DMRT1 Protein Research Articles

Liposome-Encapsulated Rec8 and Dmrt1 Plasmids Induce Red-Spotted Grouper (Epinephelus akaara) Testis Maturation.

In fish, the maturity of gonads plays an important role in the development and reproduction of the population, and it also dictates the success of captive breeding. Therefore, finding ways to promote gonadal maturation is an important goal in aquaculture. In this study, we injected recombinant dmrt1 and rec8 overexpression plasmids packaged in liposomes into the immature testis of red-spotted grouper (Epinephelus akaara) and measured the expression of Dmrt1 and Rec8 protein in vivo. Gonadosomatic index (GSI) and gonadal histology analyses showed that the testis developed from the immature to the mature state within 7days after plasmid injection. Additionally, the spermatozoa concentration and motility in plasmid-injected fish was the same as that of naturally mature fish. These results provided evidence that delivery of dmrt1 and rec8 expression plasmids into the testis via injection induced testis maturation in vivo.

Interaction between DMRT1 and PLZF protein regulates self-renewal and proliferation in male germline stem cells

Double sex and mab-3 related transcription factor 1 (DMRT1) encodes a double sex/mab-3 (DM) domain, which is the most conserved structure that involved in sex determination both in vertebrates and invertebrates. This study revealed important roles of DMRT1 in maintaining self-renewal of male germline stem cells (mGSCs). Our results showed that insufficient expression of DMRT1 in mice testes resulted in decreased number of spermatogonial cells and collapse of testicular niche in vivo. Self-renewal and proliferation of mGSCs were inhibited. Based on the bimolecular fluorescence complementation (BiFC) and co-immunoprecipitation (co-IP) assay, it was finally revealed that the interaction between DMRT1 and promyelocytic leukemia zinc finger (PLZF) protein was essential for maintaining self-renewal of mGSCs. Moreover, BTB domain of PLZF, DM and DMRT1 domain of DMRT1 were indispensable in mGSC, which were responsible for preserving the quantity of germ cells. Our research provided a new scientific basis for studying the mechanism of self-renewal and spermatogenesis in goat mGSCs.

Transcriptomic and Epigenetic Preservation of Genetic Sex Identity in Estrogen-feminized Male Chicken Embryonic Gonads.

Whereas in ovo exposure of genetically male (ZZ) chicken embryos to exogenous estrogens temporarily feminizes gonads at the time of hatching, the morphologically ovarian ZZ-gonads (FemZZs for feminized ZZ gonads) are masculinized back to testes within 1 year. To identify the feminization-resistant "memory" of genetic male sex, FemZZs showing varying degrees of feminization were subjected to transcriptomic, DNA methylome, and immunofluorescence analyses. Protein-coding genes were classified based on their relative mRNA expression across normal ZZ-testes, genetically female (ZW) ovaries, and FemZZs. We identified a group of 25 genes that were strongly expressed in both ZZ-testes and FemZZs but dramatically suppressed in ZW-ovaries. Interestingly, 84% (21/25) of these feminization-resistant testicular marker genes, including the DMRT1 master masculinizing gene, were located in chromosome Z. Expression of representative marker genes of germline cells (eg, DAZL or DDX4/VASA) was stronger in FemZZs than normal ZZ-testes or ZW-ovaries. We also identified 231 repetitive sequences (RSs) that were strongly expressed in both ZZ-testes and FemZZs, but these RSs were not enriched in chromosome Z. Although 94% (165/176) of RSs exclusively expressed in ZW-ovaries were located in chromosome W, no feminization-inducible RS was detected in FemZZs. DNA methylome analysis distinguished FemZZs from normal ZZ- and ZW-gonads. Immunofluorescence analysis of FemZZ gonads revealed expression of DMRT1 protein in medullary SOX9+ somatic cells and apparent germline cell populations in both medulla and cortex. Taken together, our study provides evidence that both somatic and germline cell populations in morphologically feminized FemZZs maintain significant transcriptomic and epigenetic memories of genetic sex.

