DM Domain Research Articles

The Dmrt1 gene: Identification and functional analysis in testicular development and spermatogenesis in the freshwater prawn, Macrobrachium nipponense

Products of the Dmrt gene family are known to harbor a zinc-finger DM domain and play crucial roles as transcription factors in various physiological processes, including sexual differentiation, gonadal development, and spermatogenesis. Despite their significance, research on the Dmrt family in Crustacea remains limited. In the present study, we identified a member of this gene family in the freshwater prawn Macrobrachium nipponense and designated it as MnDmrt1. We investigated its expression patterns across different tissues, potential target genes, and its influence on testicular development and protein interactions within the testes. Our findings revealed that MnDmrt1 encodes a protein composed of 252 amino acids that is predominantly expressed in the testes and localized in spermatogonia, spermatocytes, and spermatozoa. Silencing MnDmrt1 disrupts spermatogenesis and alters the expression levels of several key genes associated with testicular development, spermatogenesis, and sex reversal, such as Dmrt11E, Insulin receptor, and Masculinizer. Additionally, we identified 59 proteins that interact with MnDmrt1 in the testis using pull-down assays and liquid chromatography-tandem mass spectrometry. Notably, the eyestalk negatively regulates the expression level of MnDmrt1 in the testis. Collectively, these results underscore the essential role of MnDmrt1 in testicular development and spermatogenesis in M. nipponense.

Diversity of transactivation regions of DMRT1 in vertebrates

BackgroundDoublesex and mab-3 related transcription factor (DMRT) 1, commonly found in all vertebrates, regulates the transcription of genes involved in the masculinization and maintenance of gonadal somatic cells and/or germline cell development. DMRT1 has a DNA-binding domain called the DM domain and a transcription regulatory region. Unlike the former, there is little knowledge about the latter transcription regulatory region. This study aimed to identify the transcription activation regions of DMRT1 from four species: humans and mice (mammals), leopard geckos (reptiles), and medaka (teleost fish), adding perspectives on evolutionary conservation and diversity.Methods and resultsFor each species, several expression plasmids of deletion mutants were constructed, and the resultant plasmid and a DMRT1-driven luciferase reporter were co-transfected into cultured cells to measure transactivation ability. The key point of this analysis is that the transactivation ability was normalized by quantifying the expression levels of DMRT1 variants using the HiBiT tag. As a result, two to three transactivation regions were suggested to exist in the C-terminal region of the DM domain in all four species. Among seven regions in DMRT1, the fourth region from the N-terminus contributed to transactivation common to the four species, and the sixth and seventh regions on the C-terminal side differed depending on the species.ConclusionsThese findings indicated that the regions involved in the transactivation ability of DMRT1 could subtly change during evolution, indicating diversity in transactivation domains.

The potential modulation mechanisms of Dmrt-1 and Dmrt-3 affecting testicular development of Scylla paramamosain

The Dmrt gene family, with conserved functions throughout evolution, contains DM domains that play roles in various physiological processes like sex determination and gonadal differentiation. The mud crab (Scylla paramamosain), a significant marine culture crab in China, exhibits sexual dimorphism and is an important subject for reproduction research. In this study, the ORFs of the SpDmrt-1 and SpDmrt-3 genes from the mud crab gonad transcriptome database were obtained and confirmed by PCR. Both genes were expressed at the highest level in the testis among various tissues and in the T2 stage testis during different gonad developmental stages. In situ hybridization results showed that both genes were expressed during spermatogenesis. Silencing one gene led to a decrease in the expression of the other. Moreover, the expressions of some gonadal development-related genes (dsx, foxl-2, vasa, Dmrt-like, and IAG) changed significantly. Transcriptomic data indicated that SpDmrt-1 and SpDmrt-3 regulate various signaling pathways, including the Notch signaling pathway, the MAPK signaling pathway, the Hippo signaling pathway, the Calcium signaling pathway, and several other gender-related pathways, showing their involvement in male mud crab development. Furthermore, after long-term interference with SpDmrt-1 in vitro, most cells in the testis of the mud crab were at the spermatid stage, only a small amount of spermatozoa could be observed, and obvious empty plaque-like areas appeared in the tissue. Our findings provided a crucial foundation for understanding the role of the Dmrt in the process of gonadal development in crustaceans.

