Diversity of transactivation regions of DMRT1 in vertebrates.

BACKGROUND: Doublesex 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 RESULTS: For 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. CONCLUSIONS: These findings indicated that the regions involved in the transactivation ability of DMRT1 could subtly change during evolution, indicating diversity in transactivation domains.

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.

Overexpression of Kdm6b induces testicular differentiation in a temperature-dependent sex determination system.

In reptiles, such as the red-eared slider turtle ( Trachemys scripta elegans), gonadal sex determination is highly dependent on the environmental temperature during embryonic stages. This complex process, which leads to differentiation into either testes or ovaries, is governed by the finely tuned expression of upstream genes, notably the testis-promoting gene Dmrt1 and the ovary-promoting gene Foxl2. Recent studies have identified epigenetic regulation as a crucial factor in testis development, with the H3K27me3 demethylase KDM6B being essential for Dmrt1 expression in T. s. elegans. However, whether KDM6B alone can induce testicular differentiation remains unclear. In this study, we found that overexpression of Kdm6b in T. s. elegans embryos induced the male development pathway, accompanied by a rapid increase in the gonadal expression of Dmrt1 at 31°C, a temperature typically resulting in female development. Notably, this sex reversal could be entirely rescued by Dmrt1 knockdown. These findings demonstrate that Kdm6b is sufficient for commitment to the male pathway, underscoring its role as a critical epigenetic regulator in the sex determination of the red-eared slider turtle.

foxl2l is a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

Zebrafish serve as a valuable model organism for studying germ cell biology and reproductive processes. The AB strain of zebrafish is proposed to exhibit a polygenic sex determination system, where most males initially develop juvenile ovaries before committing to male fate. In species with chromosomal sex determination, gonadal somatic cells are recognized as key determinants of germ cell fate. Notably, the loss of germ cells in zebrafish leads to masculinization, implying that germ cells harbor an intrinsic feminization signal. However, the specific signal triggering oogenesis in zebrafish remains unclear. In the present study, we identified foxl2l as an oocyte progenitor-specific gene essential for initiating oogenesis in germ cells. Results showed that foxl2l-knockout zebrafish bypassed the juvenile ovary stage and exclusively developed into fertile males. Further analysis revealed that loss of foxl2l hindered the initiation of oocyte-specific meiosis and prevented entry into oogenesis, leading to premature spermatogenesis during early gonadal development. Furthermore, while mutation of the pro-male gene dmrt1 led to fertile female differentiation, simultaneous disruption of foxl2l in dmrt1 mutants completely blocked oogenesis, with a large proportion of germ cells arrested as germline stem cells, highlighting the crucial role of foxl2l in oogenesis. Overall, this study highlights the unique function of foxl2l as a germ cell-intrinsic gatekeeper of oogenesis in zebrafish.

Thyroid Hormone Regulates Postnatal Development of the Rete Testis in Mice.

Thyroid hormone regulates the rate of testis maturation in mammals. Manipulations of thyroid hormone levels in neonatal animals affect various aspects of testis biology. However, there have been no studies examining the effects of thyroid hormone on the rete testis (RT). Here, we used animal models of neonatal hyperthyroidism (injections of triiodothyronine, or T3) and hypothyroidism (goitrogen 6-propyl-2-thiouracil [PTU] treatment) and found that higher levels of thyroid hormone accelerate RT development, while lower levels of thyroid hormone delay it. T3 and PTU treatments influence RT size, proliferation of RT cells, and expression of DMRT1 and androgen receptor in the RT. T3 supplementation accelerates RT development in an organ testicular culture, which indicates the local action of thyroid hormone. Additionally, it was found that follicle-stimulating hormone could be involved in the regulation both of RT proliferation and RT size. The fact that RT cells in a cell culture do not respond to T3 suggests indirect action of thyroid hormone on the RT in vivo or the loss of the responsiveness to the hormone in vitro.

