DMRT1-mediated regulation of TOX3 modulates expansion of the gonadal steroidogenic cell lineage in the chicken embryo.

During gonadal sex determination, the supporting cell lineage differentiates into Sertoli cells in males and pre-granulosa cells in females. Recently, single cell RNA-seq data have indicated that chicken steroidogenic cells are derived from differentiated supporting cells. This differentiation process is achieved by a sequential upregulation of steroidogenic genes and downregulation of supporting cell markers. The exact mechanism regulating this differentiation process remains unknown. We have identified TOX3 as a previously unreported transcription factor expressed in embryonic Sertoli cells of the chicken testis. TOX3 knockdown in males resulted in increased CYP17A1-positive Leydig cells. TOX3 overexpression in male and female gonads resulted in a significant decline in CYP17A1-positive steroidogenic cells. In ovo knockdown of the testis determinant DMRT1 in male gonads resulted in a downregulation of TOX3 expression. Conversely, DMRT1 overexpression caused an increase in TOX3 expression. Taken together, these data indicate that DMRT1-mediated regulation of TOX3 modulates expansion of the steroidogenic lineage, either directly, via cell lineage allocation, or indirectly, via signaling from the supporting to steroidogenic cell populations.

Perturbed development of calb2b expressing dI6 interneurons and motor neurons underlies locomotor defects observed in calretinin knock-down zebrafish larvae.

Calcium binding proteins are essential for neural development and cellular activity. Calretinin, encoded by calb2a and calb2b, plays a role during early zebrafish development and has been proposed as a marker for distinct neuronal populations within the locomotor network. We generated a calb2b:hs:eGFP transgenic reporter line to characterize calretinin expressing cells in the developing spinal cord and describe morphological and behavioral defects in calretinin knock-down larvae. eGFP was detected in primary and secondary motor neurons, as well as in dI6 and V0v interneurons. Knock-down of calretinin lead to disturbed development of motor neurons and dI6 interneurons, revealing a crucial role during early development of the locomotor network. Primary motor neurons showed delayed axon outgrowth and the distinct inhibitory CoLo neurons, originating from the dI6 lineage, were absent. These observations explain the locomotor defects we observed in calretinin knock-down animals where the velocity, acceleration and coordination were affected during escapes. Altogether, our analysis suggests an essential role for calretinin during the development of the circuits regulating escape responses and fast movements within the locomotor network.

The Curious Case of Avian Sex Determination.

Ioannidis and colleagues show that the gene DMRT1 is the master regulator of testis development in the chicken. Yet, remarkably, when this gene is deleted in genetic males and gonads form ovaries, the body remains male. This debunks the notion that somatic sex is driven primarily by hormones in birds.

The microRNA miR-202 prevents precocious spermatogonial differentiation and meiotic initiation during mouse spermatogenesis.

Spermatogonial differentiation and meiotic initiation during spermatogenesis are tightly regulated by a number of genes, including those encoding enzymes for miRNA biogenesis. However, whether and how single miRNAs regulate these processes remain unclear. Here, we report that miR-202, a member of the let-7 family, prevents precocious spermatogonial differentiation and meiotic initiation in spermatogenesis by regulating the timely expression of many genes, including those for key regulators such as STRA8 and DMRT6. In miR-202 knockout (KO) mice, the undifferentiated spermatogonial pool is reduced, accompanied by age-dependent decline of fertility. In KO mice, SYCP3, STRA8 and DMRT6 are expressed earlier than in wild-type littermates, and Dmrt6 mRNA is a direct target of miR-202-5p. Moreover, the precocious spermatogonial differentiation and meiotic initiation were also observed in KO spermatogonial stem cells when cultured and induced in vitro, and could be partially rescued by the knockdown of Dmrt6. Therefore, we have not only shown that miR-202 is a regulator of meiotic initiation but also identified a previously unknown module in the underlying regulatory network.

Germline sexual fate is determined by the antagonistic action of dmrt1 and foxl3/foxl2 in tilapia.

