Doublesex And Mab-3 Related Transcription Factor 1 Research Articles

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.

A novel sorting method for the enrichment of early human spermatocytes from clinical biopsies

To determine if early spermatocytes can be enriched from a human testis biopsy using fluorescence-activated cell sorting (FACS). Potential surface markers for early spermatocytes were identified using bioinformatics analysis of single-cell RNA-sequenced human testis tissue. Testicular sperm extraction samples from three participants with normal spermatogenesis were digested into single-cell suspensions and cryopreserved. Two to four million cells were obtained from each and sorted by FACS as separate biologic replicates using antibodies for the identified surface markers. A portion from each biopsy remained unsorted to serve as controls. The sorted cells were then characterized for enrichment of early spermatocytes. A laboratory study. Three men with a diagnosis of obstructive azoospermia (age range, 30-40 years). None. Sorted cells were characterized for RNA expression of markers encompassing the stages of spermatogenesis. Sorting markers were validated by their reactivity on human testis formalin-fixed paraffin-embedded tissue. Serine protease 50 (TSP50) and SWI5-dependent homologous recombination repair protein 1 were identified as potential surface proteins specific for early spermatocytes. After FACS sorting, the TSP50-sorted populations accounted for 1.6%-8.9% of total populations and exhibited the greatest average-fold increases in RNA expression for the premeiotic marker stimulated by retinoic acid (STRA8), by 23-fold. Immunohistochemistry showed the staining pattern for TSP50 to be strong in premeiotic undifferentiated embryonic cell transcription factor 1-/doublesex and Mab-3 related transcription factor 1-/STRA8+ spermatogonia as well as SYCP3+/protamine 2- spermatocytes. This work shows that TSP50 can be used to enrich early STRA8-expressing spermatocytes from human testicular biopsies, providing a means for targeted single-cell RNA sequencing analysis and invitro functional interrogation of germ cells during the onset of meiosis. This could enable investigation into details of the regulatory pathways underlying this critical stage of spermatogenesis, previously difficult to enrich from whole tissue samples.

Deleterious variants in X-linked RHOXF1 cause male infertility with oligo- and azoospermia.

Oligozoospermia and azoospermia are two common phenotypes of male infertility characterized by massive sperm defects owing to failure of spermatogenesis. The deleterious impact of candidate variants with male infertility is to be explored. In our study, we identified three hemizygous missense variants (c.388G>A: p.V130M, c.272C>T: p.A91V, and c.467C>T: p.A156V) and one hemizygous nonsense variant (c.478C>T: p.R160X) in the Rhox homeobox family member 1 gene (RHOXF1) in four unrelated cases from a cohort of 1201 infertile Chinese men with oligo- and azoospermia using whole-exome sequencing and Sanger sequencing. RHOXF1 was absent in the testicular biopsy of one patient (c.388G>A: p.V130M) whose histological analysis showed a phenotype of Sertoli cell-only syndrome. In vitro experiments indicated that RHOXF1 mutations significantly reduced the content of RHOXF1 protein in HEK293T cells. Specifically, the p.V130M, p.A156V, and p.R160X mutants of RHOXF1 also led to increased RHOXF1 accumulation in cytoplasmic particles. Luciferase assays revealed that p.V130M and p.R160X mutants may disrupt downstream spermatogenesis by perturbing the regulation of doublesex and mab-3 related transcription factor 1 (DMRT1) promoter activity. Furthermore, ICSI treatment could be beneficial in the context of oligozoospermia caused by RHOXF1 mutations. In conclusion, our findings collectively identified mutated RHOXF1 to be a disease-causing X-linked gene in human oligo- and azoospermia.

Functional assessment of DMRT1 variants and their pathogenicity for isolated male infertility

