino acid 185. Because of a CAG shift while splicing at this site, Dmrt1 c missed an amino acid Q. This transcript was also truncated by alternative polyadenylation compared with dmrt1 a. These transcripts were further confirmed by PCR amplification and sequencing of full-length cDNAs. Alignments showed that the highest level of conservation is within the DM domain of the Dmrt1 proteins when compared with those of human and chicken (Fig. 1). The 5' region and DM domain of these dmrt1 isoforms are common to all transcripts, and alternative splicing occurs only in the 3' region, not in 5' to the DM domain (Fig.2).



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  		FIG. 1. Protein alignment of the human and chicken DMRT1, and alternatively spliced transcripts of the rice field eel dmrt1. The DM domain is underlined. The chicken Dmrt1 sequence at the 5' region is not complete. GenBank accession numbers are AF421347–AF421350 for rice field eel Dmrt1, AAF19666 for chicken, and CAB82427 for human DMRT1



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  		FIG. 2. Diagram of all alternatively spliced transcripts of rice field eel dmrt1. Splicing of dmrt1 forms four kinds of mRNAs: dmrt1 a, dmrt1 b, dmrt1 c, and dmrt1 d, which encode four proteins with 301, 196, 300, and 205 amino acids, respectively. Dmrt1 c has deleted an amino acid Q compared with that of Dmrt1 a because of alternative splicing. DM domains are indicated by shaded boxes from amino acids 24–88. Sequences from amino acids 1–133 are common among the four transcripts. Alternatively spliced regions in the 3' region are shown by different colors, and same color represents the same DNA sequence. The numbers in the end under the lines indicate nucleotide numbers of these cDNAs

Spliced Isoforms of dmrt1 Are Specifically Expressed in Adult Gonads

To analyze expression patterns of these alternatively spliced dmrt1 isoforms, we carried out RT-PCR using RNAs isolated from gonads and other tissues. All isoforms of the dmrt1 expression are detectable by RT-PCR in testis, ovotestis, and ovary of the rice field eel. The dmrt1 a and b isoforms were also weakly expressed in the brain, but not the heart, kidney, liver, or spleen of the rice field eel (Fig. 3a). We further performed Northern blot analysis of dmrt1 with dmrt1 a as the probe. Strong expression (a 1.5-kilobase of the major band is an isoform of dmrt1 a, and bands for the other isoforms are very faint) was observed in the testis, and there was slightly lower expression in the ovotestis II of intersex and in the ovary (Fig. 3b). Accurate quantitative analysis was carried out by real-time fluorescent quantitative RT-PCR, an explanation of which follows.



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  		FIG. 3. RT-PCR and Northern blot analysis of dmrt1 of the rice field eel. a) RT-PCR of dmrt1 of the rice field eel shows its expression in three kinds of gonads. There is no expression in other tissues other than a faint band observed in the brain. b) Northern blot analysis (sample load among wells is semiquantitative based on band intensity of 28S rRNA) of dmrt1 expression (the major band of 1.5 kilobase is an isoform of dmrt1 a, and bands for the other isoforms are very faint) in testis, ovotestis II (medium stage of intersex), and ovary of the rice field eel. The expression of dmrt1 is high in testis, with a slightly lower expression in the ovary and ovotestis II of intersex animals

Spliced Transcripts Are Differentially Expressed During Gonadal Transformation

Taking advantage of the sex reversal characteristic of the rice field eel, we investigated the expression of dmrt1 isoforms during sex transformation from female via intersex to male. The expression patterns of these spliced isoforms were analyzed and quantitated using real-time fluorescent quantitative RT-PCR. Based on comparisons of mean values from this RT-PCR analysis, the expression level of dmrt1 a was from low to high: ovary < ovotestis I < ovotestis II < ovotestis III < testis (Fig. 4). Although the expression level of dmrt1 b changed in a pattern similar to that of dmrt1 a, the overall expression level of dmrt1 b was much lower than that of dmrt1 a. The dmrt10 d expression not only was low, but also did not change significantly during sex transformation.