Germline sexual fate has long been believed to be determined by the somatic environment, but this idea is challenged by recent studies of foxl3 mutants in medaka. Here, we demonstrate that the sexual fate of tilapia germline is determined by the antagonistic interaction of dmrt1 and foxl3, which are transcriptionally repressed in male and female germ cells, respectively. Loss of dmrt1 rescued the germ cell sex reversal in foxl3Δ7/Δ7 XX fish, and loss of foxl3 partially rescued germ cell sex reversal but not somatic cell fate in dmrt1Δ5/Δ5 XY fish. Interestingly, germ cells lost sexual plasticity in dmrt1Δ5/Δ5 XY and foxl3Δ7/Δ7 XX single mutants, as aromatase inhibitor (AI) and estrogen treatment failed to rescue the respective phenotypes. However, recovery of germ cell sexual plasticity was observed in dmrt1/foxl3 double mutants. Importantly, mutation of somatic cell-specific foxl2 resulted in testicular development in foxl3Δ7/Δ7 or dmrt1Δ5/Δ5 mutants. Our findings demonstrate that sexual plasticity of germ cells relies on the presence of both dmrt1 and foxl3. The existence of dmrt1 and foxl3 allows environmental factors to influence the sex fate decision in vertebrates.

Familial DMRT1-related non-obstructive azoospermia: a case report.

PURPOSE: To report an exceptional case of male-to-male transmission of genetically based non-obstructive azoospermia (NOA) and varicocele through a naturally obtained pregnancy. SUBJECTS AND METHODS: A father and his son were both diagnosed with NOA after centrifugation and varicocele. The father has no other clinical concerns apart from infertility, detected after many attempts of having another child, but given his urological situation (bilateral varicocele and NOA) assisted reproductive techniques were discouraged. After genetic counseling, several genetic-chromosomal analyses were carried out in the son (karyotype, chromosome Y microdeletions, CFTR screening, NGS infertility panels, and finally array-CGH). RESULTS: After a series of inconclusive tests, array-CGH detected a deletion of 224-283 kb (del9p24.3) involving part of the KANK1 and DMRT1 genes, inherited from the father. Haploinsufficiency of DMRT1 was therefore considered the determining factor in the development of azoospermia in the family by a loss of function mechanism. CONCLUSION: The confirmation of father-to-son transmission of a deletion including DMRT1 represents an important point for clinicians dealing with male infertility, even when complete azoospermia is repetitively detected, and must be of hope for a relevant portion of men. Inclusion criteria for the access to assisted reproductive techniques may also be reconsidered and worthy of a greater number of clinical insights. Finally, since DMRT1 alterations have been associated with NOA and abnormal testicular development, but not specifically with varicocele, further studies are required to validate this issue, as varicocele may have played a crucial role in this case.

A Testis-Specific DMRT1 (Double Sex and Mab-3-Related Transcription Factor 1) Plays a Role in Spermatogenesis and Gonadal Development in the Hermaphrodite Boring Giant Clam Tridacna crocea.

The testis-specific double sex and mab-3-related transcription factor 1 (DMRT1) has long been recognized as a crucial player in sex determination across vertebrates, and its essential role in gonadal development and the regulation of spermatogenesis is well established. Here, we report the cloning of the key spermatogenesis-related DMRT1 cDNA, named Tc-DMRT1, from the gonads of Tridacna crocea (T. crocea), with a molecular weight of 41.93 kDa and an isoelectric point of 7.83 (pI). Our hypothesis is that DMRT1 machinery governs spermatogenesis and regulates gonadogenesis. RNAi-mediated Tc-DMRT1 knockdown revealed its critical role in hindering spermatogenesis and reducing expression levels in boring giant clams. A histological analysis showed structural changes, with normal sperm cell counts in the control group (ds-EGFP) but significantly lower concentrations of sperm cells in the experimental group (ds-DMRT1). DMRT1 transcripts during embryogenesis exhibited a significantly high expression pattern (p < 0.05) during the early zygote stage, and whole-embryo in-situ hybridization confirmed its expression pattern throughout embryogenesis. A qRT-PCR analysis of various reproductive stages revealed an abundant expression of Tc-DMRT1 in the gonads during the male reproductive stage. In-situ hybridization showed tissue-specific expression of DMRT1, with a positive signal detected in male-stage gonadal tissues comprising sperm cells, while no signal was detected in other stages. Our study findings provide an initial understanding of the DMRT1 molecular machinery controlling spermatogenesis and its specificity in male-stage gonads of the key bivalve species, Tridacna crocea, and suggest that DMRT1 predominantly functions as a key regulator of spermatogenesis in giant clams.

Genome-wide identification and expression analysis of Dmrt genes in bivalves.