DMRT3-mediated lncRNA OIP5-AS1 promotes the pyroptosis of bronchial epithelial cells by binding with EIF4A3 to enhance YAP mRNA stability.

Asthma is featured by persistent airway inflammation. Long noncoding RNAs (lncRNAs) are reported to play critical roles in asthma. However, the function of Opa interacting protein 5-antisense 1 (OIP5-AS1) in pyroptosis during the development of asthma remains unexplored. The blood samples of asthma patients (n = 32) as well as the baseline characteristics of asthma patients or healthy people were collected. An in vivo model of asthma was established using house dust mites (HDM). To mimic asthma in vitro, BEAS-2B cells were treated with HDM. Cell pyroptosis and apoptosis were examined by flow cytometry. The levels of interleukin-1 beta (IL-1ß) and interleukin-18 (IL-18) were detected by enzyme-linked immunosorbent assay (ELISA). The binding among messenger RNAs (mRNAs) was assessed by chromatin immunoprecipitation (ChIP), dual luciferase report assay, RNA immunoprecipitation (RIP), co-immunoprecipitation (Co-IP), and RNA pull-down assay, respectively. The cellular localization was observed by fluorescence in situ hybridization (FISH) staining. The level of OIP5-AS1 was upregulated in asthma patients. HDM induced pyroptosis and increased the levels of IL-18, IL-1ß, and lactate dehydrogenase (LDH) in BEAS-2B cells, which was obviously reversed by OIP5-AS1 knockdown. Consistently, the expressions of NOD-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), c-caspase 1, and pyroptosis-related gasdermin D-1 (GSDMD-1) in BEAS-2B cells were upregulated by HDM treatment, while these phenomena were partially abolished by silencing of OIP5-AS1. Moreover, HDM promoted the progression of asthma in vivo, which was rescued by the downregulation of OIP5-AS1. OIP5-AS1 silencing decreased HDM-induced cell pyroptosis by inactivation of NLRP3. More importantly, OIP5-AS1 promoted the mRNA stability of yes-associated protein (YAP) via binding with eukaryotic translation initiation factor 4A3 (EIF4A3), and OIP5-AS1 was transcriptionally upregulated by doublesex and mab-3 related transcription factor 3 (DMRT3). DMRT3-mediated OIP5-AS1 aggravated the progression of asthma by mediation of the EIF4A3/YAP axis, which might provide a new therapeutic strategy against asthma.

dmrt1 Is Responsible for Androgen-Induced Masculinization in Nile Tilapia.

17α-Methyltestosterone (MT) is a widely used androgen for all-male fish production in aquaculture. However, the molecular mechanism underlying MT-induced masculinization remains unclear. In this study, we aim to identify the key gene responsible for MT-induced masculinization using the Nile tilapia (Oreochromis niloticus) amhy, dmrt1, and gsdf mutants, which exhibit male-to-female sex reversal. Nile tilapia fry from these three mutant lines were treated with 50 µg/g MT from 5 to 30 days after hatching (dah). The results showed that amhy and gsdf mutants, but not dmrt1 mutants, were masculinized by the MT treatment. Gonadal transcriptome analysis revealed that genes involved in steroidogenesis and germ cell development in MT-treated dmrt1 mutants exhibited a similar expression pattern to that of the wild type (WT) XX. In addition, the dmrt1 mutants cannot be masculinized by co-treatment with MT and the aromatase inhibitor fadrozole. The MT treatment completely blocked early steroidogenic enzyme (Star2, Cyp17a2, and Cyp19a1a) expression independent of amhy, gsdf, and dmrt1. A luciferase analysis showed that MT directly suppressed basal and Sf-1-activated cyp19a1a promoter activity through ara and arb in cultured HEK293 cells. Furthermore, MT treatment inhibited germ cell proliferation in amhy and gsdf mutants but not in dmrt1 mutants. Consistently, dmrt1 expression was induced in MT-treated WT XX, -amhy, and -gsdf mutants. Taken together, these results suggest that dmrt1 is indispensable for MT-induced masculinization in Nile tilapia and that MT functions by inhibiting early steroid synthesis and activating dmrt1 to promote testis development.

miR-34 negatively regulates the expression of Dmrt and related genes in the testis of mud crab Scylla paramamosain.