ObjectiveTo study the impact of Doublesex and mab-3-related transcription factor 1 (DMRT1) gene variants on the encoded protein’s function and the variants’ pathogenic relevance for isolated male infertility caused by azoospermia. DesignThis study established a novel luciferase assay for DMRT1 missense variants using 2 different target promotors and validated the assay by analyzing previously published variants associated with differences in sex development. SettingUniversity genetics research institute and tertiary referral center for couples’ infertility. Patient(s)Eleven infertile men with severely impaired spermatogenesis resulting in crypto- or azoospermia and carrying rare heterozygous missense variants in DMRT1 were identified within the Male Reproductive Genomics study. Main Outcome Measure(s)Luciferase assays with human DMRT1 variants to test functional effects on the CYP19A1 and Stra8 target promoters. Result(s)We first developed and refined luciferase assays to reliably test the functional impact of DMRT1 missense variants. Next, the assay was validated by analyzing 2 DMRT1 variants associated with differences in sex development, of which c.240G>C p.(Arg80Ser) displayed highly significant effects on both target promoters compared with the wild-type protein (-40% and +100%, respectively) and c.331A>G p.(Arg111Gly) had a significant effect on the Stra8 promoter (-76%). We then systematically characterized 11 DMRT1 variants identified in infertile men. The de novo variant c.344T>A p.(Met115Lys) showed a pronounced loss of function in both DMRT1 target promoters (-100% and -86%, respectively). Variants c.308A>G p.(Lys103Arg) and c.991G>C p.(Asp331His) showed a significant gain of function exclusively for the CYP19A1 promoter (+15% and +19%, respectively). Based on these results, 3 variants were reclassified according to clinical guidelines. Conclusion(s)The present study highlights the importance of functionally characterizing DMRT1 variants of uncertain clinical significance. Using luciferase assays for diagnostic purposes enables an improved causal diagnosis for isolated male infertility.

Abstract 769: Genomic and transcriptomic characterization of benign and malignant struma ovarii

Abstract Background: Struma Ovarii (SO) is a rare ovarian teratoma characterized by the presence of thyroid tissue in &amp;gt;50% of the tumor. The majority of SO are benign; however, malignant transformation occurs in up to 5% of the cases. The molecular foundations of benign and malignant SO are grossly unknown. Therefore, the goal of this study was to perform the first comprehensive genomic and transcriptomic analysis of the benign and malignant SO. Material and Method: We performed whole-exome sequencing (WES) and targeted RNA-sequencing (seq) on the DNA and RNA extracted from formalin-fixed paraffin-embedded SO tumor tissue samples. WES library was prepared using Agilent’s SureSelect XT HS2 kit, with 4 samples failing the quality assessment (QA). Variants were called from GATK processed WES data and annotated using VEP (with ClinVar and COSMIC databases). The targeted RNA-Seq library was prepared using the TruSight RNA Pan-Cancer Panel kit covering 1385 cancer genes, with all samples passing QA. The clinical characteristics of the study cohort were summarized by percentages for categorical variables and medians with 25-75% interquartile ranges for continuous variables. Results: The study included 31 tissue samples - 21 benign and 10 malignant, including 6 cases of papillary thyroid cancer (PTC), 3 of follicular variant of PTC, and 1 of follicular thyroid cancer. Patients with benign SO were characterized by the median age at diagnosis of 39 years [33-54], tumor size of 3.1 cm [2.5-5.8], while the patients with malignant SO presented at age of 45 [28-54], tumor size of 6 cm [0.85-14] and metastatic disease in 30% (3/10) - 2 patients with peritoneal metastases and 1 patient with pelvic lymph node metastases. The E1A Binding Protein P300, EP300 (6/27), and Isocitrate dehydrogenase, IDH2 (5/27) were the topmost mutated genes in the SO samples. Malignant SO samples were characterized with the presence of pathogenic variants of KRAS (pQ61L and pG12V), NRAS (pQ61R), TP53 (splice site) mutations, and Nuclear Receptor Binding SET Domain Protein 1 (NSD1) fusion as the most common molecular drivers. Among benign SO samples, the most common driver was Thyroglobulin (TG) fusion with either Guanine Nucleotide binding protein (GNAS) or Rac Family Small GTPase 1 (RAC1). Differential expression analysis showed that the member of tumor suppressor family - tumor protein 63 (TP63) was the most downregulated (Log2FC = -3), while Double-sex and Mab-3 Related Transcription Factor 1 (DMRT1), implicated in the development of germ cell tumors, was the most upregulated gene in malignant SO samples over benign (Log2FC = 2.1; padj&amp;lt;0.05). Conclusions: In contrast to cancer arising from the thyroid gland, characterized by BRAFV600E as the most common mutation, malignant SO belongs to RAS-like tumors. The downregulation of tumor suppressors and upregulation of DMRT1 might be implicated in the malignant transformation of SO. Citation Format: Shilpa Thakur, Darryl Nousome, Kshama Aswath, Stephanie Cardenas, Sonam Kumari, Ruth Adewale, Maria Merino, Esra Dikoglu, Padmasree Veeraraghavan, Sriram Gubbi, Joanna Klubo-Gwiezdzinska. Genomic and transcriptomic characterization of benign and malignant struma ovarii [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 769.