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  		FIG. 4. Real-time fluorescent quantitative RT-PCR analysis. The relative expression of dmrt1 isoforms to beta-actin in ovary, ovotestis I (preintersex stage, most of the gonad is ovary; testis beginning to develop), ovotestis II (medium intersex stage), ovotestis III (postintersex stage, most of the gonad is testis), and testis measured by real-time RT-PCR. Each sample (testis, 6; ovary, 4; ovotestis, 3) was analyzed three to six times. Data were produced by Rotor-gene software, version 4.6. A mean percentage of the dmrt1 to beta-actin expression of each sex was obtained. The error bars represent ± SEM

Expression of dmrt1 Is Primarily Restricted to the Somatic Layer of Gonadal Epithelium

To determine where dmrt1 is expressed in gonads of the rice field eel, we performed dmrt1 in situ hybridization in gonad sections. The dmrt1 expression is restricted primarily to the outer layer (primarily somatic cells) of gonadal epithelium, and undifferentiated germ cells, whereas it is not found in differentiated germ cells in male, intersex, and female animals (Fig. 5, a–c).



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  		FIG. 5. Expression analysis of dmrt1 of the rice field eel by in situ hybridization to gonadal sections of female, intersex, and male rice field eel. a–c) Antisense probed for dmrt1 shows expression of these transcripts in gonadal lamellae or epithelium (white arrowheads) of female, intersex, and male, respectively (the square is an amplification of each area of the white arrowhead). Sense probing as control (d–f) and hematoxylin and eosin staining (g–i) in the gonad samples of the three sexes are shown below. T, seminiferous tubules; O, ova. Original magnification a–i x200; insets in a–c x400

Expression of dmrt1 Isoforms Is Regulated by Their 3' UTRs

To investigate the roles of different 3' UTRs in the regulation of dmrt1 isoform expression, we examined gfp expression levels using different 3' UTRs of dmrt1 isoforms. As shown in Figure 6, the 3' UTR of dmrt1 a has a positive effect on gene expression, whereas the 3' UTR of dmrt1 c, which has same sequence as dmrt1 a but was truncated because of alternative polyadenylation, reduced gfp expression by about 15%. However, the 3' UTR of dmrt1 b that was alternatively spliced has a negative effect on gfp expression (Fig. 6a). Intracellular localization of the GFP with different 3' UTR was further observed to determine whether there is any difference in localization. Expression of gfp showed a similar fluorescence pattern, primarily nuclear fluorescence with a diffuse cytosolic distribution (Fig. 6b). We further analyzed secondary structures of 3' UTRs of dmrt1 a and b (3' UTRs of both dmrt1 c and d are too short to form secondary structures). Complex secondary structures with high minimal free energy of delta G = –66.7 kcal/mol (dmrt1 a) and –91.75 kcal/mol (dmrt1 b) were observed (Fig. 7).



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  		FIG. 6. Expression regulation of dmrt1 isoforms by their 3' UTRs. a) Expression levels of egfp are shown as geometric means of GFP fluorescence of M1 range of cells by flow cytometry analysis. GFP a, CMV-egfp-dmrt1 a 3' UTR; GFP b, CMV-egfp-dmrt1 b 3' UTR; GFP c, CMV-egfp-dmrt1 c 3' UTR; GFP o, CMV-egfp without 3' UTR as a control. The error bars represent ± SEM. b) Intracellular localization of the constructions GFP a (A), GFP b (B), and GFP c (C) in COS-7 cells. Original magnification x600



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  		FIG. 7. Predicted folding form of the 3' UTRs of the dmrt1 a (a) and dmrt1 b (b) mRNAs of the rice field eel. The dmrt1 mRNA 3' UTRs are folded (A-U, G-C pairs form shape of ladder) according to the GeneQuest program of DNAStar software to yield a structure of minimum free energy


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The rice field eel, an economically important freshwater fish of Southeast Asia, exhibits natural sex reversal from female via intersex to male during its life cycle. Its chromosomes are all telocentric (2n = 24) and its haploid genome size of 600 Mb is among the smallest of the vertebrates. These features make it an ideal vertebrate model for studying molecular mechanisms and evolution of sex reversal [32]. Taking advantage of the sex reversal characteristic of the rice field eel, we cloned the dmrt1 cDNA based on DM domain sequence information that we had previously reported [31]. We found that multiple alternative splicing of the gene occurred in testis, ovotestis, and ovary. We demonstrated differential expression of these dmrt1 isoforms during gonad transformation from female via intersex to male and characterized the roles of 3' UTRs of dmrt1 isoforms in the regulation of dmrt1 expression.