The mud crab (Scylla paramamosain) is a commercially significant marine decapod crustacean. Due to its obvious sexual dimorphism, the mechanism of sex differentiation and gonadal development has attracted significant research interest. The Dmrt (double-sex and mab-3 related transcription factor) genes are vital in animal gonadal development and sex differentiation. In the present study, miR-34 was predicted to target the 3' end of Dmrt-1, idmrt-2, Dmrt-3, Dsx and Dmrt-like genes by prediction software, and the interactions between miR-34 and these Dmrt genes were validated by in vivo and in vitro experiments. Dual luciferase assay results indicated that miR-34 mimics/inhibitors co-transfected with plasmid vectors with 3' end of Dmrt-1, idmrt-2, Dmrt-3, Dsx and Dmrt-like, respectively, led to a significant decrease/increase of fluorescence activity in HEK293T cells. In vivo experiments showed that injection of agomir-34 significantly inhibited Dmrt-1, idmrt-2, Dsx and Dmrt-like expression, while injection of antagomir-34 caused the opposite result. However, Dmrt-3 expression was not affected by injection of miR-34 reagents. Meanwhile, the expression of spermatogenesis and testicular development-related molecular marker genes (IAG, foxl2 and vasa) in mud crabs was significantly changed after injecting the miR-34 reagent in vivo. Furthermore, the result of immunoblotting proved that the expression level of Dmrt-like protein can be regulated by miR-34. These results imply that miR-34 is indirectly involved in sex differentiation and testicular development of S. paramamosain by regulating Dmrt-1, idmrt-2, Dsx and Dmrt-like genes.

Medaka (Oryzias latipes) Dmrt3a Is Involved in Male Fertility.

Research across various species has demonstrated that the doublesex and mab-3-related transcription factor 3 (dmrt3) plays pivotal roles in testis development. However, the precise molecular mechanisms of dmrt3 remain unclear. In this study, we investigated the role of dmrt3 (dmrt3a) in testis development using the model organism medaka (Oryzias latipes). SqRT-PCR and ISH analyses revealed that dmrt3a is predominantly expressed in the testis, especially in the spermatid and spermatozoon. Using CRISPR/Cas9, we generated two dmrt3a homozygous mutants (-8 bp and -11 bp), which exhibited significantly reduced fertilization rates and embryo production. Additionally, the number of germ cells and sperm motility were markedly decreased in the dmrt3a mutants, manifesting as the symptoms of asthenozoospermia and oligozoospermia. Interestingly, RNA-Seq analysis showed that the deficiency of dmrt3a could lead to a significant downregulation of numerous genes related to gonadal development and severe disruptions in mitochondrial function. These results suggested that dmrt3a is essential for spermatogenesis and spermatozoa energy production. This paper provides new insights and perspectives for further exploring the molecular mechanisms underlying spermatogenesis and addressing male reproductive issues.

A New Investigation to Discriminate Sexes in Alive Nile Tilapia (Oreochromis niloticus) Using Cyp19a1a and Dmrt1 Gene Expression in Tail Fin Tissues.