NFS-29. DEVELOPMENTAL SCREENING AND MONITORING IN NEUROFIBROMATOSIS-1

Abstract BACKGROUND Pilot data showed developmental screening at Neurofibromatosis-1 (NF1) appointments is feasible to identify those needing neuropsychology or other referrals. Outcomes for this larger sample, many re-screened and/or seen for a neuropsychology visit, allow us to describe evolving clinical management. METHODS Screenings are offered annually (ages 2-17) at NF1 clinic appointments by the psychosocial team. Forty-eight early childhood patients (ages 2-5) were screened (54% male, 66.7% White,) while 35 were ineligible (e.g., recent neuropsychology visit) or experienced logistical barriers to screening. These groups did not differ in race/ethnicity, sex, glioma status, or childhood opportunity index. Parents completed well-validated measures including the PEDS: Developmental Milestones – Assessment Level (PEDS:DM), Modified Checklist for Autism in Toddlers, and/or an ADHD Rating for preschoolers. RESULTS On initial screens, 73.9% showed a delay of 25% or more on at least one PEDS:DM domain. Measures often aligned with clinical impressions (81.3%); however, when findings were contextualized with record review, there were false positives (8.3%) and negatives (10.4%). Screening identified emerging concerns in attention, fine-motor, and academic skills. Nearly half (47.9%) of patients had services at the time of screening, and new/modified recommendations were offered for 70.8%. One third were referred to our Education Coordinator or Social Worker for educational advocacy, support through Early Intervention/school evaluation, or outpatient referrals. Twenty-two (45.8%) were referred to Neuropsychology and 18 have now been seen (consultation, targeted testing, or comprehensive evaluation) to confirm or rule-out neurodevelopmental diagnoses (e.g., autism, ADHD, language or global delay) and further guide educational or intervention management. Follow-up screens (N = 21) have monitored developmental progress, previously subclinical symptoms rising to a significant level, and need for re-evaluation or additional services. CONCLUSIONS Multipronged systematic screening results in referrals for diagnostic clarity, guides transition from Early Intervention to school services, and supports dynamic management of changing concerns.

Identification and characterization of the Dmrt1B gene in the oriental river prawn, Macrobrachium nipponense.

Dmrt (doublesex and mab-3 related transcription factor) is a protein family of transcription factors implicated in sexual regulation. Dmrt proteins are widely conserved and known for their involvement in sex determination and differentiation across species, from invertebrates to humans. In this study, we identified a novel gene with a DM (doublesex/Mab-3)-domain gene in the river prawn, Macrobrachium nipponense, which we named MniDmrt1B due to its similarities and close phylogenetic relationship with Dmrt1B in Macrobrachium rosenbergii. Through amino acid alignments and structural predictions, we observed conservation and identified putative active sites within the DM domain. qRT-PCR analysis revealed that MniDmrt1B exhibited high expression levels in the testis, with consistently higher expression in males compared to females during development. Additionally, similar to other sex-regulated genes, the MniDmrt1B gene exhibited high expression levels during the sex differentiation-sensitive periods in M. nipponense. These results strongly indicated that MniDmrt1B probably plays an important role in testis development and sex differentiation in M. nipponense.

Genome-wide identification and expression analysis of Dmrt gene family and their role in gonad development of Pacific oyster (Crassostrea gigas).

