Sertoli and granulosa cells, the initial differentiated somatic cells in bipotential gonads, play crucial roles in directing male and female gonad development, respectively. The transcription factor Foxo1 is involved in diverse cellular processes, and its expression in gonadal somatic cells is sex-dependent. While Foxo1 is abundantly expressed in ovarian granulosa cells, it is notably absent in testicular Sertoli cells. Nevertheless, its function in gonadal somatic cell differentiation remains elusive. In this study, we find that ectopic expression of Foxo1 in Sertoli cells leads to defects in testes development. Further study uncovers that the ectopic expression of Foxo1 induces the abundant expression of Foxl2 in Sertoli cells, along with the upregulation of other female-specific genes. In contrast, the expression of male-specific genes is reduced. Mechanistic studies indicate that Foxo1 directly binds to the promoter region of Foxl2, inducing its expression. Our findings highlight that Foxo1 serves as a key regulator for the lineage maintenance of ovarian granulosa cells. This study contributes valuable insights into understanding the regulatory mechanisms governing the lineage maintenance of gonadal somatic cells.

Non-obstructive azoospermia, a severe form of male infertility caused by spermatogenic failure (SPGF), has a largely unknown genetic basis across ancestries. To our knowledge, this is the first trans-ethnic meta-analysis of genome-wide association studies on SPGF, involving 2255 men with idiopathic SPGF and 3608 controls from European and Asian populations. Using logistic regression and inverse variance methods, we identify two significant genetic associations with Sertoli cell-only (SCO) syndrome, the most extreme SPGF phenotype. The G allele of rs34915133, in the major histocompatibility complex class II region, significantly increases SCO risk (P = 5.25E-10, OR = 1.57), supporting a potential immune-related cause. Additionally, the rs10842262 variant in the SOX5 gene region is also a genetic marker of SCO (P = 5.29E-09, OR = 0.72), highlighting the key role of this gene in the male reproductive function. Our findings reveal shared genetic factors in male infertility across ancestries and provide insights into the molecular mechanisms underlying SCO.

Abundant direct and in-direct genes are involved in regulating sexual hormones, and reproductive process under nitrite antioxidant plants in rabbit feed. However, there is not enough information about the role of chia seeds and matcha tea as anti-oxidative plants inhibit some direct and in-direct genes related to puberty of growing male rabbits. In this study New-Zealand White (NZW) rabbits treated with chia seeds and matcha tea in water from age after weaning to marketing age about two months ago and determent some sexual hormones, direct and in-direct genes related to puberty and reproduction process. Our data showed total testosterone measured by ELISA increased significantly in chia rabbits compared to control. ELISA analysis revealed that there were no alterations in the levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in the treated groups. Direct genes such as doublesex and mab-3 related transcription factor 1 (DMRT1), sex-determining region Y protein (SRY), and gonadotropin-releasing hormone 1 (GnRH1) determent by qPCR show up-regulating in matcha groups comparable to control group. While in-direct genes follicle stimulating hormone receptor (FSHR) and estrogen receptor 1 (ESR1) detected by qPCR showed up-regulated in matcha rabbits compared to control rabbits. But, luteinizing hormone receptor (LHR) gene was down-regulated in matcha group, and it was up-regulated in chia seeds groups. The prolactin receptor (PRLR) gene investigates down-regulation in all treatment groups. Collectively, matcha tea as one of antioxidant plants were involved those genes studied and activated via hypothalamic- pituitary gonadal axis and led to early puberty in growing male NZW rabbits.