Translatome changes in acute myeloid leukemia cells post exposure to pegcrisantaspase and venetoclax

The clinical outcomes of patients with acute myeloid leukemia (AML) treated with available therapy remain unsatisfactory. We recently reported that the BCL-2 inhibitor venetoclax synergizes with pegcrisantaspase (Ven-PegC) and exhibits remarkable in vivo efficacy in a preclinical model of AML with complex karyotype. The Ven-PegC combination blocks synthesis of proteins in AML cells by inhibiting cap-dependent translation of mRNA. To further explore the impact of Ven-PegC on protein translation, we used polysome profiling and high-throughput RNA sequencing to characterize Ven-PegC-dependent changes to the translatome. Here we report that the translation of five mRNAs, including two microRNAs, one rRNA, and two mitochondrial genes, was altered after exposure to all three treatments (Ven, PegC, and Ven-PegC). We focused our translatome validation studies on six additional genes related to translational efficiency that were modified by Ven-PegC. Notably, Ven-PegC treatment increased the RNA translation and protein levels of Tribbles homologue 3 (TRIB3), eukaryotic translation initiation factor 3 subunit C (eIF3C), doublesex and mab-3-related transcription factor 1 (DMRT1), and salt-inducible kinase 1 (SIK1). We validated the observed changes in gene/protein expression in vitro and confirmed our cell line-based studies in the bone marrow of an AML patient-derived xenograft model after Ven-PegC treatment. These results support examining alterations in the translatome post chemotherapy to offer insight into the drug's mechanism of action and to inform future therapeutic decisions.

MiRNA target prediction of avian Z-linked DMRT1 gene during sex determination in chicken (G. Gallus)

Sex determination in dimorphic animal, such as chicken (G. Gallus), is controlled by the expression of doublesex and mab-3 related transcription factor 1 (DMRT1) gene. This gene act as sex determination switch by critically needed for testis differentiation and as antagonist of ovarian development. miRNA, is belongs to short noncoding RNA which modulate gene expression in specific or board targeted genes. This study was aimed to predict miRNA(s) candidate targeted to DMRT1 expression in chicken. In silico method was employed to mining miRNA targeted to DMRT1 using three online databases namely miRDB, TargetScan, and microT-CDS. Following prediction, clustering was performed to select common miRNA(s) in minimal two databases for gene ontology (GO) analysis. Totally 78 miRNAs were targeted to DMRT1 3’UTR, and eight miRNA(s) were found in minimal two databases. The GO analysis found seven distinct biological functions in membrane, cytoplasm, protein binding, nucleus, integral component of membrane and molecular function, and all are related to the cell growth namely cell proliferation. According to the result, it shows the possibility to use selected candidate of miRNA(s) targeted to DMRT1 to reveal the sex determination mechanism at early stage of chicken development.

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.

Histone deacetylase inhibition leads to regulatory histone mark alterations and impairs meiosis in oocytes