Doublesex and Mab-3-related transcription factor (Dmrt) is a type of transcription factor with a zinc-finger DM structural domain, which plays a significant role in sex determination and differentiation in animals. Although Dmrt has been reported in many vertebrates and invertebrates, it has rarely been studied in bivalves. In this study, a total of three members of the Dmrt gene family were identified and characterized in Crassostrea gigas, and all these CgDmrt genes contained a conserved DM domain. Analysis of the phylogenetic tree and gene structure revealed that Dmrt genes clustered on one branch may have similar functions in bivalves. Expression profiling of CgDmrt mRNA in different tissues and stages of gonad development indicated that CgDmrt3 exhibited sexually dimorphic expression and played an important role in the development of the male gonad in C. gigas. Furthermore, analysis of CgDmrt mRNA expression between fertile triploids and sterile triploids showed that CgDmrt3 may be involved in sperm production. Collectively, the systematic analysis of the CgDmrt genes will provide potential insights into the function of these genes in gonadal development.

Analyses of the Dmrt family in a decapod crab, Eriocheir sinensis uncover new facets on the evolution of DM domain genes.

DM domain genes are a group of transcription factors that are integral to sexual development and its evolution in metazoans. Their functions and regulatory mechanisms are not well understood in Malacostraca (crabs and crayfish) while these sex regulators have been widely identified in the past decade. In this study, the Dmrt family was investigated in the decapod crab, Eriocheir sinensis. We find that most members of the EsDmrt family begin to enrich around the juvenile 1 stage. In reproductive organs, EsDsx1, EsDsx2, EsiDMY and EsiDmrt1a highly express in the male-specific androgenic gland (AG), while EsDmrt-like, EsDsx-like, EsDmrt11E, and EsiDmrt1b show relatively high expression in testis. Also, we find the highly aberrant expression of EsiDMY and EsiDmrt1a in the chimeric AG, strongly indicating their function in AG development. Moreover, RNA interference of EsDsx1, EsiDMY, and EsiDmrt1a results in a significant decrease in transcription of the Insulin-like androgenic hormone (IAG), respectively. Our findings suggest that Dmrt genes in E. sinensis primarily function in male sexual differentiation, especially in AG development. Besides, this study identifies two unique groups of Dmrt genes in Malacostraca: Dsx and iDmrt1. In Malacostraca Dsx, we uncover a cryptic mutation in the eight zinc motif-specific residues, which were firmly believed to be invariant across the Dmrt family. This mutation sets the Malacostraca Dsx apart from all the other Dmrt genes and implies a different way of transcriptional regulation. Genes from the iDmrt1 group show phylogenetical limitation to the malacostracan species and underwent positive selection, suggesting their highly specialized gene function to this class. Based on these findings, we propose that Dsx and iDmrt1 in Malacostraca have developed unique transcriptional regulation mechanisms to facilitate AG development. We hope that this study would contribute to our understandings of sexual development in Malacostraca and provide new insights into the evolutionary history of the Dmrt family.

Genome-wide identification and comparative analysis of Dmrt genes in echinoderms

The Dmrt (Doublesex-mab3-related transcription factor) gene family is a class of crucial transcription factors characterized by one or several conserved DM (Doublesex/Mab-3) domains. Dmrt family genes can participate in various physiological developmental processes, especially in sex determination/differentiation. Echinoderms are extremely important research objects in various fields, such as sex determination/differentiation and neuroscience. However, to date, the genome-wide characterization and analysis of Dmrt genes in echinoderms have not been investigated. In this study, the identification and analysis of Dmrt genes in 11 representative echinoderms were performed using bioinformatics methods. A total of 43 Dmrt genes have been found in the studied echinoderms, and the number of Dmrt genes in different species ranges from 2 to 5. The phylogenetic tree showed that all Dmrt genes from echinoderms can be subdivided into 5 classes, the Dmrt2-like class, Dmrt3-like class, Dmrt4/5-like class, Dsx-like class, and a novel Dmrt (starfish-specific) class. Furthermore, selective pressure assessment suggested that the Dmrt genes underwent purifying selection pressure. In general, this study provides a molecular basis for echinoderm Dmrt genes and may serve as a reference for in-depth phylogenomics.

Identification and characterization of the Doublesex gene and its mRNA isoforms in the brine shrimp Artemia franciscana.