In recent years, some common themes in the development of sex-specific traits in different animal lineages have started to emerge since the discovery of the Dmrt (doublesex-mab3-related transcription factor gene) genes. Bivalves are characterized by a diversity of sexual systems, including simultaneous hermaphroditism, sequential hermaphroditism, and strict gonochorism. However, to date, no research has focused on the genome-wide characterization and analysis of Dmrt genes in bivalves. In this study, the identification and analysis of Dmrt genes in 15 bivalves were performed using bioinformatics methods. A total of 55 Dmrt genes were retrieved in the studied bivalve genomes. The number of Dmrt genes in different species ranged from 3 to 5. The phylogenetic tree showed that Dmrt genes in bivalves can be subdivided into 5 classes: the Dmrt2-like class, Dmrt3-like class, Dmrt4/5-like class, Dsx-like class, and scallop-specific Dmrt class. The Ka/Ks ratios suggested that all Dmrt classes underwent purifying selection pressure. Furthermore, the spatiotemporal expression of Dmrt genes in four bivalve species suggested that different Dmrt genes may have different functions, and scallop-specific Dmrt genes may play a key role in sex determination/differentiation. In general, this study provides a molecular basis for in-depth examination of the functions of Dmrt genes and phylogenomic analyses in bivalves.

Disruption of dmrt1 rescues the all-male phenotype of the cyp19a1a mutant in zebrafish - a novel insight into the roles of aromatase/estrogens in gonadal differentiation and early folliculogenesis.

Sex determination and differentiation are complex processes controlled by many different factors; however, the relationships among these factors are poorly understood. Zebrafish gonadal differentiation exhibits high plasticity involving multiple factors and pathways, which provides an excellent model for investigating the interactions between them. Ovarian aromatase (cyp19a1a) and dmrt1 are key factors in directing vertebrate ovary and testis differentiation, respectively. Knockout of zebrafish cyp19a1a leads to all-male offspring, whereas the loss of dmrt1 results in a female-biased sex ratio. In the present study, we established dmrt1-/- ;cyp19a1a-/- double mutant zebrafish and discovered that the introduction of the dmrt1 mutation into the cyp19a1a mutant could rescue the all-male phenotype of the latter. Interestingly, despite the lack of aromatase/estrogens, the follicles in the ovary of the rescued cyp19a1a mutant could develop normally up to the previtellogenic stage. Further evidence suggested the ovarian aromatase directed ovarian differentiation by suppressing dmrt1 expression via nuclear estrogen receptors (nERs). Our results provide solid evidence for an interaction between cyp19a1a and dmrt1 in zebrafish gonadal differentiation, and for the dispensability of estrogens in controlling early folliculogenesis.

Loss of dmrt1 restores zebrafish female fates in the absence of cyp19a1a but not rbpms2a/b.

Sex determination and differentiation is a complex process regulated by multiple factors, including factors from the germline or surrounding somatic tissue. In zebrafish, sex-determination involves establishment of a bipotential ovary that undergoes sex-specific differentiation and maintenance to form the functional adult gonad. However, the relationships among these factors are not fully understood. Here, we identify potential Rbpms2 targets and apply genetic epistasis experiments to decipher the genetic hierarchy of regulators of sex-specific differentiation. We provide genetic evidence that the crucial female factor rbpms2 is epistatic to the male factor dmrt1 in terms of adult sex. Moreover, the role of Rbpms2 in promoting female fates extends beyond repression of Dmrt1, as Rbpms2 is essential for female differentiation even in the absence of Dmrt1. In contrast, female fates can be restored in mutants lacking both cyp19a1a and dmrt1, and prolonged in bmp15 mutants in the absence of dmrt1. Taken together, this work indicates that cyp19a1a-mediated suppression of dmrt1 establishes a bipotential ovary and initiates female fate acquisition. Then, after female fate specification, Cyp19a1a regulates subsequent oocyte maturation and sustains female fates independently of Dmrt1 repression.

Comprehensive molecular analysis identifies eight novel variants in XY females with disorders of sex development.