BackgroundPanobinostat (PB), a histone deacetylase (HDAC) inhibitor drug, is clinically used in the treatment of cancers. We investigated the effects of PB on murine ovarian functions in embryos and adult animals.MethodsC57BL/6J mice were treated with 5 mg/kg PB on alternate days from embryonic day (E) 6.5 to E15.5. We analysed the effects of PB on the ovaries by using immunofluorescence, gene expression analysis and DNA methylation analysis techniques.ResultsAt E15.5, we observed increases in histone H3K9Ac, H4Ac and H3K4me3 marks, while the level of the silencing H3K9me3 mark decreased. Synaptonemal complex examination at E15.5, E17.5 and E18.5 showed a delay in meiotic progression characterized by the absence of synaptonemal complexes at E15.5 and the persistence of double-strand breaks (DSBs) at E17.5 and E18.5 in PB-exposed oocytes. We found that exposure to PB led to changes in the expression of 1169 transcripts at E15.5. Genes regulated by the male-specific factors SRY-Box Transcription Factor 9 (SOX9) and Doublesex and Mab-3-related Transcription factor 1 (DMRT1) were among the most upregulated genes in the ovaries of PB-exposed mice. In contrast, PB treatment led to decreases in the expression of genes regulated by the WNT4 pathway. Notably, we observed 119 deregulated genes encoding Zn-finger proteins. The observed alterations in epigenetic marks and gene expression correlated with decreases in the numbers of germ cells at E15.5. After birth, PB-exposed ovaries showed increased proliferation of primary and secondary follicles. We also observed decreases in the numbers of primordial, primary and secondary follicles in adult ovaries from mice that were exposed to PB in utero. Finally, epigenetic alterations such as decreased H3K4me3 and increased H4 acetylation levels were also detected in somatic cells surrounding fully grown oocytes.ConclusionOur data suggest that inhibition of histone deacetylase by PB during a critical developmental window affects reprogramming and germ cell specification via alteration of epigenetic marks.

Primary sex determination in birds depends on DMRT1 dosage, but gonadal sex does not determine adult secondary sex characteristics

In birds, males are the homogametic sex (ZZ) and females the heterogametic sex (ZW). Primary sex determination is thought to depend on a sex chromosome gene dosage mechanism, and the most likely sex determinant is the Z chromosome gene Doublesex and Mab-3-Related Transcription factor 1 (DMRT1). To clarify this issue, we used a CRISPR-Cas9-based monoallelic targeting approach and sterile surrogate hosts to generate birds with targeted mutations in the DMRT1 gene. The resulting chromosomally male (ZZ) chicken with a single functional copy of DMRT1 developed ovaries in place of testes, demonstrating the avian sex-determining mechanism is based on DMRT1 dosage. These ZZ ovaries expressed typical female markers and showed clear evidence of follicular development. However, these ZZ adult birds with an ovary in place of testes were indistinguishable in appearance to wild-type adult males, supporting the concept of cell-autonomous sex identity (CASI) in birds. In experiments where estrogen synthesis was blocked in control ZW embryos, the resulting gonads developed as testes. In contrast, if estrogen synthesis was blocked in ZW embryos that lacked DMRT1, the gonads invariably adopted an ovarian fate. Our analysis shows that DMRT1 is the key sex determination switch in birds and that it is essential for testis development, but that production of estrogen is also a key factor in primary sex determination in chickens, and that this production is linked to DMRT1 expression.

Comparative transcriptome analysis of three gonadal development stages reveals potential genes involved in gametogenesis of the fluted giant clam (Tridacna squamosa)

BackgroundGonad development and differentiation is an essential function for all sexually reproducing species, and many aspects of these developmental processes are highly conserved among the metazoa. However, the mechanisms underlying gonad development and gametogenesis remain unclear in Tridacna squamosa, a large-size bivalve of great ecological value. They are protandrous simultaneous hermaphrodites, with the male gonad maturing first, eventually followed by the female gonads. In this study, nine gonad libraries representing resting, male and hermaphrodite stages in T. squamosa were performed to identify the molecular mechanisms.ResultsSixteen thousand four hundred ninety-one unigenes were annotated in the NCBI non-redundant protein database. Among the annotated unigenes, 5091 and 7328 unigenes were assigned to Gene Ontology categories and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database, respectively. A total of 4763 differentially expressed genes (DEGs) were identified by comparing male to resting gonads, consisting of 3499 which were comparatively upregulated in males and 1264 which were downregulated in males. Six hundred-ninteen DEGs between male and hermaphroditic gonads were identified, with 518 DEGs more strongly expressed in hermaphrodites and 101 more strongly expressed in males. GO (Gene Ontology) and KEGG pathway analyses revealed that various biological functions and processes, including functions related to the endocrine system, oocyte meiosis, carbon metabolism, and the cell cycle, were involved in regulating gonadal development and gametogenesis in T. squamosa. Testis-specific serine/threonine kinases 1 (TSSK1), TSSK4, TSSK5, Doublesex- and mab-3-related transcription factor 1 (DMRT1), SOX, Sperm surface protein 17 (SP17) and other genes were involved in male gonadal development in Tridacna squamosal. Both spermatogenesis- (TSSK4, spermatogenesis-associated protein 17, spermatogenesis-associated protein 8, sperm motility kinase X, SP17) and oogenesis-related genes (zona pellucida protein, Forkhead Box L2, Vitellogenin, Vitellogenin receptor, 5-hydroxytryptamine, 5-hydroxytryptamine receptor) were simultaneously highly expressed in the hermaphroditic gonad to maintain the hermaphroditism of T. squamosa.ConclusionAll these results from our study will facilitate better understanding of the molecular mechanisms underlying giant clam gonad development and gametogenesis, which can provided a base on obtaining excellent gametes during the seed production process for giant clams.