Unlike many other developmental processes (e.g., body axis establishment), the sex-determining mechanism varies considerably among phyla. A potential exception to this lack of conservation is the recent finding of structural and functional similarity among the sexual regulators mab-3 of C. elegans, doublesex of Drosophila, and dmrt/dmy of vertebrates [9, 33]. The DM genes share a conserved cysteine-rich DNA-binding domain (DM) [34], and each plays similar roles in male-specific sexual development. However, outside the DM domain of these genes, there is poor conservation between species, and there is also a lack of data to show any conservation of regulation of these genes, the targets they act on among phyla. These observations make it difficult to infer from the model organisms where vertebrate DM factors are located in a gene regulatory pathway. Evolution and function of the DM genes need further exploration. Here we have shown that the dmrt genes have another characteristic conserved among phyla: a special alternative splicing. The alternative splicing events occurred 3' to the DM domain in diverse species, including Drosophila (dsx), zebrafish (dmrt1) [35], and the rice field eel (dmrt1). Alternative splicing is known to increase diversity of the proteomic world, evolutionary and functional significance for the vast majority of alternative splicing events is unknown [36]. However, it is rare that the alternative splicing events consistently occur in the 3' region of genes (e.g., DM genes) and play a similar role in sexual development across species in different phyla.

Different kinds of alternative splicing in DM genes have also been observed in human DMRT2 [37], dmrt1 of Tetraodon nigroviridis and lizard [38, 39], and a DM gene (ADM1) of coral (Acropora millepora) [40], and the doublesex homologue Bmdsx of the silkmoth [41]. Diverse isoforms of the dmrt1 generated by alternative splicing at 3' termini in the rice field eel may also provide potential diverse targets for different upstream and downstream interacting factors in sexual regulation. Few targets of regulation by DM factors have yet been clearly identified, and most of the data have come from dsx of Drosophila and mab-3 of C. elegans [42, 43], which were focused on the yolk protein gene (Yp). In Drosophila, the dsx gene is considered the last sex-determination regulatory gene in its branch of the hierarchy because its proteins bind to and directly regulate the transcription of two of the genes encoding terminal sexual differentiation proteins, the Yp-1 and Yp-2 genes [44, 45]. DSXF (an alternatively spliced female-form of dsx) activates Yp transcription by interaction with IX protein (encoded by intersex gene), but DSXM represses Yp transcription, as the IX does not interact with DSXM [46, 47]. Further identification of interactive factors with dmrt1 in vertebrates will determine how these spliced dmrt1 genes operate, although a few other partners have been reported [48, 49].

Differential expression of these dmrt1 isoforms in gonadal epithelium with bipotential differentiation capacity during gonad transformation in the rice field eel provides new insight into the role of alternative splicing of dmrt1 in sexual differentiation. Actually, typical roles of the DM-related gene, dsx of Drosophila, were performed by an alternatively spliced isoform of dsxM and dsxF. More studies have also shown that the Sry, other Sox, and Wt1 factors play a role in pre-mRNA splicing in mammalian cells [50– 55]. All of these results suggest important roles of regulation at the level of splicing in governing the sex-differentiation cascade. Further studies are required to identify genes that are subject to regulation at the posttranscriptional level, which will help in understanding molecular mechanisms acting in the sex-determination process.


   ACKNOWLEDGMENTS
 
We thank Professor Terrence Tiersch for commenting on the manuscript and making corrections to it.


   FOOTNOTES
 
1 Supported by the National Natural Science Foundation of China, the National Key Basic Research project (2004CB117400) and by a Key Project of the Chinese Ministry of Education (2004.28). GenBank accession numbers are AF421347–AF421350 for rice field eel Dmrt1. Back

2 Correspondence. FAX: 0086 27 67856253; rjzhou{at}public.wh.hb.cn Back

3 Correspondence. FAX: 0086 27 67856253; hhcheng{at}whu.edu.cn Back

Received: 15 March 2005.

First decision: 11 April 2005.

Accepted: 11 July 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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A. Shirak, E. Seroussi, A. Cnaani, A. E. Howe, R. Domokhovsky, N. Zilberman, T. D. Kocher, G. Hulata, and M. Ron
Amh and Dmrta2 Genes Map to Tilapia (Oreochromis spp.) Linkage Group 23 Within Quantitative Trait Locus Regions for Sex Determination
Genetics, November 1, 2006; 174(3): 1573 - 1581.
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