DNA methylation is a common epigenetic modification of DNA levels in the genome of eukaryotic cells, and an aberrant elevation of DNA methylation in gene promoter regions can inhibit gene expression. DNA methyltransferases (DNMTs) are involved in genomic DNA methylation, divided into maintenance DNA methyltransferases and de novo methylases, which are expressed to different degrees in the testis and ovaries. 5-aza-2'-deoxycytidine (5-aza-dC) is a cytidine analog with a strong methylation inhibition. In this experiment, medaka fish fries were treated with 5-aza-dC at 0 μg/L, 50 μg/L, and 100 μg/L. It was found that 100 g/L concentration of 5-aza-dC inhibited both body length and body weight of the adult fish, while 50 g/L concentration had no significant difference. In addition, paraffin section observation and gonad index statistics showed that after 100 g/L concentration of 5-aza-dC treatment, the gonad index of female fish increased significantly, but the gonad index of male fish had no significant difference. And the development of sperms and ovaries was normal without significant difference. Finally, we found that 5-aza-dC not only significantly decreased the transcription levels of dnmt1 and dnmt3bb.1, but also significantly increased the expression levels of female-related genes such as foxl2, cyp19a1 and wnt4, and significantly decreased the expression levels of male-related genes such as dmrt1, sox9a and amh. The DNA methylation patterns of foxl2 and dmrt1 genes were altered. This work provides more references for understanding the mechanism of DNA methylation affecting sex determination in fish.

Somatic sex identity must be maintained throughout adulthood for tissue function. Adult somatic stem cells in the Drosophila testis (i.e., CySCs) lacking the transcription factor Chinmo are reprogrammed to their ovarian counterparts by induction of female-specific Tra F , but this is not mechanistically understood. Pioneer factors play central roles in direct reprogramming, and many upregulated genes in chinmo -/- CySCs contain binding sites for the pioneer factor Zelda (Zld). microRNAs repress zld mRNA in wild type CySCs, but they are downregulated after Chinmo loss, allowing for zld mRNA translation. Zld depletion from chinmo -/- CySCs suppresses feminization, and ectopic Zld induces Tra F and feminizes wild-type CySCs. qkr58E-2 and ecdysone receptor ( EcR ), direct Zld targets in the embryo, are female-biased in adult gonads and upregulated in chinmo -/- CySCs. The RNA-binding protein Qkr58E-2 produces Tra F , while EcR promotes female-biased gene expression. Ectopic Zld feminizes adult male adipose tissue, demonstrating that Zld can instruct female and override male identity in adult XY tissues.

zld mRNA is repressed by microRNAs in XY somatic gonadal cells Zld is upregulated in and required for sex reversal of XY chinmo -/- cells Zld induces Qkr58E-2 and EcR, which cause Tra F and female-biased transcription Zld feminizes XY adipose cells by inducing Tra F and downregulating Chinmo.

The most severe form of male infertility is idiopathic non-obstructive azoospermia (NOA), a complete sperm absence in the ejaculate. We performed exome sequencing in the Croatian infertile brothers with NOA and found a variant in DMRT1 (Doublesex and mab-3 related transcription factor 1) gene that was further assessed by the EMSA assay and molecular dynamic simulations. We additionally screened for DMRT1 mutations in 1940 infertile men diagnosed with spermatogenic failure, 644 normozoospermic controls, and 105 females with primary ovarian insufficiency (POI) recruited to the GEnetics of Male INfertility Initiative (GEMINI) or Estonian Andrology (ESTAND) cohorts. DMRT1 p.Pro74Leu (chr9:g.842059C > T) variant was detected in infertile brothers in the highly conserved position within the DNA binding DM domain of the protein. EMSA assay showed reduced DNA binding of DMRT1P74L and molecular dynamic simulations showed differences in structural and dynamical properties between the wild type protein and DMRT1P74L. Plausible disease-causing DMRT1 variants were only identified in infertile men (13/1940; 0.67%), and none in 639 fertile controls. Burden testing showed an excess of rare deleterious DM domain mutations in the infertility cohort compared to gnomAD v.4.0 population-based controls (Fisher's exact test, p = 1.44 x 10-5). Three rare deleterious variants in DMRT1 were found in 104 cases of POI. The findings of this study strengthen the evidence of DMRT1 variants being a causal factor for male infertility and provide the distribution of likely pathogenic variants across the gene. This is also the first study to suggest that DMRT1 variants may also be linked to POI.

Karyotype 46, XY female disorders of sex development (46, XY female DSD) are congenital conditions due to irregular gonadal development or androgen synthesis or function issues. Genes significantly influence DSD; however, the underlying mechanisms remain unclear. This study identified a Chinese family with 46, XY female DSD due to the CUL4B gene.