Disorders of sex development (DSD) are a group of clinical conditions with variable presentation and genetic background. Females with or without development of secondary sexual characters and presenting with primary amenorrhea (PA) and a 46,XY karyotype are one of the classified groups in DSD. In this study, we aimed to determine the genetic mutations in 25 females with PA and a 46,XY karyotype to show correlations with their phenotypes. Routine Sanger sequencing with candidate genes like SRY, AR, SRD5A2, and SF1, which are mainly responsible for 46,XY DSD in adolescent females, was performed. In a cohort of 25 patients of PA with 46,XY DSD, where routine Sanger sequencing failed to detect the mutations, next-generation sequencing of a targeted gene panel with 81 genes was used for the molecular diagnosis. The targeted sequencing identified a total of 21 mutations including 8 novel variants in 20 out of 25 patients with DSD. The most frequently identified mutations in our series were in AR (36%), followed by SRD5A2 (20%), SF1 (12%), DHX37 (4%), HSD17B3 (4%), and DMRT2 (4%). We could not find any mutation in the DSD-related genes in five (20%) patients due to complex molecular mechanisms in 46,XY DSD, highlighting the possibility of new DSD genes which are yet to be discovered in these disorders. In conclusion, genetic testing, including cytogenetics and molecular genetics, is important for the diagnosis and management of 46,XY DSD cases.

Identification and characterization of single nucleotide polymorphisms in DMRT3 gene in Indian horse (Equus caballus) and donkey (Equus asinus) populations.

Equines' ability in racing and riding as well as gaitedness have influenced the human civilization. Aim of this study was to identify and characterize the novel polymorphisms or SNPs in DMRT3 gene in Indian horse and donkey breeds. In this study, the DMRT3 gene was sequenced and characterized in 72 Indian horses' and 33 Indian donkeys' samples. One SNP (A > C) at 878 was found in studied horses while identical SNPs (A > C) at two different nucleotide positions i.e., 878 and 942 in DMRT3 gene (chromosome 23) were observed in studied Indian donkey breeds. Horses and donkeys both have a non-synonymous mutation (A > C) at nucleotide 878 (codon 61) that converts a Stop codon (TAG > TCG) to coding codon Serine, whereas donkeys have a synonymous mutation at nucleotide 942 (codon 82) that converts Serine (TCA > TCC) into Serine. A phylogenetic tree indicated that the DMRT3 gene was equally distributed among the equine breeds. Most of the donkey breeds have been shown high levels of genetic diversity while horse breeds and Halari donkey showed the least genetic diversity. Mutation in DMRT3 has a major impact on gaitedness in horses and is presented at a high frequency in gaited breeds and in horses breed for harness racing.

Transposon-induced epigenetic silencing in the X chromosome as a novel form of dmrt1 expression regulation during sex determination in the fighting fish.

BACKGROUND: Fishes are the one of the most diverse groups of animals with respect to their modes of sex determination, providing unique models for uncovering the evolutionary and molecular mechanisms underlying sex determination and reversal. Here, we have investigated how sex is determined in a species of both commercial and ecological importance, the Siamese fighting fish Betta splendens. RESULTS: We conducted association mapping on four commercial and two wild populations of B. splendens. In three of the four commercial populations, the master sex determining (MSD) locus was found to be located in a region of ~ 80 kb on LG2 which harbours five protein coding genes, including dmrt1, a gene involved in male sex determination in different animal taxa. In these fish, dmrt1 shows a male-biased gonadal expression from undifferentiated stages to adult organs and the knockout of this gene resulted in ovarian development in XY genotypes. Genome sequencing of XX and YY genotypes identified a transposon, drbx1, inserted into the fourth intron of the X-linked dmrt1 allele. Methylation assays revealed that epigenetic changes induced by drbx1 spread out to the promoter region of dmrt1. In addition, drbx1 being inserted between two closely linked cis-regulatory elements reduced their enhancer activities. Thus, epigenetic changes, induced by drbx1, contribute to the reduced expression of the X-linked dmrt1 allele, leading to female development. This represents a previously undescribed solution in animals relying on dmrt1 function for sex determination. Differentiation between the X and Y chromosomes is limited to a small region of ~ 200 kb surrounding the MSD gene. Recombination suppression spread slightly out of the SD locus. However, this mechanism was not found in the fourth commercial stock we studied, or in the two wild populations analysed, suggesting that it originated recently during domestication. CONCLUSIONS: Taken together, our data provide novel insights into the role of epigenetic regulation of dmrt1 in sex determination and turnover of SD systems and suggest that fighting fish are a suitable model to study the initial stages of sex chromosome evolution.