The genomic structure of the highly-conserved dmrt1 gene in Solea senegalensis (Kaup, 1868) shows an unexpected intragenic duplication.

Knowing the factors responsible for sex determination in a species has significant theoretical and practical implications; the dmrt1 gene (Doublesex and Mab-3 (DM)-related Transcription factor 1) plays this role in diverse animal species. Solea senegalensis is a commercially important flat fish in which females grow 30% faster than males. It has 2n = 42 chromosomes and an XX / XY chromosome system for sex determination, without heteromorph chromosomes but with sex proto-chromosome. In the present study, we are providing the genomic structure and nucleotide sequence of dmrt1 gene obtained from cDNA from male and female adult gonads. A cDNA of 2027 containing an open-reading frame (ORF) of 1206 bp and encoding a 402 aa protein it is described for dmrt1 gene of S. senegalensis. Multiple mRNA isoforms indicating a high variable system of alternative splicing in the expression of dmrt1 of the sole in gonads were studied. None isoforms could be related to sex of individuals. The genomic structure of the dmrt1 of S. senegalensis showed a gene of 31400 bp composed of 7 exons and 6 introns. It contains an unexpected duplication of more than 10399 bp, involving part of the exon I, exons II and III and a SINE element found in the sequence that it is proposed as responsible for the duplication. A mature miRNA of 21 bp in length was localized at 336 bp from exon V. Protein-protein interacting networks of the dmrt1 gene showed matches with dmrt1 protein from Cynoglossus semilaevis and a protein interaction network with 11 nodes (dmrt1 plus 10 other proteins). The phylogenetic relationship of the dmrt1 gene in S. senegalensis is consistent with the evolutionary position of its species. The molecular characterization of this gene will enhance its functional analysis and the understanding of sex differentiation in Solea senegalensis and other flatfish.

Spatiotemporal Expression of Foxl2 and Dmrt1 before, during, and after Sex Determination in the Sea Turtle Lepidochelys olivacea

The sex of sea turtles is determined by temperature during egg incubation. Thus, climate change affects the sex ratio, exacerbating their vulnerability to extinction. Understanding spatiotemporal effects of temperature on sex determination at the gonadal level may facilitate the design of strategies to mitigate the effects of global warming. Here, we used qRT-PCR and immunofluorescence to analyze the spatiotemporal expression of Dmrt1 and Foxl2 in developing gonads of Lepidochelys olivacea incubated at male-producing temperature (MPT, 26°C) or female-producing temperature (FPT, 33°C). Although both transcription factors are expressed in bipotential gonads up to stage 25, the timing of their sexually dimorphic regulation differs. Whereas the dimorphic expression of Dmrt1 protein initiates at stage 24, Foxl2 protein was expressed specifically in females at stage 25. Interestingly, whereas Dmrt1 colocalizes with Sox9 in cell nuclei of primary medullary cords to form the testis cords, Foxl2 protein is first detected in Sox9-negative cells of primary medullary cords, prior to its substantial expression in the ovarian cortex. Thus, results suggest that the temperature-dependent regulation of sexual pathways is stochastic among the cells of primary medullary cords in undifferentiated bipotential gonads of the olive ridley.

Molecular characterization, computational analysis and expression profiling of Dmrt1 gene in Indian major carp, Labeo rohita (Hamilton 1822)

Sexual dimorphism of fish morphology, physiology and behavior is diverse and complex in nature. Doublesex and mab-3 related transcription factor (Dmrt) is a large protein family whose function is sexual development and differentiation in vertebrates. Here, we report a full-length cDNA sequence of Labeo rohita (rohu) Dmrt1 of 907 bp length having 798 bp of open reading frame encoding 265 amino acids. The molecular weight of rohu DMRT1 protein was found to be 28.74 KDa and isoelectric point was 7.53. DMRT1 protein contains 23 positively and 24 negatively charged amino acids with a GRAVY score of -0.618. A characteristic DM domain was found in DMRT1 protein, which is a novel DNA-binding domain. Phylogenetic analysis showed maximum similarity with Cyprinus carpio when compared with DMRT1 of other vertebrates. Molecular docking study identified active sites to be targeted for drug designing. Rohu DMRT1 was observed to interact with other proteins such as FOXL2, CYP19a1a, AMH and SOX9a. Differential expression study revealed higher expression in testis tissue implying its role in male sex differentiation and testicular development. The information generated in the present work could facilitate further research to resolve the issues related to gonadal maturation and reproduction of commercially important aquaculture species.