The proband medical history and pedigree were investigated. Whole-exome sequencing was performed to analyze different variations. Transiently transfected testicular teratoma (NT2/D1), KGN ovarian cells with either mutant or wild-type CUL4B gene, and knock-in Cul4b mouse models were confirmed. The expression levels of sex-related genes were analyzed.

A 9.5-year-old girl was diagnosed with 46, XY DSD. A hemizygous variant c.838 T > A of the CUL4B gene was detected. The mRNA and protein levels of WNT4 and FOXL2 genes were higher than those in the wild-type group; however, CTNNB1, SOX9, and DMRT1 were lower in the wild-type group in NT2/D1 cells. In KGN ovarian cells of the mutant group, the mRNA and protein levels for WNT4 and CTNNB1 were elevated. Damaged testicular vasculature and underdeveloped seminal vesicles were observed in Cul4bL337M mice.

A missense CUL4B variant c.838 T > A associated with 46, XY female DSD was identified, and may activate the Wnt4/β-catenin pathway. Our findings provide novel insights into the molecular mechanisms of 46, XY female DSD.

Abnormalities in spermatogenesis, a fundamental component of male reproductive function, can cause male infertility. Somatic cells constituting the testis microenvironment are essential for controlling normal spermatogenesis. Although testicular somatic cells are thought to sense and respond to germ cells to ensure proper spermatogenesis, the details of this signaling mechanism are unknown. Here, we investigated somatic cell dynamics in testicular tissue lacking spermatogenesis using the mice with deletion of the testis-specific histone H3 variant gene H3t. Testicular tissue sections of H3tΔ/Δ mice exhibited an increased interstitial area compared with those of wild-type mice, which was primarily attributed to an increase in Leydig cell numbers. Furthermore, this increase in Leydig cells led to increased testosterone synthesis, which occurred alongside cellular senescence-associated β-galactosidase activity. These findings suggest that Leydig cells monitor the progress of spermatogenesis and possess a mechanism to promote functional germ cell formation.

Zebrafish sex differentiation is a complicated process and the detailed mechanism has not been fully understood. Here we characterized a transcription factor, Foxl2l, which participates in female oogenesis. We show that it is expressed specifically in proliferating germ cells in juvenile gonads and mature ovaries. We have used CRISPR-Cas9 to generate zebrafish deficient in foxl2l expression. Zebrafish with foxl2l-/- are all males, and this female-to-male sex reversal cannot be reversed by tp53 mutation, indicating this sex reversal is unrelated to cell death. We have generated transgenic fish expressing GFP under the control of foxl2l promoter to track the development of foxl2l + -germ cells; these cells failed to enter meiosis and accumulated as cystic cells in the foxl2l-/- mutant. Our RNA-seq analysis also showed the reduced expression of genes in meiosis and oogenesis among other affected pathways. All together, we show that zebrafish Foxl2l is a nuclear factor controlling the expression of meiotic and oogenic genes, and its deficiency leads to defective meiotic entry and the accumulation of premeiotic germ cells.

Animal sex chromosomes typically carry the upstream sex-determining gene that triggers testis or ovary development and, in some species, are regulated by global dosage compensation in response to functional decay of the Y chromosome. Despite the importance of these pathways, they exhibit striking differences across species, raising fundamental questions regarding the mechanisms underlying their evolutionary turnover. Recent studies of non-model organisms, including insects, reptiles and teleosts, have yielded a broad view of the diversity of sex chromosomes that challenges established theories. Moreover, continued studies in model organisms with recently developed technologies have characterized the dynamics of sex determination and dosage compensation in three-dimensional nuclear space and at single-cell resolution. Here, we synthesize recent insights into sex chromosomes from a variety of species to review their evolutionary dynamics with respect to the canonical model, as well as their diverse mechanisms of regulation.

