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Background Disorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously. Results We analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management. Conclusions Our massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.

BackgroundSea urchin gonads of both sexes, commonly termed “roe”, are highly valued seafood delicacies, and Strongylocentrotus intermedius is considered one of the tastiest sea urchins. In order to produce high-quality gonads for consumption and clarify the mechanism of gonad growth and development of the sea urchin, more genetic information, especially at the transcriptome level, is needed.ObjectiveA more thorough understanding of sea urchin gonad growth and development in both sexes could enable regulation of these processes at several stages with the aim of suppressing gametogenesis in order to produce high-quality gonads for consumption.MethodsThe adult sea urchins S. intermedius were cultured for 3 months, and were sampled for the gonadal transcriptome analysis which has been performed on the RNAs of three male and female adults of S. intermedius in each gonad development stage.ResultsIllumina sequencing raw sequence data was deposited in the NCBI Sequence Read Archive (SRA) database (PRJNA532998). It generated 560,196,356 raw reads and 548,956,944 clean reads were acquired, which were assembled into 107,850 transcripts with 44,124 genes. Comparative analysis showed the differentially expressed genes (DEGs) from 114 to 2566. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used to determine the functional significance of these DEGs. We have selected 9 genes related to growth and 12 genes related to fatty acid biosynthesis and metabolism in sea urchin gonads.ConclusionThese data for sea urchins were intended to provide markers for gonad growth and development that can be accumulated for use in aquaculture applications.

Human embryonic stem cells are differentiated to cells similar to the embryonic aorta-gonad-mesonephros, which gives rise to hematopoietic stem cells. The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34 + cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34 + cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial ( SOX17 ) to hematopoietic ( RUNX1C ) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34 + hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17 + vessels from which RUNX1C + blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34 + hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

Cadherins are a group of membrane proteins responsible for cell adhesion. They are crucial for cell sorting and recognition during the morphogenesis, but they also play many other roles such as assuring tissue integrity and resistance to stretching, mechanotransduction, cell signaling, regulation of cell proliferation, apoptosis, survival, carcinogenesis, etc. Within the cadherin superfamily, E- and N-cadherin have been especially well studied. They are involved in many aspects of sexual development and reproduction, such as germline development and gametogenesis, gonad development and functioning, and fertilization. E-cadherin is expressed in the primordial germ cells (PGCs) and also participates in PGC migration to the developing gonads where they become enclosed by the N-cadherin-expressing somatic cells. The differential expression of cadherins is also responsible for the establishment of the testis or ovary structure. In the adult testes, N-cadherin is responsible for the integrity of the seminiferous epithelium, regulation of sperm production, and the establishment of the blood–testis barrier. Sex hormones regulate the expression and turnover of N-cadherin influencing the course of spermatogenesis. In the adult ovaries, E- and N-cadherin assure the integrity of ovarian follicles and the formation of corpora lutea. Cadherins are expressed in the mature gametes and facilitate the capacitation of sperm in the female reproductive tract and gamete contact during fertilization. The germ cells and accompanying somatic cells express a series of different cadherins; however, their role in gonads and reproduction is still unknown. In this review, we show what is known and unknown about the role of cadherins in the germline and gonad development, and we suggest topics for future research.

The divergence of distinct cell populations from multipotent progenitors is poorly understood, particularly in vivo. The gonad is an ideal place to study this process, because it originates as a bipotential primordium where multiple distinct lineages acquire sex-specific fates as the organ differentiates as a testis or an ovary. To gain a more detailed understanding of the process of gonadal differentiation at the level of the individual cell populations, we conducted microarrays on sorted cells from XX and XY mouse gonads at three time points spanning the period when the gonadal cells transition from sexually undifferentiated progenitors to their respective sex-specific fates. We analyzed supporting cells, interstitial/stromal cells, germ cells, and endothelial cells. This work identified genes specifically depleted and enriched in each lineage as it underwent sex-specific differentiation. We determined that the sexually undifferentiated germ cell and supporting cell progenitors showed lineage priming. We found that germ cell progenitors were primed with a bias toward the male fate. In contrast, supporting cells were primed with a female bias, indicative of the robust repression program involved in the commitment to XY supporting cell fate. This study provides a molecular explanation reconciling the female default and balanced models of sex determination and represents a rich resource for the field. More importantly, it yields new insights into the mechanisms by which different cell types in a single organ adopt their respective fates.