Dmrt1 (doublesex and mab-3-related transcription factor 1) expression during gonadal development and spermatogenesis in the Japanese eel

Dmrt1, doublesex- and mab-3-related transcription factor-1, has been suggested to play critical roles in male gonadogenesis, testicular differentiation and development, including spermatogenesis, among different vertebrates. Vasa is a putative molecular marker of germ cells in vertebrates. In this study, we cloned the full-length dmrt1 cDNA from Japanese eel, and the protein comprised 290 amino acids and presented an extremely conserved Doublesex and Mab-3 (DM) domain. Vasa proteins were expressed in gonadal germ cells in a stage-specific manner, and were expressed at high levels in PGC and spermatogonia, low levels in spermatocytes, and were absent in spermatids and spermatozoa of Japanese eels. Dmrt1 proteins were abundantly expressed in spermatogonia B cells, spermatocytes, spermatids, but not in spermatozoa, spermatogonia A and Sertoli cells. To our knowledge, this study is the first to show a restricted expression pattern for the Dmrt1 protein in spermatogonia B cells, but not spermatogonia A cells, of teleosts. Therefore, Dmrt1 might play vital roles at the specific stages during spermatogenesis from spermatogonia B cells to spermatids in the Japanese eel. Moreover, the Dmrt1 protein exhibited a restricted localization in differentiating oogonia in the early differentiating gonad (ovary-like structure) of male Japanese eels and in E2-induced feminized Japanese eels. We proposed that dmrt1 may be not only required for spermatogenesis but might also play a role in oogenesis in the Japanese eel.

DMRT1 repression using a novel approach to genetic manipulation induces testicular dysgenesis in human fetal gonads.