In humans, the X and Y chromosomes determine the biological sex, XX specifying for females and XY for males. The long noncoding RNA X-inactive specific transcript (lncRNA XIST) plays a crucial role in the process of X chromosome inactivation (XCI) in cells of the female, a process that ensures the balanced expression of X-linked genes between sexes. Initially, it was believed that XIST can be expressed only from the inactive X chromosome (Xi) and is considered a typically female-specific transcript. However, accumulating evidence suggests that XIST can be detected in male cells as well, and it participates in the development of cancers and other human diseases by regulating gene expression at epigenetic, chromatin remodeling, transcriptional, and translational levels. XIST is abnormally expressed in many sexually dimorphic diseases, including autoimmune and neurological diseases, pulmonary arterial hypertension (PAH), and some types of cancers. However, the underlying mechanisms are not fully understood. Escape from XCI and skewed XCI also contributes to sex-biased diseases and their severity. Interestingly, in humans, similar to experimental animal models of human disease, the males with the XIST gene activated display the sex-biased disease condition at a rate close to females, and significantly greater than males who had not been genetically modified. For instance, the men with supernumerary X chromosomes, such as men with Klinefelter syndrome (47, XXY), are predisposed toward autoimmunity similar to females (46, XX), and have increased risk for strongly female biased diseases, compared to 46, XY males. Interestingly, chromosome X content has been linked to a longer life span, and the presence of two chromosome X contributes to increased longevity regardless of the hormonal status. In this review, we summarize recent knowledge about XIST structure/function correlation and involvement in human disease with focus on XIST abnormal expression in males. Many human diseases show differences between males and females in penetrance, presentation, progression, and survival. In humans, the X and Y sex chromosomes determine the biological sex, XX specifying for females and XY for males. This numeric imbalance, two X chromosomes in females and only one in males, known as sex chromosome dosage inequality, is corrected in the first days of embryonic development by inactivating one of the X chromosomes in females. While this "dosage compensation" should in theory solve the difference in the number of genes between sexes, the expressed doses of X genes are incompletely compensated by X chromosome inactivation in females. In this review we try to highlight how abnormal expression and function of XIST, a gene on the X chromosome responsible for this inactivation process, may explain the sex differences in human health and disease. A better understanding of the molecular mechanisms of XIST participation in the male-female differences in disease is highly relevant since it would allow for improving the personalization of diagnosis and sex-specific treatment of patients.

The process of sexual development in animals is modulated by a variety of mechanisms. Some species respond to environmental cues, while, in others, sex determination is thought to be controlled by a single 'master regulator' gene. However, many animals respond to a combination of environmental cues (e.g., temperature) and genetic factors (e.g., sex chromosomes). Even among species in which genetic factors predominate, there is a continuum between monofactorial and polygenic systems. The perception that polygenic systems are rare may result from experiments that lack the statistical power to detect multiple loci. Intellectual biases against the existence of polygenic sex determination (PSD) may further arise from misconceptions about the regulation of developmental processes and a misreading of theoretical results on the stability of polygenic systems of sex determination.

Smoc1 and Smoc2, members of the SPARC family of genes, encode signaling molecules downstream of growth factors such as the TGF-β, FGF, and PDGF families. Smoc1 has been implicated in playing a crucial role in microphthalmia with limb anomalies in humans and mice, while Smoc2 deficiency causes dental developmental defects. Although developmental cytokines/growth factors including TGF-β superfamily have been shown to play critical roles in postnatal spermatogenesis, there are no reports analyzing the spatial and temporal expression of Smoc1 and Smoc2 in the postnatal testis. In this study, we investigated the mRNA and protein expression of Smoc1 and Smoc2 in neonatal, juvenile, and adult mouse testes by RNA in situ hybridization, immunofluorescence, and single-cell RNA-seq analysis. We show that Smoc1 and Smoc2 have distinct expression patterns in male germ cells: Smoc1 is more highly expressed than Smoc2 in the germline. In contrast, Smoc2 is highly expressed in testicular somatic cells from neonatal to juvenile stages. The Smoc2-expressing cells then switch from somatic cells to germ cells in adults. Thus, although SMOC1 and SMOC2 proteins are structurally very similar, their spatial and temporal expression patterns in the postnatal testis differ significantly, suggesting their distinct roles in reproduction.