The Nile Tilapia (Oreochromis niloticus), a gonochoristic teleost fish with a XX/XY sex-determination system, is an ideal model for investigating gonadal sex differentiation. During gonadal differentiation, the expression of cyp19a1a in XX gonads and dmrt1 in XY gonads are required for undifferentiated tissues to develop into ovary or testis. In this study, quantitative real-time RT-PCR assessed the expression of cyp19a1a and dmrt1 genes in gonads and tail fin tissues. Differences in gene expression mean among sexually differentiated fish were analyzed using two-way analysis of variance (ANOVA) and validation of mixed model using discriminant analysis (DA) for morphometric traits and the gene expression in gonads and tail fin tissues used to validate and utilize them in discriminating sexes in sex-differentiated Nile Tilapia fish. The results revealed that, cyp19a1a gene expression in female ovaries was more significant than dmrt1 in male testis. In the other hand, the dmrt1 gene expression in the tail fin was higher in males than females. Both, cyp19a1a and dmrt1 genes, can discriminate fish sexes by 100% by using their expression in tail fin tissues. In conclusion, the cyp19a1a and dmrt1 genes could be used as a genetic marker to discriminate between the Nile Tilapia sexes, whereas used as an indicator for ovarian or testis differentiation in sexually differentiated Nile Tilapia using tail fin tissues. It is worth mentioning that this is the first investigation for using cyp19a1a and dmrt1 genes from Nile Tilapia tail fin tissues in sex determination.

The Chinese medaka (Oryzias sinensis) dmrt1 gene converts females to males in medaka (Oryzias latipes).

BACKGROUND: Chinese medaka (Oryzias sinensis) is widely distributed in freshwater rivers in China. Similar to the medaka (Oryzias latipes), Chinese medaka has the characteristics of small size, rapid reproductive cycle, and strong adaptability, which makes it suitable as a model organism for studies in basic biology and environmental toxicology. Chinese medaka exhibits distinct sexual dimorphism. However, due to the lack of complete genomic information, the regulation of sex determination and differentiation-related genes in Chinese medaka remains unclear. METHODS: Chinese medaka dmrt1 (Osdmrt1) was cloned by PCR, and transgenic individuals of medaka [Tg(CMV:Osdmrt1)] overexpressing Osdmrt1 were generated to investigate the role of Osdmrt1 in sex determination. Western blot was used to validate the integration of the Osdmrt1 into the medaka genome. Tissue sectioning and HE staining were used to identify Tg(CMV:Osdmrt1) physiological gender and phenotype. qRT-PCR was used to analyze the expression of gonad-specific genes. RESULTS: Osdmrt1 was cloned and identified, and it shared similar evolutionary relationships with medaka dmrt1. Tg(CMV:Osdmrt1) exhibited partial sex reversal from female to male in the F2 generation, with genetically female individuals developing testes and producing functional sperm. Additionally, the secondary sexual characteristics of the transgenic females also changed to males. CONCLUSION: The Chinese medaka dmrt1 gene could convert females to males in medaka. GENERAL SIGNIFICANCE: These results not only elucidate the function of Chinese medaka dmrt1, but also accumulate knowledge for studying the function of economically important fish genes in model fish by transgenic technology.

Genome-wide identification and expressional profile of the Dmrt gene family in the swimming crab (Portunus trituberculatus).

The swimming crab, Portunus trituberculatus is one of crucial aquaculture crabs with significant differences in growth and economic performance between male and female swimming crabs. Consequently, the culture of female populations presents higher economic value. The doublesex and mab-3 related transcription factor (Dmrt) gene family are known to play crucial role in gonad differentiation and development. However, there is limited information about this gene family in Portunus trituberculatus. In this study, we identified seven members of the Dmrt gene family in P. trituberculatus based on the published transcriptome and genome data and designated as Ptdmrt-1, Ptdoublesex (Ptdsx), Ptidmrt-1, Ptdmrt-11E, Ptidmrt-2, Ptdmrt-99B, and Ptdmrt-3 based on the homology analysis results, respectively. These Ptdmrt genes distributed across 6 chromosomes and were predicted to encode 283 aa, 288 aa, 529 aa, 436 aa, 523 aa, 224 aa, and 435 aa protein precursors, respectively. The expression patterns of these dmrt genes were characterized by qRT-PCR and gonad transcriptome data. The results showed that five members (Ptdmrt-99B, Ptdsx, Ptdmrt-1, Ptdmrt-3, and Ptdmrt-11E) were differentially expressed between the testis and ovary. In addition, their expression patterns from zoea 2 to juvenile 1 were characterized by published transcriptome data and the results showed that they were lowly expressed and did not exhibit notable difference except for Ptdsx during early development. Overall, majority of Ptdmrt genes were involved in gonad differentiation in the swimming crab. Current findings provide a solid foundation for further exploration of the roles of these genes in gonad development and differentiation in P. trituberculatus.