STUDY QUESTIONDoes loss of DMRT1 in human fetal testis alter testicular development and result in testicular dysgenesis?SUMMARY ANSWERDMRT1 repression in human fetal testis alters the expression of key testicular and ovarian determining genes, and leads to focal testicular dysgenesis.WHAT IS KNOWN ALREADYTesticular dysgenesis syndrome (TDS) is associated with common testicular disorders in young men, but its etiology is unknown. DMRT1 has been shown to play a role in the regulation of sex differentiation in the vertebrate gonad. Downregulation of DMRT1 in male mice results in trans-differentiation of Sertoli cells into granulosa (FOXL2+) cells resulting in an ovarian gonadal phenotype.STUDY DESIGN, SIZE, DURATIONTo determine the effect of DMRT1 repression on human fetal testes, we developed a novel system for genetic manipulation, which utilizes a Lentivral delivered miRNA during short-term in vitro culture (2 weeks). A long-term (4–6 weeks) ex vivo xenograft model was used to determine the subsequent effects of DMRT1 repression on testicular development and maintenance. We included first and second-trimester testis tissue (8–20 weeks gestation; n = 12) in the study.PARTICIPANTS/MATERIALS, SETTING, METHODSHuman fetal testes were cultured in vitro and exposed to either of two DMRT1 miRNAs (miR536, miR641), or to scrambled control miRNA, for 24 h. This was followed by a further 14 days of culture (n = 3–4), or xenografting (n = 5) into immunocompromised mice for 4–6 weeks. Tissues were analyzed by histology, immunohistochemistry, immunofluorescence and quantitative RT-PCR. Endpoints included histological evaluation of seminiferous cord integrity, mRNA expression of testicular, ovarian and germ cell genes, and assessment of cell number and protein expression for proliferation, apoptosis and pluripotency factors. Statistical analysis was performed using a linear mixed effect model.MAIN RESULTS AND THE ROLE OF CHANCEDMRT1 repression (miR536/miR641) resulted in a loss of DMRT1 protein expression in a sub-population of Sertoli cells of first trimester (8–11 weeks gestation) human fetal testis; however, this did not affect the completion of seminiferous cord formation or morphological appearance. In second-trimester testis (12–20 weeks gestation), DMRT1 repression (miR536/miR641) resulted in disruption of seminiferous cords with absence of DMRT1 protein expression in Sertoli (SOX9+) cells. No differences in proliferation (Ki67+) were observed and apoptotic cells (CC3+) were rare. Expression of the Sertoli cell associated gene, SOX8, was significantly reduced (miR536, 34% reduction, P = 0.031; miR641 36% reduction, P = 0.026), whilst SOX9 expression was unaffected. Changes in expression of AMH (miR536, 100% increase, P = 0.033), CYP26B1 (miR641, 38% reduction, P = 0.05) and PTGDS (miR642, 30% reduction, P = 0.0076) were also observed. Amongst granulosa cell associated genes, there was a significant downregulation in R-spondin 1 expression (miR536, 76% reduction, P < 0.0001; miR641, 49% reduction, P = 0.046); however, there were no changes in expression of the granulosa cell marker, FOXL2. Analysis of germ cell associated genes demonstrated a significant increase in the expression of the pluripotency gene OCT4 (miR536, 233%, P < 0.001). We used the xenograft system to investigate the longer-term effects of seminiferous cord disruption via DMRT1 repression. As was evident in vitro for second-trimester samples, DMRT1 repression resulted in focal testicular dysgenesis similar to that described in adults with TDS. These dysgenetic areas were devoid of germ cells, whilst expression of FOXL2 within the dysgenetic areas, indicated trans-differentiation from a male (Sertoli cell) to female (granulosa cell) phenotype.LIMITATIONS, REASONS FOR CAUTIONHuman fetal testis tissue is a limited resource; however, we were able to demonstrate significant effects of DMRT1 repression on the expression of germ and somatic cell genes, in addition to the induction of focal testicular dysgenesis, using these limited samples. In vitro culture may not reflect all aspects of human fetal testis development and function; however, the concurrent use of the xenograft model which represents a more physiological system supports the validity of the in vitro findings.WIDER IMPLICATIONS OF THE FINDINGSOur findings have important implications for understanding the role of DMRT1 in human testis development and in the origin of testicular dysgenesis. In addition, we provide validation of a novel system that can be used to determine the effects of repression of genes that have been implicated in gonadal development and associated human reproductive disorders.STUDY FUNDING/COMPETING INTEREST(S)This project was funded by a Wellcome Trust Intermediate Clinical Fellowship (Grant No. 098522) awarded to RTM. LBS was supported by MRC Programme Grant MR/N002970/1. RAA was supported by MRC Programme Grant G1100357/1. RMS was supported by MRC Programme Grant G33253. This work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. The funding bodies had no input into the conduct of the research or the production of this manuscript. The authors have declared no conflicts of interest.

Expression and cellular localization of double sex and mab-3 related transcription factor 1 in testes of postnatal Small-Tail Han sheep at different developmental stages