Background Takifugu rubripes is an economically valuable fish species in Asia. The implementation of all-male culture for T. rubripes is highly anticipated in aquaculture. Aromatase inhibitor (AI, letrozole) treatment was found to be an efficient method to induced masculinization in T. rubripes , as reported in our previous study. Here, to further explore the underlying regulation mechanism of AI-induced masculinization, a whole-transcriptome analysis comparing was conducted between AI-induced masculinized XX (AI-XX) gonads and control (Con) gonads in T. rubripes . Results In Con-XX/Con-XY comparison, 1,172 differential expression (DE) mRNAs, 129 DEmiRNAs, 210 DElncRNAs, and 4 DEcircRNAs were identified. In the Con-XX/AI-XX comparison, 1,329 DEmRNAs, 174 DEmiRNAs, 6 DEcircRNAs and 280 DElncRNAs were found. Con-XX/Con-XY and Con-XX/AI-XX comparisons shared 690 DEmRNAs, 50 DEmiRNAs, 3 DEcircRNAs, and 105 DElncRNAs. The analyses of protein-protein interaction (PPI) and competitive endogenous RNA (ceRNA) network identified interactions among these shared DERNAs. Kcnh2b , trim27 , cnnm2b , reln , cckbra , pkd1l2 , steap4 , gsg1l , hamp , and foxg1c were predicted as the top ten of hub genes. miRNAs included miRNA-27 family and miRNA-489 family showed targeting relationship with hub genes. GO and KEGG functional enrichment analysis showed that the targeted genes were mainly enriched in GO:0065008 regulation of biological quality and TGF-beta signaling pathway. qPCR validation confirmed the differential expression of selected mRNAs, and ncRNAs. Conclusions This research comprehensively reveals the potential regulatory effects of ncRNAs on cellular motility, fate regulation, and hormonal regulation during gonadal masculinization in T. rubripes . It may provide significant insights into the regulation mechanisms underlying sex reversal in fish.

Studies in non-teleost vertebrates have found microRNAs (miRNAs) to be essential for proper gonadal development. However, comparatively little is known about their role during gonadal development in teleost fishes. So far in zebrafish, a model teleost, transcript profiling throughout gonadal development has not been established because of a tiny size of an organ in juvenile stages and its poor distinguishability from surrounding tissues. We performed small RNA sequencing on isolated gonads of See-Thru-Gonad line, from the undifferentiated state at 3 weeks post fertilization (wpf) to fully mature adults at 24 wpf. We identified 520 gonadal mature miRNAs; 111 of them had significant changes in abundance over time, while 50 miRNAs were either testis- or ovary-enriched significantly in at least one developmental stage. We characterized patterns of miRNA abundance over time including isomiR variants. We identified putative germline versus gonadal somatic miRNAs through differential small RNA sequencing of isolated gametes versus the whole gonads. This report is the most comprehensive analysis of the miRNA repertoire in zebrafish gonads during the sexual development to date and provides an important database from which functional studies can be performed.

Background GATA1 transcription factors, traditionally recognized for their role in hematopoiesis, have recently been implicated in reproduction in vertebrates. However, their function in invertebrate gonadal development remains largely unknown. Results In this study, we identified and characterized GATA1 (designated as Ag GATA1) in the blood clam Anadara granosa and explored its potential role in gonadal development. Phylogenetic analysis revealed that Ag GATA1 exhibited a high degree of conservation across animal species. Furthermore, Ag GATA1 was most highly expressed in the gonads, where it was predominantly expressed in oogonia and spermatogonia, whereas its expression was not observed in oocytes and spermatocyte. In addition, knockdown of the Ag GATA1 gene reduced the number of germ cells in the testis through down-regulation of gonadogenesis-related genes ( FOXL2 , WNT , and Sox9 ). Conclusions Collectively, these findings provide the first functional evidence that GATA1 is involved in molluscan gonadal development, expanding its known role beyond hematopoiesis and offering new insights into the molecular mechanisms underlying reproductive regulation in invertebrates.

All identified genes on the avian W chromosome have homologous counterparts on the Z chromosome, with protein-coding genes showing over 90% homology. These W-linked genes are typically dose-sensitive and conserved, ensuring balanced expression between sexes during development. This study explores the sequence characteristics, expression patterns, and potential functions of the RASA1 gene in chicken embryos. In gonads, RASA1-Z was found to be more highly expressed in males, but total mRNA levels ( RASA1-C ) were higher in females due to RASA1-W compensation. However, RASA1 protein levels were significantly higher in males, suggesting limited contribution of RASA1-W. Knockdown of RASA1-Z reduced both mRNA and protein levels, whereas overexpression of RASA1-W increased mRNA but not protein levels. Sex reversal experiments showed no significant changes in RASA1 expression, indicating it is not involved in gonadal sex differentiation. However, higher expression in the left female gonad suggests a role in gonadal development. Functional studies using CCK8 proliferation assays revealed that RASA1-Z promotes cell proliferation, while RASA1-W has minimal impact. This study indicates that RASA1 primarily functions in cell proliferation and angiogenesis rather than sex differentiation, with RASA1-W contributing to transcript balance but not protein output.

Developing in vitro protocols for non-model species poses challenges, and yet are essential for advancing molecular biology studies. This is particularly true for sex determination research that relies on being able to functionally demonstrate the role of genes in determining sex and guiding the process of sex differentiation. Reptile species are attractive models for sex determination research as many species display thermolabile sex systems, allowing for exploration of gene-environment interactions. The first in vitro gonad culture technique for a turtle was published almost 35 years ago in 1990, but these techniques have seen limited use. This is likely because of challenges inherent to the system, where cultures need to be maintained for prolonged periods for gonadal differentiation to occur. All published techniques for long-term cultures involve removing the gonad from the surrounding mesonephros, which causes issues for proper testes development. Here we present the first protocol developed in the reptile model, Pogona vitticeps , that allows the long-term culture of the whole urogenital system supporting differentiation from biopotential gonads to ovaries or testes. Gross morphology is well maintained, and the gonad can be dissected from the mesonephros even after a culture period of up to 19 days. The cultured gonads display sex specific gene expression and morphology. This protocol will facilitate research in sex determination by providing an effective and low-cost alternative to existing protocols and expand capacity for functional manipulation studies in non-model species.