Doublesex (DSX) proteins are members of the Doublesex/mab-3-related (DMRT) protein family and play crucial roles in sex determination and differentiation among the animal kingdom. In the present study, we identified two Doublesex (Dsx)-like mRNA isoforms in the brine shrimp Artemia franciscana (Kellogg 1906), which are generated by the combination of alternative promoters, alternative splicing and alternative polyadenylation. The two transcripts exhibited sex-biased enrichment, which we termed AfrDsxM and AfrDsxF. They share a common region which encodes an identical N-terminal DNA-binding (DM) domain. RT-qPCR analyses showed that AfrDsxM is dominantly expressed in male Artemia while AfrDsxF is specifically expressed in females. Expression levels of both isoforms increased along with the developmental stages of their respective sexes. RNA interference with dsRNA showed that the knockdown of AfrDsxM in male larvae led to the appearance of female traits including an ovary-like structure in the original male reproductive system and an elevated expression of vitellogenin. However, silencing of AfrDsxF induced no clear phenotypic change in female Artemia. These results indicated that the male AfrDSXM may act as inhibiting regulator upon the default female developmental mode in Artemia. Furthermore, electrophoretic mobility shift assay analyses revealed that the unique DM domain of AfrDSXs can specifically bind to promoter segments of potential downstream target genes like AfrVtg. These data show that AfrDSXs play crucial roles in regulating sexual development in Artemia, and further provide insight into the evolution of sex determination/differentiation in sexual organisms.

A testis-specific gene doublesex is involved in spermatogenesis and early sex differentiation of the Chinese mitten crab Eriocheir sinensis

The doublesex (Dsx) is a DM domain gene involved in sex determination and/or differentiation in insects. In this study, a novel sex-specific Dsx homolog, designated as EsDsx-like, was identified from the Chinese mitten crab Eriocheir sinensis. The full-length cDNA of EsDsx-like was 921 bp, encoding a protein of 215 amino acids. The EsDsx-like mRNA and protein were detected exclusively in the testis. During spermatogenesis, the EsDsx-like mRNA were continuously expressed in spermatogonium, spermatocyte, spermatid and spermatozoon. In situ hybridization analysis showed that the EsDsx-like mRNA was mainly detected in the nucleus of spermatogonium and spermatocyte, and then concentrated in a dot on the edge of the nucleus in spermatid. In spermatozoa, the hybridization signal was localized to the acrosomal tubule. Immunofluorescence analysis revealed that the EsDsx-like protein was detected mainly in the cytoplasm around the nucleus in spermatocyte and spermatid, and then appeared as a dot immuno-signal in the acrosomal tubule and distributed in the cup-shaped nucleus of the spermatozoa. Together, these results suggest a potential role for EsDsx-like in the formation of acrosome complex and nuclear morphogenesis during sperm maturation. In the early development, similar with the male sex regulator, insulin-like androgenic gland hormone (EsIAG), the EsDsx-like mRNA was specifically detected in all male individuals tested at juvenile III stage just before the first appearance of the petasma in morphological sex differentiation at juvenile IV. Further, RNAi knockdown of EsDsx-like significantly downregulated the expression of EsIAG in adult and juvenile-III males. The direct interaction between the EsDsx-like protein and EsIAG promoter was confirmed by dual-luciferase reporter assays, indicating that EsDsx-like could act as an upstream regulatory gene of EsIAG in sex differentiation. This study provided valuable insights into the role of sex-specific gene associated with morphological sex differentiation and might contribute to development of sex control techniques in the Chinese mitten crab.

Characterization, expression profiling, and estradiol response analysis of DMRT3 and FOXL2 in clam Cyclina sinensis