Double sex and mab-3 related transcription factor 1 (Dmrt1), an evolutionarily conserved gene, is a sex-related gene expressed in male gonads, that is involved in the regulation of sex differentiation, testicular development and reproductive function maintenance. Until now, functional studies on the Dmrt1 gene in sheep (Ovis aries) have been lacking. In this study, testis, heart, liver, spleen, lung, kidney and longissimus dorsi muscle tissues were collected from Small-Tail Han sheep at 0, 2, 5, 12 and 24months after birth (mab). Dmrt1 expression and cellular localization were detected in various testicular tissues by quantitative real time PCR (qRT-PCR), western blot and immunohistochemistry methods. The morphological structures of testicular tissues at different developmental stages were observed by hematoxylin & eosin (HE) staining. The Dmrt1 mRNA expression levels in 12 and 24 mab sheep were significantly higher than those in 0 and 2 mab sheep (P<0.05), and Dmrt1 protein expression showed a similar trend. The qRT-PCR results in various tissues at 12 mab showed that Dmrt1 mRNA was predominantly expressed in testes. Immunohistochemical staining in testes at different developmental stages showed that Dmrt1 protein immunoreactive responses were mainly localized in Sertoli cells and gonocytes at 0, 2 and 5 mab, while they were localized in spermatocytes, sperm cells and some spermatogonia and Sertoli cells at 12 and 24 mab. We speculate that the Dmrt1 gene plays a vital role in postnatal sheep spermatogenesis, perhaps by regulating the maturation and functional maintenance of Sertoli cells, the proliferation and differentiation of gonocytes in prepubertal sheep testes, and the mitosis and meiosis of germ cells in adult sheep, but the specific mechanisms underlying these phenomena must be further studied and verified. AbbreviationsImage 1BCAbicinchoninic acidcDNADNA complementary to RNADABdiaminobenzidineDmrt1double sex and mab-3 related transcription factor 1FOXL2forkhead transcription factor gene 2HEhematoxylin-eosinHRPhorseradish peroxidasePBSphosphate buffer salinePlzfpromyelocytic leukemia zinc fingerPVDFpolyvinylidene fluorideqRT-PCRquantitative real time PCRRAretinoic acidRspo1R-spondin1SDS-PAGEsodium dodecyl sulfate polyacrylamide gel electrophoresisSohlh1spermatogenesis and oogenesis-specific basic helix-loop-helix1SOX9sex determining region Y-box 9Srysex-determining region of the Y chromosomeSSCsspermatogonial stem cellsStra8stimulated by retinoic acid 8

Sox9 and Sox8 protect the adult testis from male-to-female genetic reprogramming and complete degeneration

The new concept of mammalian sex maintenance establishes that particular key genes must remain active in the differentiated gonads to avoid genetic sex reprogramming, as described in adult ovaries after Foxl2 ablation. Dmrt1 plays a similar role in postnatal testes, but the mechanism of adult testis maintenance remains mostly unknown. Sox9 and Sox8 are required for postnatal male fertility, but their role in the adult testis has not been investigated. Here we show that after ablation of Sox9 in Sertoli cells of adult, fertile Sox8(-/-) mice, testis-to-ovary genetic reprogramming occurs and Sertoli cells transdifferentiate into granulosa-like cells. The process of testis regression culminates in complete degeneration of the seminiferous tubules, which become acellular, empty spaces among the extant Leydig cells. DMRT1 protein only remains in non-mutant cells, showing that SOX9/8 maintain Dmrt1 expression in the adult testis. Also, Sox9/8 warrant testis integrity by controlling the expression of structural proteins and protecting Sertoli cells from early apoptosis. Concluding, this study shows that, in addition to its crucial role in testis development, Sox9, together with Sox8 and coordinately with Dmrt1, also controls adult testis maintenance.

Molecular characterization and expression pattern of dmrt1 in the immature Chinese sturgeon Acipenser sinensis.

In this study, the cDNA of dmrt1 gene from the Chinese sturgeon Acipenser sinensis was isolated and its expression pattern was characterized in different tissues of immature A. sinensis. By real-time quantitative PCR (qrtPCR) analysis, the A. sinensis dmrt1 mRNA was detected mainly in gonad and with a higher level in the testis than the ovary, especially in 3 and 4 year-old samples. This indicated that the dmrt1 expression exhibited gradual testis specificity with development. The subcellular localization analysis indicated that the Dmrt1 protein exists only in germ cells and not in somatic cells. These results suggest that A. sinensis dmrt1 might be a highly specific sex differentiation gene for testis development and spermatogenesis.