Early-Onset Gonadoblastoma in a 13-Month-Old Infant with 46,XY Complete Gonadal Dysgenesis Identified with Prenatal Testing: A Case of Chromosome 9P Deletion

Individuals with 46,XY complete gonadal dysgenesis (CGD) are at high risk of developing gonadal neoplasms. Chromosome 9p monosomy with deletion of the DMRT1 gene, a key transcription factor in testicular development, is one of the known causes of 46,XY CGD. Noninvasive prenatal testing (NIPT) is being increasingly used, and can identify disorders of sexual development (DSDs). We report the case of a 46,XY infant with phenotypically female external genitalia, müllerian structures including uterus and fallopian tubes, and bilateral streak gonads who was found to have unilateral gonadoblastoma at 13 months. 46,XY DSD was suggested prenatally when discordance between NIPT and fetal ultrasound was noted. Genetic investigation revealed a deletion of 12.5 million base pairs at chromosome 9p24.3, which includes the doublesex and MAB-3-related transcription factor-1 (DMRT1) gene. Current guidelines recommend gonadectomy at the time of diagnosis in cases of 46,XY CGD, and our patient had gonadoblastoma at 13 months. 46,XY DSD, including rare disorders such as CGD, will be increasingly identified before birth with more widespread use of NIPT, raising the question about the appropriate timing of gonadectomy in prenatal diagnoses. Our case supports the current recommendation to perform gonadectomy as early as possible after diagnosis.

Transcriptional effects in the estuarine guppy Poecilia vivipara exposed to sanitary sewage in laboratory and in situ

The urban growth has increased sanitary sewage discharges in coastal ecosystems, negatively affecting the aquatic biota. Mangroves, one of the most human-affected coastal biomes, are areas for reproduction and nursing of several species. In order to evaluate the effects of sanitary sewage effluents in mangrove species, this study assessed the hepatic transcriptional responses of guppy fish Poecilia vivipara exposed to sanitary sewage 33% (v:v), using suppressive subtraction hybridization (SSH), high throughput sequencing of RNA (Ion-proton) and quantification of transcript levels by qPCR of some identified genes in fish kept in a sewage-contaminated environment. Genes identified are related predominantly to xenobiotic biotransformation, immune system and sexual differentiation. The qPCR results confirmed the induction of cytochrome P450 1A (CYP1A), glutathione S transferase A-like (GST A-like) methyltransferase (MET) and UDP glycosyltransferase 1A (UDPGT1A), and repression of complement component C3 (C3), doublesex and mab-3 related transcription factor 1 (DMRT1), and transferrin (TF) in the laboratory experiment. In the field exposure, the transcript levels of CYP1A, DMRT1, MET, GST A-like and UDPGT1A were higher in fishes exposed at the contaminated sites compared to the reference site. Chemical analysis in fish from the laboratory and in situ experiments, and surface sediment from the sewage-contaminated sites revealed relevant levels of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyl (PCBs) and linear alkylbenzenes (LABs). These data reinforce the use of P. vivipara as a sentinel for monitoring environmental contamination in coastal regions.

The Sertoli cell marker FOXD1 regulates testis development and function in the chicken.