The clam Cyclina sinensis is one of the important economical aquaculture shellfish in China. However, the mechanisms of sex determination and differentiation in C. sinensis have not been fully studied. In this study, full-length cDNAs of DMRT3 and FOXL2 were cloned and functionally characterized. The ORF region of CsDMRT3 consists of 1137 nucleotides, which encode 378 amino acids contains a conserved DM domain of DMRT family. The ORF region of CsFOXL2 is 1245 bp, encodes 414 amino acids, and contains a conserved FH domain. Tissue-specific expression results showed that the higher expression level of CsDMRT3 and CsFOXL2 was found in the ovary and testis of C. sinensis. The expression levels of CsDMRT3 and CsFOXL2 also peaked at the maturation stage of male and female gonadal development, respectively. Moreover, the expression levels of CsDMRT3 and CsFOXL2 were significantly higher in the trochophore and D-larval stages than in other stages. The transcript levels of CsDMRT3 reached the highest level at 11 months of age, while the CsFOXL2 reached the highest level at 7 months of age. In estradiol-treated experiments, the expression levels of CsDMRT3 and CsFOXL2 in the gonads were highest at 5 µg/L estradiol treatment, and histologically, it was observed that the oocytes diameters became larger with increasing estradiol concentration. These results suggest that CsDMRT3 and CsFOXL2 play an important role in gonadal development and sex differentiation of C. sinensis.

Dmrtb1 is involved in the testicular development in Larimichthys crocea.

dmrtb1 performs critical functions in sex determination/differentiation and gonadal development in many organisms, but its role in teleost is rarely studied. Through gene cloning, in situ hybridization, and RNA interference technology, the function of dmrtb1 in testicular development of large yellow croaker (Larimichthys crocea) was studied; our study will be helpful in understanding further the molecular regulation mechanism of Lcdmrtb1/Lcdmrt6 in testicular development in L. crocea, and our results enrich the theory of fish dmrts involved in reproductive regulation and provide a new idea for sex control breeding of L. crocea by manipulating reproductive pathway. Doublesex- and mab-3-related transcription factor B1 (dmrtb1/dmrt6) belongs to one of the members of DMRT family, which performs critical functions in sex determination and differentiation, gonadal development, and functional maintenance. However, knowledge of its exact mechanism remains unclear in teleost. Very little is known about the role of dmrtb1 in the gonad development of Larimichthys crocea. In this study, a dmrtb1 homolog in L. crocea named as Lcdmrtb1 with the full-length cDNA was isolated and characterized. Except for the conserved DM domain, the other regions had low homology. Of the tissues sampled, Lcdmrtb1 was only found to be highly expressed in the testis. In situ hybridization of testis revealed Lcdmrtb1 in both spermatogonia and spermatocytes. After Lcdmrtb1 interference in the testis cells (LYCT) of L. crocea, the expression levels of Lcdmrtb1 and Lcdmrt1 were significantly decreased; subsequently, testicular cell morphology changed from fibrous to round and their growth rate slowed. Similarly, the expression levels of Lcdmrtb1, Lcdmrt1, sox9a/b, and amh were significantly decreased after RNAi in the testis. Furthermore, it was discovered that the spermatogonia had disappeared, and the Sertoli cells had been reduced. The results of immunohistochemistry showed that the expression of Sox9 protein in the testis was not detected after dmrtb1 was knocked down. These results indicated that the absence of Lcdmrtb1 not only greatly inhibited cell growth and destroyed the morphology of testis cells but also down-regulated Lcdmrt1 expression in the testis. This study will be helpful in understanding further the molecular regulation mechanism of Lcdmrtb1/Lcdmrt6 in testicular development in L. crocea.

The function and evolution of a genetic switch controlling sexually dimorphic eye differentiation in honeybees

Animals develop sex-specific morphological structures that are diverse between organisms. However, understanding the developmental and evolutionary mechanisms governing these traits is still limited and largely restricted to DM domain genes, which are conserved, sex-specific developmental regulators identified in genetic models. Here, we report a sex-specific developmental regulator gene, glubschauge (glu) that selectively regulates sexually dimorphic eye differentiation in honeybees. We found that the sex determination gene feminizer (fem) controls sex-specific splicing of glu transcripts, establishing a genetic switch in which Glu proteins with a zinc finger (ZnF) domain are only expressed in females. We showed that female coding sequence was essential and sufficient for partial feminization. Comparative sequence and functional studies revealed that the evolutionary origination of the genetic switch was followed by the mutational origin of the essential ZnF domain. Our results demonstrate that glu is a newly evolved sex-specific genetic switch for region-specific regulation of a dimorphic character.