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.

Sexual dimorphism in the tardigrade Paramacrobiotus metropolitanus transcriptome.

BACKGROUND: In gonochoristic animals, the sex determination pathway induces different morphological and behavioral features that can be observed between sexes, a condition known as sexual dimorphism. While many components of this sex differentiation cascade show high levels of diversity, factors such as the Doublesex-Mab-3-Related Transcription factor (DMRT) are widely conserved across animal taxa. Species of the phylum Tardigrada exhibit remarkable diversity in morphology and behavior between sexes, suggesting a pathway regulating this dimorphism. Despite the wealth of genomic and zoological knowledge accumulated in recent studies, the sexual differences in tardigrades genomes have not been identified. In the present study, we focused on the gonochoristic species Paramacrobiotus metropolitanus and employed omics analyses to unravel the molecular basis of sexual dimorphism. RESULTS: Transcriptome analysis between sex-identified specimens revealed numerous differentially expressed genes, of which approximately 2,000 male-biased genes were focused on 29 non-male-specific genomic loci. From these regions, we identified two Macrobiotidae family specific DMRT paralogs, which were significantly upregulated in males and lacked sex specific splicing variants. Furthermore, phylogenetic analysis indicated all tardigrade genomes lack the doublesex ortholog, suggesting doublesex emerged after the divergence of Tardigrada. In contrast to sex-specific expression, no evidence of genomic differences between the sexes was found. We also identified several anhydrobiosis genes that exhibit sex-biased expression, suggesting a possible mechanism for protection of sex-specific tissues against extreme stress. CONCLUSIONS: This study provides a comprehensive analysis for analyzing the genetic differences between sexes in tardigrades. The existence of male-biased, but not male-specific, genomic loci and identification of the family specific male-biased DMRT subfamily provides the foundation for understanding the sex determination cascade. In addition, sex-biased expression of several tardigrade-specific genes which are involved their stress tolerance suggests a potential role in protecting sex-specific tissue and gametes.

linc-ADAIN, a human adipose lincRNA, regulates adipogenesis by modulating KLF5 and IL-8 mRNA stability.

Adipose tissue remodeling and dysfunction, characterized by elevated inflammation and insulin resistance, play a central role in obesity-related development of type 2 diabetes (T2D) and cardiovascular diseases. Long intergenic non-coding RNAs (lincRNAs) are important regulators of cellular functions. Here, we describe the functions of linc-ADAIN (adipose anti-inflammatory), an adipose lincRNA that is downregulated in white adipose tissue of obese humans. We demonstrate that linc-ADAIN knockdown (KD) increases KLF5 and interleukin-8 (IL-8) mRNA stability and translation by interacting with IGF2BP2. Upregulation of KLF5 and IL-8, via linc-ADAIN KD, leads to an enhanced adipogenic program and adipose tissue inflammation, mirroring the obese state, in vitro and in vivo. KD of linc-ADAIN in human adipose stromal cell (ASC) hTERT adipocytes implanted into mice increases adipocyte size and macrophage infiltration compared to implanted control adipocytes, mimicking hallmark features of obesity-induced adipose tissue remodeling. linc-ADAIN is an anti-inflammatory lincRNA that limits adipose tissue expansion and lipid storage.

Curcumin Inhibits the Progression of Non-small Cell Lung Cancer by Regulating DMRT3/SLC7A11 Axis.