FOXD1, one of the transcription factors of the FOX family, has been shown to be important for mammalian reproduction but little is known about its function in avian species. In the present study, we identified the expression pattern and location of FOXD1 in chicken tissues and testis by performing quantitative polymerase chain reaction, immunohistochemistry and immunofluorescence, and further investigated the regulatory relationship of FOXD1 with genes involved in testis development by RNA interference. Our results showed that FOXD1 is confirmed to be significantly male-biased expressed in the brain, kidney and testis of adults as well as in embryonic gonads, and it is localised in the testicular Sertoli cell in chicken, consistent with its localisation in mammals. After knock-down of FOXD1 in chicken Sertoli cells, the expression of anti-Müllerian hormone (AMH), sex-determining region Y-box 9 (SOX9) and PKA regulatory subunits type I α (RIα) was significantly downregulated, expression of androgen receptor (AR) was notably increased whereas double-sex and MAB-3-related transcription factor 1 (DMRT1) showed no obvious change in expression. These results suggest that FOXD1 is an essential marker for Sertoli cells upstream of SOX9 expression and a potential regulator of embryonic testis differentiation and development and of normal testis function in the chicken.

Knockdown of DEAD-box helicase 4 (DDX4) decreases the number of germ cells in male and female chicken embryonic gonads.

DEAD-box helicase 4 (DDX4; also known as vasa) is essential for the proper formation and maintenance of germ cells. Although DDX4 is conserved in a variety of vertebrates and invertebrates, its roles differ between species. This study investigated the function of DDX4 in chicken embryos by knocking down its expression using retroviral vectors that encoded DDX4-targeting microRNAs. DDX4 was effectively depleted invitro and invivo via this approach. Male and female gonads of DDX4-knockdown embryos contained a decreased number of primordial germ cells, indicating that DDX4 is essential to maintain a normal level of these cells in chicken embryos of both sexes. Expression of doublesex and mab-3 related transcription factor 1 (DMRT1) and sex determining region Y-box 9 (SOX9), which are involved in testis determination and differentiation, was normal in male gonads of DDX4-knockdown embryos. In contrast, expression of cytochrome P450 family 19 subfamily A member 1 (CYP19A1), which encodes aromatase and is essential for ovary development, was significantly decreased in female gonads of DDX4-knockdown embryos. Expression of forkhead box L2 (FOXL2), which plays an important role in ovary differentiation, was also slightly reduced in DDX4-knockdown embryos, but not significantly. Based on several pieces of evidence FOXL2 was hypothesised to regulate aromatase expression. The results of this study indicate that aromatase expression is also regulated by several additional pathways.

Protein expression of the transcription factors DMRT1, TCLF5, and OCT4 in selected germ cell neoplasms of the testis

In the present study, we investigated protein expression of the transcription factors mammalian doublesex and mab-3 related transcription factor 1 (DMRT1), basic helix-loop-helix transcription factor-like 5 (TCLF5), and octamer-binding transcription factor 4 (OCT4) in normal human spermatogenesis, testicular mixed germ cell-sex cord stromal tumor (MGC-SCST), spermatocytic tumor, and seminoma. In normal human spermatogenesis, DMRT1 is expressed in the nuclei of spermatogonia but not in those of more mature germ cells. By way of contrast, TCLF5 is expressed in the nuclei of some clusters of primary spermatocytes that have entered meiosis 1, in secondary spermatocytes, and in round (early) spermatids in the seminiferous tubules of adults during the reproductive years. OCT4 is expressed in primordial germ cells but not in the seminiferous tubules of the normal adult testis during the reproductive years. DMRT1 is expressed in the germ cells of both testicular MGC-SCST and spermatocytic tumor, whereas TCLF5 is not expressed in either neoplasm. These low-grade neoplasms, however, differ histologically in that all the germ cell nuclei of testicular MGC-SCST resemble spermatogonia, whereas in spermatocytic tumor, the nuclei of the medium-sized and large cells resemble those of primary spermatocytes. Both neoplasms lack expression of OCT4. By way of contrast, in seminoma, a fully malignant testicular germ cell tumor, the germ cell nuclei express OCT4 but do not express either DMRT1 or TCLF5.

Molecular cloning and expression analysis of the aqp1aa gene in half-smooth tongue sole (Cynoglossus semilaevis).