DMRT1 is required for Müllerian duct formation in the chicken embryo

DMRT1 is a conserved transcription factor with a central role in gonadal sex differentiation. In all vertebrates studied, DMRT1 plays an essential function in testis development and/or maintenance. No studies have reported a role for DMRT1 outside the gonads. Here, we show that DMRT1 is expressed in the paired Müllerian ducts in the chicken embryo, where it is required for duct formation. DMRT1 mRNA and protein are expressed in the early forming Müllerian ridge, and in cells undergoing an epithelial to mesenchyme transition during duct morphogenesis. RNAi-mediated knockdown of DMRT1 in ovo causes a greatly reduced mesenchymal layer, which blocks caudal extension of the duct luminal epithelium. Critical markers of Müllerian duct formation in mammals, Pax2 in the duct epithelium and Wnt4 in the mesenchyme, are conserved in chicken and their expression disrupted in DMRT1 knockdown ducts. We conclude that DMRT1 is required for the early steps of Müllerian duct development. DMRT1 regulates Müllerian ridge and mesenchyme formation and its loss blocks caudal extension of the duct. While DMRT1 plays an important role during testis development and maintenance in many vertebrate species, this is the first report showing a requirement for DMRT1 in Müllerian duct development.

Molecular Cloning and Expression Pattern of &amp;lt;italic&amp;gt;Dmrt1&amp;lt;/italic&amp;gt; and Its Expression Change in Sex Reversal in &amp;lt;italic&amp;gt;Pelodiscus sinensis&amp;lt;/italic&amp;gt;

In many vertebrate species, <italic>Dmrt1</italic> gene is required for male sexual differentiation. In this study, the mRNA and protein expression of <italic>Dmrt1</italic> gene and its cellular localization in gonads of different developmental stages of both sexes were examined to investigate the involvement of <italic>Dmrt1</italic> in sexual differentiation in <italic>Pelodiscus sinensis</italic>. RACE results showed that the complete cDNA sequence of <italic>P. sinensis</italic> <italic>Dmrt1</italic> was composed of 230 bp of 5′ un-translated region, 1072 bp of 3′ un-translated region and 1107 bp of open reading frame, bearing a highly conserved DM domain, which encodes 368 amino acids. Real-Time PCR and immunofluorescence analysis revealed that <italic>Dmrt1</italic> gene initiated expression in male embryonic gonad of stage 16 before gonadal differentiation, prior to <italic>Sox9</italic> and <italic>Amh</italic> gene. During the gonadal development, Dmrt1 protein localized in the nuclei of pre-sertoli cells, however, no Dmrt1 signal was detected in female gonad. Moreover, <italic>Dmrt1</italic> expression was significantly decreased in estrogen-induced male to female sex reversal. Whereas, <italic>Dmrt1</italic> was trigged to express in aromatase inhibitor-induced female to male sex reversal. In conclusion, this study indicates that <italic>Dmrt1</italic> gene is a male-specific gene and participates in the male gonadal development, which is likely to play a key role during primary male sex determination in <italic>P. sinensis</italic>.

Absence of inactivating mutations and deletions in the DMRT1 and FGF9 genes in a large cohort of 46,XY patients with gonadal dysgenesis

Despite advances in our understanding of the mechanisms involved in sex determination and differentiation, the specific roles of many genes in these processes are not completely understood in humans. Both DMRT1 and FGF9 are among this group of genes. Dmrt1 controls germ cell differentiation, proliferation, migration and pluripotency and Sertoli cell proliferation and differentiation. Fgf9 has been considered a critical factor in early testicular development and germ cell survival in mice. We screened for the presence of DMRT1 and FGF9 mutations in 33 patients with 46,XY gonadal dysgenesis. No deletions in either DMRT1 or FGF9 were identified using the MLPA technique. Eight allelic variants of DMRT1 were identified, and in silico analysis suggested that the novel c.968-15insTTCTCTCT variant and the c.774G>C (rs146975077) variant could have potentially deleterious effects on the DMRT1 protein. Nine previously described FGF9 allelic variants and six different alleles of the 3’ UTR microsatellite were identified. However, none of these DMRT1 or FGF9 variants was associated with increased 46,XY gonadal dysgenesis. In conclusion, our study suggests that neither DMRT1 nor FGF9 abnormalities are frequently involved in dysgenetic male gonad development in patients with non-syndromic 46,XY disorder of sex development.