Non-small cell lung cancer (NSCLC) is a fatal malignancy all over the world. Emerging studies have shown that curcumin might repress NSCLC progression by regulating ferroptosis, but the underlying mechanism remains unclear. 16HBE, LK-2, and H1650 cell viability was detected using Cell Counting Kit-8 assay. LK-2 and H1650 cell proliferation, apoptosis, and angiopoiesis were measured using 5-ethynyl-2'-deoxyuridine, flow cytometry, and tube formation assay. Superoxide dismutase, Malondialdehyde, Glutathione, and lactate dehydrogenase levels in LK-2 and H1650 cells were examined using special assay kits. Fe+ level was assessed using an iron assay kit. Doublesex and Mab-3 related Transcription Factor 3 (DMRT3) and solute carrier family 7 member 11 (SLC7A11) protein levels were detected using western in NSCLC tissues, adjacent matched normal tissues, 16HBE cells, LK-2 cells, H1650 cells, and xenograft tumor tissues. Glutathione peroxidase 4, Acyl-CoA Synthetase Long Chain Family Member 4, and transferrin receptor 1 protein levels in LK-2 and H1650 cells were examined by western blot assay. DMRT3 and SLC7A11 levels were determined using real-time quantitative polymerase chain reaction. After JASPAR prediction, binding between DMRT3 and SLC7A11 promoter was verified using Chromatin immunoprecipitation and dual-luciferase reporter assays in LK-2 and H1650 cells. Role of curcumin on NSCLC tumor growth was assessed using the xenograft tumor model in vivo. Curcumin blocked NSCLC cell proliferation and angiopoiesis, and induced apoptosis and ferroptosis. DMRT3 or SLC7A11 upregulation partly abolished the suppressive role of curcumin on NSCLC development. In mechanism, DMRT3 was a transcription factor of SLC7A11 and increased the transcription of SLC7A11 via binding to its promoter region. Curcumin inhibited NSCLC growth in vivo by modulating DMRT3. Curcumin might constrain NSCLC cell malignant phenotypes partly through the DMRT3/SLC7A11 axis, providing a promising therapeutic strategy for NSCLC.

Gonadal transcriptome sequencing reveals sexual dimorphism in expression profiling of sex-related genes in Asian arowana (Scleropages formosus).

Asia arowana (Scleropages formosus) is an ornamental fish with high economic value, while its sex determination mechanism is still poorly understood. By far, no morphological evidence or molecular marker has been developed for effective distinguishment of genders, which poses a critical challenge to our captive breeding efforts. In this study, we sequenced gonadal transcriptomes of adult Asian arowanas and revealed differential expression profiling of sex-related genes. Based on the comparative transcriptomics analysis of testes (n = 3) and ovaries (n = 3), we identified a total of 8,872 differentially expressed genes (DEGs) and 18,490 differentially expressed transposable elements (TEs) between male and female individuals. Interestingly, the expression of TEs usually has been more significantly testis-biased than related coding genes. As expected, several genes related to females (such as foxl2 and cyp19a1a) are significantly transcribed in the ovary, and some genes related to male gonad development (such as dmrt1, gsdf and amh) are highly expressed in the testis. This sexual dimorphism is valuable for ascertaining the differential expression patterns of sex-related genes and enriching the genetic resources of this economically important species. These valuable genetic materials thereby provide instructive references for gender identification and one-to-one breeding practices so as to expand fish numbers for a rapid elevation of economic value.

Allantoic Fluid-Based qPCR for Early Onset In Ovo Sexing.