Aquaporin 1 (AQP1) is a member of the transmembrane water channel family of proteins with special structural features, and two AQP1 paralogous genes (aqp1aa and aqp1ab) are reported in teleosts. In the present study, the aqp1aa gene of half-smooth tongue sole (Cynoglossus semilaevis) was cloned and characterized. The full-length cDNA of aqp1aa is 1411 bp with a 786 bp open reading frame encoding a 261-amino acid putative protein with a characteristic structure consisting of 6 membrane-spanning α-helical domains and two highly conserved asparagine—proline—alanine motifs. Real-time quantitative PCR revealed that aqp1aa mRNA is expressed predominantly in the testis of males and pseudo-males, while its expression is low in the ovary and lowest in doublesex and mab-3-related transcription factor 1(DMRT1) knock out fish and triploid males. In situ hybridization indicated that aqp1aa mRNA is expressed mainly in the germ cells of males and pseudo-males, especially in spermatozoa and spermatids. These results suggest that the aqp1aa may play a role in spermatogenesis of C. semilaevis.

Contribution of the coelomic epithelial cells specific to the left testis in the chicken embryo.

The left male gonad in the chicken embryo has a thickened cortical layer, but it eventually becomes flattened after the onset of testicular development. Because the destination of the cortical cells migrating from the left gonad remains unclear, we examined this issue herein. The testis-inducing gene doublesex- and mab-3-related transcription factor 1 (DMRT1) was detected in a proportion of the columnar and cubic epithelial cells in the cortex of the left testis as well as Sertoli cells in both testes. Interestingly, some of the DMRT1-expressing cortical cells were contiguous with Sertoli cells in the testis cord. Some cortical cells exhibited a vimentin-positive cytoplasm that was elongated all the way to the medulla. In addition, a desmosome-like structure was observed between the elongated cytoplasm in these cells and the adjacent Sertoli cell. After the organ culture, a few cells labeled with a fluorescent dye that stained only the cortical cells at the beginning of the culture were located in the testis cord of the left testis. Some cortical cells expressing DMRT1 were suggested to contribute to the Sertoli cells in the testis cord only after the onset of testicular development and only in the left testis. Developmental Dynamics 246:148-156, 2017. © 2016 Wiley Periodicals, Inc.

Generation of gene edited birds in one generation using sperm transfection assisted gene editing (STAGE).

Generating transgenic and gene edited mammals involves in vitro manipulation of oocytes or single cell embryos. Due to the comparative inaccessibility of avian oocytes and single cell embryos, novel protocols have been developed to produce transgenic and gene edited birds. While these protocols are relatively efficient, they involve two generation intervals before reaching complete somatic and germline expressing transgenic or gene edited birds. Most of this work has been done with chickens, and many protocols require in vitro culturing of primordial germ cells (PGCs). However, for many other bird species no methodology for long term culture of PGCs exists. Developing methodologies to produce germline transgenic or gene edited birds in the first generation would save significant amounts of time and resource. Furthermore, developing protocols that can be readily adapted to a wide variety of avian species would open up new research opportunities. Here we report a method using sperm as a delivery mechanism for gene editing vectors which we call sperm transfection assisted gene editing (STAGE). We have successfully used this method to generate GFP knockout embryos and chickens, as well as generate embryos with mutations in the doublesex and mab-3 related transcription factor 1 (DMRT1) gene using the CRISPR/Cas9 system. The efficiency of the method varies from as low as 0% to as high as 26% with multiple factors such as CRISPR guide efficiency and mRNA stability likely impacting the outcome. This straightforward methodology could simplify gene editing in many bird species including those for which no methodology currently exists.

MicroRNA-224 regulates self-renewal of mouse spermatogonial stem cells via targeting DMRT1.

MicroRNAs (miRs) play a key role in the control of gene expression in a wide array of tissue systems, where their functions include the regulation of self‐renewal, cellular differentiation, proliferation and apoptosis. However, the function and mechanisms of individual miRs in regulating spermatogonial stem cell (SSC) homeostasis remain unclear. In the present study, we report for the first time that miR‐224 is highly expressed in mouse SSCs. Functional assays using miRNA mimics and inhibitors reveal that miR‐224 is essential for differentiation of SSCs. Mechanistically, miR‐224 promotes differentiation of SSCs via targeting doublesex and Mab‐3‐related transcription factor 1 (DMRT1). Moreover, WNT/β‐catenin signalling pathway is involved in miR‐224‐mediated regulation of SSCs self‐renewal. We further demonstrate that miR‐224 overexpression increases the expression of GFRα1 and PLZF, accompanied by the down‐regulation of DMRT1 in mouse testes. Our findings provide novel insights into molecular mechanisms regulating differentiation of SSCs and may have important implications for regulating male reproduction.