A novel ncRNA gene from mouse chromosome 5 trans-splices with Dmrt1 on chromosome 19

Dmrt1 ( Dsx- and Mab3- related transcription factor- 1), a conserved transcription factor in different phyla, is a key regulator in sex determination. Here, we report the novel ncRNA gene Dmr ( Dm rt1- related gene), from mouse chromosome 5 that trans-splices with Dmrt1 from chromosome 19 to generate a Dmrt1 protein that lacks the C-terminus. Dmr is mouse and rat specific, and the surrounding genes are also conserved in both species. Dmr is alternatively spliced, and three isoforms, Dmr a, b and c, are detected in the testis. Further, Dmr serves mainly as a 3′ UTR, promotes trans-splicing and down-regulates the Dmrt1 protein. These results suggest that Dmr might play a negative regulatory role for Dmrt1 in male sexual development.

Identification of multiple dmrt1s in catfish: localization, dimorphic expression pattern, changes during testicular cycle and after methyltestosterone treatment

The double sex and mab-3 related (DM) transcription factor 1 (dmrt1) plays an important role in testicular differentiation. Here, we report cloning of multiple dmrt1s, a full-length and two alternative spliced forms from adult catfish (Clarias gariepinus) testis, which encode predicted proteins of 287 (dmrt1a), 253 (dmrt1b) and 233 (dmrt1c) amino acid residues respectively. Interestingly, dmrt1c lacks the majority of the DM domain. Multiple dmrt1s (dmrt1a and dmrt1c) were obtained from Clarias batrachus as well. Tissue distribution (transcript and protein) of catfish dmrt1 revealed exclusive expression in testis. Semi-quantitative RT-PCR revealed the presence of multiple dmrt1s with high levels of dmrt1a in adult testis but not in ovary. Real-time RT-PCR analysis during testicular cycle showed higher levels of dmrt1 transcripts in preparatory and pre-spawning when compared with spawning and post-spawning phases. Immunocytochemical and immunofluorescence localization revealed the presence of catfish Dmrt1 protein in spermatogonia and spermatocytes, which indicates plausible role in spermatogenesis. Histological analysis indicated initiation of gonadal sex differentiation in catfish around 40-50 days after hatching. The potential role for dmrt1 in testicular differentiation is evident from its stage-dependent elevated expression in developing testis. Furthermore, dimorphic expressions of dmrt1s were evident at different stages of gonadal development or recrudescence in catfish. Treatment of methyl testosterone (MT) during early stages of gonadal sex differentiation resulted in adult males. Interestingly, we also obtained MT-treated fishes having ova-testis gonads. Analysis of dmrt1, sox9a, foxl2 and cyp19a1 expression patterns in MT-treated gonads revealed tissue-specific pattern. These results together suggest that multiple dmrt1s are testis-specific markers in catfish.

Vertebrate DM domain proteins bind similar DNA sequences and can heterodimerize on DNA