The day-old male chick culling remains a welfare issue in the poultry industry. Several governments have prohibited this practice, pushing hatcheries to seek alternatives. Although different solutions exist for solving this problem, sex determination during the embryo's incubation (in ovo sexing) is considered the most suitable one among the consumers and industry. However, to be industrialized, in ovo sexing technologies must meet several requirements: compatibility with all egg colors and early developmental stages while maintaining a high hatchability rate and accuracy at low cost and high throughput. To meet these requirements, we studied the use of the sexual genes HINTW (female-specific) and DMRT-1 (both sexes) at incubation days 6-9. By utilizing the quantitative polymerase chain reaction in allantoic fluid (AF) samples, our study confirmed female-specific HINTW detection on all days without any significant detrimental effects on embryo development. We achieved 95% sexing accuracy using the HINTW cycle threshold (Ct) alone and 100% accuracy rate when using Δλ values (difference between the HINTW and DMRT-1 Ct). In conclusion, the developed assay can provide information about AF as a sample for in ovo sexing and open new industrial possibilities for faster and cheaper assays.

Gonadal transcriptome analysis of sex-biased gene and genome-wide investigation of dmrt gene family in Acanthogobius ommaturus.

Acanthogobius ommaturus is one of the largest goby fish, and widely distributed in the Northwestern Pacific as an annual benthic fish. This study aims to report the gonadal transcriptome of A. ommaturus and identify differentially expressed genes (DEGs) between sexes. A total of 5460 (27.94 %) DEGs were detected from genome, with 3301 (16.89 %) biased towards males and 2159 (11.05 %) towards females. Analysis of 76 known vertebrate sex-related genes revealed multiple key genes, including the male-biased genes dmrt1 (Doublesex and Mab-3 related transcription factor 1) and amh (Anti-Mullerian Hormone), and the female-biased genes foxl2 (Forkhead Box L2) and cyp19a1a (Cytochrome P450 Aromatase 19 Subfamily A1). Furthermore, a genome-wide gene family analysis focused on the most significantly differentially expressed male-biased gene, dmrt1, was conducted using the chromosomal-level genome. Six Aodmrt genes were identified and subjected to phylogenetic and protein interaction network analyses. To validate the expression pattern, quantitative real-time PCR (qRT-PCR) was performed and compared with gonadal transcriptome data. The results showed that only dmrt1 exhibited significant male-bias, while the expression levels and sex differences of other dmrt genes in the gonads were inconclusive. Interestingly, the other dmrt genes displayed higher expression levels in other tissues, suggesting currently unknown functions. In conclusion, this study provides valuable genetic information contributing to the understanding of the sex determination mechanism of A. ommaturus and bony fish.

Genome-wide identification and expression profiles of sex-related gene families in the Pacific abalone Haliotis discus hannai.

In recent years, members of the Dmrt family, TGF-ß superfamily and Sox family have been recognized as crucial genes for sex determination/differentiation across diverse animal species. Nevertheless, knowledge regarding the abundance and potential functions of these genes in abalone remains limited. In this study, a total of 5, 10, and 7 members of the Dmrt family, the TGF-ß superfamily and the Sox family, respectively, were identified in the Pacific abalone Haliotis discus hannai. Sequence characteristics, phylogenetic relationships and spatiotemporal expression profiles of these genes were investigated. Notably, HdDmrt-04 (Dmrt1/1L-like) emerged as a potential mollusc-specific gene with a preponderance for expression in the testis. Interestingly, none of the TGF-ß superfamily members exhibited specific or elevated expression in the gonads, highlighting the need for further investigation into their role in abalone sex differentiation. The Sox proteins in H. discus hannai were categorized into 7 subfamilies: B1, B2, C, D, E, F, and H. Among them, HdSox-07 (SoxH-like) was observed to play a crucial role in testis development, while HdSox-03 (SoxB1-like) and HdSox-04 (SoxC-like) probably cooperate in abalone ovary development. Taken together, the results of the present study suggested that HdDmrt-04 and HdSox-07 can be used as male-specific markers for gonad differentiation in H. discus hannai and imply conservation of their functions across invertebrates and vertebrates. Our findings provide new insights into the evolution and genetic structure of the Dmrt family, the TGF-ß superfamily and the Sox family in abalone and pave the way for a deeper understanding of sex differentiation in gastropods.