The Sex-Specific Splicing of Doublesex in Brine Shrimp Artemia franciscana.

The understanding of sex determination and differentiation in animals has recently made remarkable strides through the use of advanced research tools. At the gene level, the Mab-3-related transcription factor (Dmrt) gene family, which encodes for the typical DNA-binding doublesex/Mab-3 (DM) domain in their protein, is known for its contribution to sex determination and differentiation in insects. In this study, DNA-binding DM domain screening has identified eight transcripts from Artemia franciscana transcriptomic that encode proteins containing one conserved DNA-binding DM domain. The genome mapping confirmed that these eight transcripts are transcribed from six different loci on the A. franciscana genome assembly. One of those loci, the Af.dsx-4 locus, is closely related to Doublesex, a gene belonging to the Dmrt gene family. This locus could be transcribed into three alternative transcripts, namely Af.dsx4, Af.dsxF and Af.dsxM. While Af.dsx4 and Af.dsxF could putatively be translated to form an identical Af.dsxF protein of 186 aa long, Af.dsxM translates for an Af.dsxM protein of 289 aa long but shares a DNA-binding DM domain. Interestingly, Af.dsxF and Af.dsxM are confirmed as sex-specific transcripts, Af.dsxF is only present in females, and Af.dsxM is only present in male individuals. The results suggest that the sex-specific splicing mechanism of the doublesex described in insects is also present in A. franciscana. Af.dxs-4 locus can be used in further studies to clarify the sex determination pathways in A. fracnciscana.

Cloning, expression, and function of the Spdmrt-like gene in Scylla paramamosain.

The mud crab Scylla paramamosain is an economically important species for aquaculture in China and has sexually dimorphic between females and males. Understanding sex differentiation in this species is essential for the development of monosex aquaculture. The Dmrt genes play a vital role in sex differentiation in animals. In this study, two dmrt-like transcript variants, Spdmrt-like-tv1 and Spdmrt-like-v2, were cloned. SpDmrt-like-tv1 contained a DM domain, while SpDmrt-like-tv2 contained a DM and a DMA domain. Spdmrt-like-tv1 and Spdmrt-like-tv2 were both specifically expressed in testis. During testicular development, the expression level of Spdmrt-like-tv1 increased from stage I to stage II (P > 0.05) and then decreased from stage II to stage III (P < 0.05). The expression level of Spdmrt-like-tv2 in stages I and II was significantly higher than that in stage III (P < 0.05). During embryonic development, the expression level of Spdmrt-like-tv1 was higher in the mid-embryonic stage compared with the early and late stages, but the differences were not significant. Moreover, the expression level of Spdmrt-like-tv2 was stable and remained high throughout embryonic development. Furthermore, the expression level of Spdmrt-like-tv2 was significantly higher than that of Spdmrt-like-tv1. Knockdown of Spdmrt-like variants indicated that the regulative target gene of Spdmrt-like-tv1 was Spsox21, and the regulative target genes of Spdmrt-like-tv2 were Spfoxl2 and Spsox21. Combined with the results in our previously published peer-reviewed articles that the expression of Spfoxl2 in the testis was significantly higher than that in the ovary, and Spfoxl2 negatively regulated Spvtg expression. Spsox21 played a role in the development and maintenance of testis as well as in the process of neural development and regulation of body segmentation. Therefore, we suggest that Spdmrt-like-tv1 and Spdmrt-like-tv2 might be involved in testicular development and embryonic development, and Spdmrt-like-tv2 might play more important roles in these two developmental processes by regulating the expression of Spfoxl2 and Spsox21 due to its high expression.

Genome-Wide Identification, Phylogeny, and Expression Profile of the Dmrt (Doublesex and Mab-3 Related Transcription Factor) Gene Family in Channel Catfish (Ictalurus punctatus).