Background:The DM domain is a zinc finger-like DNA binding motif first identified in the sexual regulatory proteins Doublesex (DSX) and MAB-3, and is widely conserved among metazoans. DM domain proteins regulate sexual differentiation in at least three phyla and also control other aspects of development, including vertebrate segmentation. Most DM domain proteins share little similarity outside the DM domain. DSX and MAB-3 bind partially overlapping DNA sequences, and DSX has been shown to interact with DNA via the minor groove without inducing DNA bending. DSX and MAB-3 exhibit unusually high DNA sequence specificity relative to other minor groove binding proteins. No detailed analysis of DNA binding by the seven vertebrate DM domain proteins, DMRT1-DMRT7 has been reported, and thus it is unknown whether they recognize similar or diverse DNA sequences.Results:We used a random oligonucleotide in vitro selection method to determine DNA binding sites for six of the seven proteins. These proteins selected sites resembling that of DSX despite differences in the sequence of the DM domain recognition helix, but they varied in binding efficiency and in preferences for particular nucleotides, and some behaved anomalously in gel mobility shift assays. DMRT1 protein from mouse testis extracts binds the sequence we determined, and the DMRT proteins can bind their in vitro-defined sites in transfected cells. We also find that some DMRT proteins can bind DNA as heterodimers.Conclusion:Our results suggest that target gene specificity of the DMRT proteins does not derive exclusively from major differences in DNA binding specificity. Instead target specificity may come from more subtle differences in DNA binding preference between different homodimers, together with differences in binding specificity between homodimers versus heterodimers.

Differential and spermatogenic cell-specific expression of DMRT1 during sex reversal in protogynous hermaphroditic groupers

DMRT1 has been suggested to play different roles in sex determination and gonad differentiation, because different expression patterns have been reported among different vertebrates. The groupers, since their gonads first develop as ovary and then reverse into testis, have been thought as good models to study sex differentiation and determination. In this study, we cloned the full-length cDNAs of DMRT1 gene from orange-spotted grouper ( Epinephelus coioides), and prepared corresponding anti- EcDMRT1 antiserum to study the relationship of DMRT1 to sex reversal. One important finding is that the grouper DMRT1 is not only differentially expressed in different stage gonads, but also restricted to specific stages and specific cells of spermatogenesis. Grouper DMRT1 protein exists only in spermatogonia, primary spermatocytes and secondary spermatocytes, but not in the supporting Sertoli cells. Moreover, we confirmed that EcSox3 is expressed not only in oogonia and different stage oocytes, but also in Sertoli cells and spermatogonia, and EcSox9 is expressed only in Sertoli cells. The data suggested that grouper DMRT1 might be a more specific sex differentiation gene for spermatogenesis, and play its role at the specific stages from spermatogonia to spermatocytes. In addition, no introns were found in the grouper DMRT1, and no duplicated DMRT1 genes were detected. The finding implicates that the intronless DMRT1 that is able to undergo rapid transcriptional turnover might be a significant gene for stimulating spermatogenesis in the protogynous hermaphroditic gonad.

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

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.

Expression of Dmrt1 protein in developing and in sex-reversed gonads of amphibians

Many genes are known to be involved in gonadal differentiation in vertebrates. Dmrt1, a gene that encodes a transcription factor with a DM-domain, is considered to be one of the essential genes controlling testicular differentiation in mammals, birds, reptiles, amphibians and fish. However, it still remains unknown which testicular cells of animals other than mice and chicks express Dmrt1 protein. For an explanation of its role(s) in testicular differentiation in vertebrates, the expression of the Dmrt1 protein needs to be studied. For this purpose, we conducted an immunohistochemical study of this protein in an amphibian by using an antibody specific for Dmrt1. No positive signal was found in the indifferent gonad of tadpoles of Rana rugosa at early stages. However, in the testis of tadpoles at later stages (XV–XXV) and in frogs one month after metamorphosis, this protein was expressed in interstitial cells and Sertoli cells. In the testis of adult frogs, germ cells also expressed Dmrt1 protein. RT-PCR analysis revealed that the gene for this protein was not transcribed at any time during ovarian development, but was expressed in the female to male sex-reversed gonad. This was true when immunohistological studies were performed. In addition, Southern blot analysis showed Dmrt1 to be an autosomal gene. Taken together, our findings indicate that Dmrt1 protein is expressed by interstitial cells, Seroli cells and germ cells in the testis of R. rugosa. Dmrt1 may thus be very involved in the testicular differentiation of amphibians.