Doublesex controls specification and maintenance of the gonad stem cell niches in Drosophila.

Sex-specific development of the gonads is a key aspect of sexual dimorphism that is regulated by Doublesex/Mab3-related transcription factors (DMRTs) in diverse animal species. We find that in mutants for Drosophila dsx, important components of the male and female gonad stem cell niches (hubs and terminal filaments/cap cells, respectively) still form. Initially, gonads in all dsx mutants (both XX and XY) initiate the male program of development, but later half of these gonads switch to form female stem cell niche structures. One individual can have both male-type and female-type gonad niches; however, male and female niches are usually not observed in the same gonad, indicating that cells make a 'group decision' about which program to follow. We conclude that dsx does not act in an instructive manner to regulate male versus female niche formation, as these structures form in the absence of dsx function. Instead, dsx acts to 'tip the balance' between the male or female programs, which are then executed independently of dsx We show that bric a brac acts downstream of dsx to control the male versus female niche decision. These results indicate that, in both flies and mammals, the sexual fate of the somatic gonad is remarkably plastic and is controlled by a combination of autonomous and non-autonomous cues.

DMRT1 gene disruption alone induces incomplete gonad feminization in chicken.

Compared with the well-described XY sex determination system in mammals, the avian ZW sex determination system is poorly understood. Knockdown and overexpression studies identified doublesex and mab-3-related transcription factor 1 (DMRT1) as the testis-determining gene in chicken. However, the detailed effects of DMRT1 gene disruption from embryonic to adult development are not clear. Herein, we have generated DMRT1-disrupted chickens using the clustered regularly interspaced short palindromic repeats-associated protein 9 system, followed by an analysis of physiological, hormonal, and molecular changes in the genome-modified chickens. In the early stages of male chicken development, disruption of DMRT1 induced gonad feminization with extensive physiological and molecular changes; however, functional feminine reproductivity could not be implemented with disturbed hormone synthesis. Subsequent RNA-sequencing analysis of the DMRT1-disrupted chicken gonads revealed gene networks, including several novel genes linearly and non-linearly associated with DMRT1, which are involved in gonad feminization. By comparing the gonads of wild type with the genome-modified chickens, a set of genes were identified that is involved in the ZW sex determination system independent of DMRT1. Our results extend beyond the Z-dosage hypothesis to provide further information about the avian ZW sex determination system and epigenetic effects of gonad feminization.

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

BACKGROUND: 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. METHODS AND RESULTS: 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. CONCLUSION: 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.

Snow Parameters Inversion from Passive Microwave Remote Sensing Measurements by Deep Convolutional Neural Networks.

This paper proposes a novel inverse method based on the deep convolutional neural network (ConvNet) to extract snow's layer thickness and temperature via passive microwave remote sensing (PMRS). The proposed ConvNet is trained using simulated data obtained through conventional computational electromagnetic methods. Compared with the traditional inverse method, the trained ConvNet can predict the result with higher accuracy. Besides, the proposed method has a strong tolerance for noise. The proposed ConvNet composes three pairs of convolutional and activation layers with one additional fully connected layer to realize regression, i.e., the inversion of snow parameters. The feasibility of the proposed method in learning the inversion of snow parameters is validated by numerical examples. The inversion results indicate that the correlation coefficient (R2) ratio between the proposed ConvNet and conventional methods reaches 4.8, while the ratio for the root mean square error (RMSE) is only 0.18. Hence, the proposed method experiments with a novel path to improve the inversion of passive microwave remote sensing through deep learning approaches.

Transcriptome Profiling and Expression Localization of Key Sex-Related Genes in a Socially-Controlled Hermaphroditic Clownfish, Amphiprion clarkii.

Clownfish can be an excellent research model for investigating the socially-controlled sexual development of sequential hermaphrodite teleosts. However, the molecular cascades underlying the social cues that orchestrate the sexual development process remain poorly understood. Here, we performed a comparative transcriptomic analysis of gonads from females, males, and nonbreeders of Amphiprion clarkii, which constitute a complete social group, allowing us to investigate the molecular regulatory network under social control. Our analysis highlighted that the gonads of nonbreeders and males exhibited high similarities but were far from females, both in global transcriptomic profiles and histological characteristics, and identified numerous candidate genes involved in sexual development, some well-known and some novel. Significant upregulation of cyp19a1a, foxl2, nr5a1a, wnt4a, hsd3b7, and pgr in females provides strong evidence for the importance of steroidogenesis in ovarian development and maintenance, with cyp19a1a playing a central role. Amh and sox8 are two potential key factors that may regulate testicular tissue development in early and late stages, respectively, as they are expressed at higher levels in males than in females, but with slightly different expression timings. Unlike previous descriptions in other fishes, the unique expression pattern of dmrt1 in A. clarkii implied its potential function in both male and female gonads, and we speculated that it might play promoting roles in the early development of both testicular and ovarian tissues.