The Dmrt (Doublesex and Mab-3 related transcription factor) gene family is a class of crucial transcription factors characterized by a conserved DM domain related to sex determination and differentiation, which has been systematically described in various teleost fish, but less in channel catfish (Ictalurus punctatus), an important global aquaculture species in the US and China. In this study, seven Dmrt genes from channel catfish genome were identified and analyzed using bioinformatics methods. Seven IpDmrt genes were distributed unevenly across five chromosomes. Synteny analysis revealed that Dmrt1, Dmrt2a, Dmrt2b, Dmrt3, Dmrt4, and Dmrt5 were relatively conserved in teleost fish. Tissue distribution analysis showed that IpDmrt1, IpDmrt2b, IpDmrt5, and IpDmrt6 exhibited sexually dimorphic expression patterns and, among them, IpDmrt1 and IpDmrt6 had high expression levels in the testes, while IpDmrt2b and IpDmrt5 had more significant expression levels in the ovaries than in other tissues. After 17β-estradiol treatment, IpDmrt2b and IpDmrt5 were significantly up regulated, while the expression of IpDmrt1 and IpDmrt6 was significantly repressed in XY channel catfish ovaries compared with XX channel catfish ovaries. The present study provides a comprehensive insight into the Dmrt gene family of channel catfish. The results suggest that IpDmrt1 and IpDmrt6 may play an important role in testis differentiation/development, while IpDmrt2b and IpDmrt5 are critical in ovary development in this species.

Identification and functional analysis of the doublesex gene in the mud crab Scylla paramamosain

Doublesex (Dsx) is a crucial member of the Dmrt gene family and plays a vital role in sex determination and differentiation among the animal kingdom. In the present study, a doublesex (designated as Spdsx) gene was identified and characterized for the first time in the mud crab, Scylla paramamosain. The Spdsx cDNA contains an 801 bp open reading frame (ORF) encoding 266 amino acids with a conserved DM domain. Meanwhile, to elucidate the conservation of Dsx, its orthologus were identified in several crustacean species as well. In addition, the expression pattern of Spdsx in various adult tissues and during embryo development was analyzed with qRT-PCR technology. Finally, the roles of Spdsx might play in the testis, androgenic gland, and ovary were analyzed by RNAi technology. The main results are as follows: (1) the Spdsx gene widely existed in analyzed crustacean species, and the multiple sequences alignment result indicated the conservation of Dsx was low except for the DM domain; (2) only one dsx gene was identified in analyzed crab and lobster, while 2 dsx genes (dsx-1 and dsx-2) were identified in shrimps; (3) the Spdsx gene was widely expressed in analyzed tissues, and the expression level in androgenic gland was obviously higher than that in other tissues. Interestingly, the expression level of Spdsx in the ovary was significantly higher than that in testis (p < 0.05); (4) The expression pattern of Spdsx during embryo development was divided into two groups: remained stable from blastula stage to 5 pairs of appendages stage; after 5 pairs of appendages stage, the expression level increased and remained stable from 7 pairs of appendages stage to hatching stage; (5) After the silencing of Spdsx, the expression level of marker genes in testis, ovary, and androgenic gland significantly changed, among which the expression level of vtg and vtgR in ovary down-regulated, the dmrt-like and dmrt-1a (exclusively expressed in testis) in testis up-regulated and the IAG in androgenic gland down-regulated. All the results above demonstrated that the Spdsx play crucial roles in regulating the reproduction system development of mud crab.

Characterization, expression and CpG methylation analysis of Dmrt1 and its response to steroid hormone in blotched snakehead (Channa maculata).

Dmrt1 is an important transcriptional regulator that plays critical role in male gonadogenesis, testicular differentiation and development. In this study, Dmrt1 was cloned from blotched snakehead (Channa maculata), which is designated as CmDmrt1. CmDmrt1 encoded a putative protein with 293 amino acids and presented an extremely conserved DM domain. It was nearly expressed in the gonads, and the expression was more than 15 times higher in the testis than in the ovary. 1851bp promoter sequence of CmDmrt1 was characterized and the methylation levels of the CpG sites were analyzed to detect sex-related differences. A significant negative correlation between CmDmrt1 expression and CpG methylation level of its promoter was found in the testis and ovary. During gonadal development, CmDmrt1 transcription displayed strong male-biased expression patterns, increased with the maturation of testis and reached the peak at 195days after hatching (dah), which indicates a significant role of Dmrt1 in spermatogenesis. Steroid treatment could influence CmDmrt1 expression, and long-term 17β-estradiol (E2) treatment could induce the male-to-female secondary sex reversal (SSR), which resulted in the differentiated testis transformed to ovary or ovotestis. Meanwhile, CmDmrt1 expression was down-regulated to fairly low level in the ovary of the SSR XY fish, which was similar to that in normal XX females ovary. Our research illustrates that Dmrt1 is linked to testis differentiation and spermatogenesis in blotched snakehead, providing information for functional studies on sex differentiation and gonadal development of C. maculata, and scientific basis for the production practice of all-male snakehead breeding.

Molecular characterization and functional analysis of DMRT11E in black tiger shrimp (Penaeus monodon)

As a highly conserved transcription factor family, DMRTs (Doublesex and Mab-3-related transcription factors) are involved in sex determination and sexual differentiation in numerous animals. In the present study, we identified a DM (Doublesex/Mab-3)-domain gene in Penaeus monodon, which we named PmDMRT11E because it shared many similarities with DMRT11E homologs and has a close relationship in the phylogeny of different species. The three-dimensional structure and amino acid alignments indicated the stability and conservation of the DM domain. Real-time PCR analysis revealed that PmDMRT11E was highly and preferentially expressed in the testis of sub-adult shrimp. During embryogenesis, the expression level of PmDMRT11E was higher at the nauplius than at other periods. PmDMRT11E dsRNA injection significantly decreased the transcription factor Sox9 and increased ovarian marker Foxl2 expression, implying its important regulatory role in the mechanism of male determination. Moreover, dsDMRT11E caused abnormal sperm development, indicating that PmDMRT11E may be closely related to gonadal development and spermatogenesis.

Variance in expression and localization of sex-related genes CgDsx, CgBHMG1 and CgFoxl2 during diploid and triploid Pacific oyster Crassostrea gigas gonad differentiation.

Several evolutionarily conserved classes of transcriptional regulators were involved in diverse sex determination and differentiation pathways across taxa, whereas their roles in most mollusks is still limited. The Pacific oyster Crassostrea gigas, a dioecious bivalve with sex reversal, could be an ideal model for this issue because of its complex sexuality and potential disruption of sex differentiation in triploid individuals. Here, two mRNA splicing isoforms of a DM domain gene CgDsx and two isoforms of a novel sex-related CgBHMG1 (ortholog of BHMG1 in mammals) were identified in C. gigas. Real time PCR showed that two isoforms of CgDsx and one isoform of CgBHMG1 displayed male-specific expression in diploid oysters, opposite with the female-specific CgFoxl2 (a potential factor of female gonadic differentiation). Interestingly, the four sex-specific transcripts in diploid oyster were expressed in triploid oysters with opposite sex, triploid hermaphrodites and individuals at stage I that sex could not be determined. Subsequent in situ hybridization analysis on gonads of diploid oysters revealed predominant expression of CgDsx in spermatogonia of testes, CgBHMG1 in spermatocytes of testes and follicle cells of ovaries, and CgFoxl2 in follicle cells of ovaries and some male germ cells in testes. And aberrant co-expression of the three genes in triploid oysters was localized in gonadal tubules of gonads at stage I, ovarian follicle cells and undetermined gonial cells in nontypical hermaphroditic gonads with rare female materials. From the above, temporal and spatial expression of sex-related genes in diploid and triploid gonads indicated that CgDsx and CgFoxl2 might mainly function in C. gigas sex differentiation, and CgBHMG1 appeared as a factor involved in meiosis. This work will help to illuminate the gene network of sex differentiation in bivalves and provides new sight on this issue from comparison between diploid and triploid individuals.