Full-length gonad transcriptome analysis of Amur sturgeon Dmrt family genes: identification, characterization, and expression patterns during gonadal differentiation.

The regulatory mechanisms that govern sex differentiation in sturgeon are still poorly understood. The doublesex and Mab-3-related transcription factor (Dmrt) gene family is known for its extensive roles in sex determination and differentiation across vertebrates. This study aimed to identify new members of sturgeon Dmrt family genes and core actors in the gonadal differentiation of Amur sturgeon. A full-length gonad transcriptome database was exploited to identify Dmrt gene orthologs. Analyses of phylogenetic relationships and selection pressure were performed, and tissue expression profiles and spatiotemporal expression patterns in gonads were then analyzed using real-time PCR. In total, five Dmrt family genes were identified from the full-length gonad transcriptome, including Dmrt2, DmrtA1, DmrtA2, DmrtB1a, and DmrtB1b. Phylogenetic analysis showed that these genes were clustered into clades corresponding to the doublesex/Mav-3 (DM) genes of vertebrates. Furthermore, the analysis of evolutionary selective pressure indicated that DmrtB1a and DmrtB1b were subject to positive selection, suggesting the existence of adaptive evolution in sturgeon. The extensive tissue expression profiling of each Dmrt family gene revealed typical characteristics. Remarkably, according to a spatiotemporal expression pattern analysis, in later stages, DmrtB1b expression increased in testes and was significantly higher in testes than in ovaries at 24 months after hatching (M) and 36 M. This study provides a genetic resource of full-length Dmrt family genes and increases the understanding of Dmrt functions in sex differentiation in sturgeon.

The molecular regulation mechanism of dmrt1-based on the establishment of the testis cell line derived from two-spot puffer Takifugu bimaculatus.

The establishment of fish cell lines can provide an important in vitro model for developmental biology, pathology, and genetics and also an effective tool to investigate the interactions and related functions of genes. Two-spot puffer Takifugu bimaculatus is a high economic and nutritional value marine fish in Fujian in recent years. Nevertheless, dmrt1 plays a key role in the male differentiation from invertebrates to vertebrates. To understand the molecular regulatory mechanisms of dmrt1 in T. bimaculatus, a testis cell line called TBTc from a juvenile testis of this organism was established with modified Leibovitz's L-15 medium supplemented with 20% FBS, fish serum, embryo extract, and other growth factors. The TBTc with a stable karyotype can be passaged continuously, which was composed of fibroblast-like cells and expressed the marker genes of male-special cells, dmrt1, and amh, and the absence of vasa expression may rule out the possibility of the presence of germ cells. Therefore, TBTc appeared to consist of the mixture of the Sertoli cell and germ cell of the testis. The dmrt1 was significantly expressed in the testes and slightly expressed in the late embryonic development, illustrating that the dmrt1 may participate in the molecular regulation of gonads development and sex differentiation. With the high transfection efficiency of TBTc by electroporation, the cell lines could be used effectively in the study for the expression of exogenous and endogenous genes. Meanwhile, after the knockdown of dmrt1, the morphological changes and survival rates of cells proved that dmrt1 could affect the growth of testicular cells. Furthermore, with the loss of dmrt1, the expression of male-bias genes amh, sox9, and cyp11a was significantly decreased, and the expression of female-bias genes foxl2, sox3, and cyp19a was increased, which suggested that dmrt1 upregulates amh, sox9, and cyp11a and downregulates foxl2, sox3, and cyp19a to participate in the testis development. As a first fish gonadal cell lines of T. bimaculatus, which can be a more convenient, efficient, and rapid model for the investigation of the expression and function of genes, the results will lay a foundation for the next study of the molecular regulation mechanism in gonadal development and sex determination of fish in the future.