Hexafluoropropylene oxide trimer acid (HFPO-TA), a novel alternative to perfluorooctanoic acid (PFOA), has been widely used and ubiquitously detected in aquatic environments. However, its potential effects on sex differentiation of aquatic organisms are not well known. Therefore, in this study, zebrafish were exposed to HFPO-TA at different development stages (0-21, 21-42, and 42-63 dpf) to investigate the effects on sex differentiation and its underlying mechanisms. All three exposures to HFPO-TA resulted in the feminization of zebrafish, and the impact of Stage II was most significant. The transcription levels of key genes related to female differentiation (bpm15, cyp19a1a, esr1, vtg1, and sox9b) were up-regulated, while those of key genes related to male differentiation (dmrt1, gata4, amh, and sox9a) were down-regulated, which could lead to the feminization. In addition, it was found that the dysregulations of these genes were prolonged in adult zebrafish even through a long recovery, which could cause sex imbalance in populations. Therefore, HFPO-TA might not be a safe alternative to PFOA, and more evidences from multi- and transgenerational toxicology are warranted.

Epigenetic modifications regulate chromatin accessibility, gene expression, cell differentiation and tissue development. As epigenetic modifications can be inherited via mitotic and meiotic cell divisions, they enable a heritable memory of cell identity and function and can alter inherited characteristics in the next generation. Tight regulation of epigenetic information is critical for normal cell function and is often disrupted in diseases including cancer, metabolic, neurological and inherited congenital conditions. The ovary performs critical functions in female reproductive health and fertility, including oocyte and sex-hormone production. Oocytes undergo extensive epigenetic programming including the establishment of maternal genomic imprints, which are critical for offspring health and development. Epigenetic modifiers also regulate ovarian somatic cells, such as granulosa and theca cells which support oocytes and produce hormones. While ovarian dysfunction contributes to serious ovarian conditions such as primary ovarian insufficiency (POI), polycystic ovary syndrome (PCOS) and ovarian cancers, the roles of epigenetic modifications in the ovary and their contribution to ovarian dysfunction are not properly understood. Here we review recent advancements in understanding Polycomb proteins, important epigenetic modifiers that have emerging roles in ovarian development and maternal epigenetic inheritance. Polycomb group proteins (PcGs) contribute to the faithful establishment of epigenetic information in oocytes, a process essential for normal offspring development in mice. Emerging evidence also indicates that PcGs regulate ovarian function and female fertility. Understanding these and similar mechanisms will provide greater insight into the epigenetic regulation of ovarian and oocyte function, and how its disruption can impact reproductive health and maternal inheritance.

Normal ovarian function requires the expression of estrogen receptors α (ESR1) and β (ESR2) in distinct cell types within the ovary. The double estrogen receptor knockout (αβERKO) ovary had the appearance of seminiferous tubule-like structures that expressed SOX9; this phenotype was lost when the animals were repeatedly backcrossed to the C57BL/6J genetic background. A new line of ERKO mice, Ex3αβERKO, was developed for targeted disruption on a mixed genetic background. Histological examination of the ovaries in the Ex3αβERKO showed the appearance of seminiferous tubule-like structures in mice aged 6 to 12 months. These dismorphogenic regions have cells that no longer express granulosa cell-specific FOXL2, while other cells express Sertoli cell-specific SOX9 as examined by immunohistochemistry. Whole ovarian gene expression analysis in Ex3αERKO, Ex3βRKO, and Ex3αβERKO found many genes differentially expressed compared to controls with one Esr1 and Esr2 allele. The genes specific to the Ex3αβERKO ovary were compared to other models of postnatal ovarian transdifferentiation, identifying 21 candidate genes. To examine the genetic background contributions, DNA was isolated from αβERKO mice that did not show ovarian transdifferentiation and compared to DNA from Ex3αβERKO using Mouse Diversity Array. A genomic region putatively associated with transdifferentiation was identified on Chr18 (5-15 M) and genes in this region were compared to the genes differentially expressed in models of ovarian transdifferentiation. This work demonstrates the importance of ESRs in maintaining granulosa cell differentiation within the ovary, identifies several potential gene candidates, and suggests that genetic background can be a confounding factor.

Vertebrate spermatogonial stem cells maintain sperm production over the lifetime of an animal, but fertility declines with age. Although morphological studies have informed our understanding of typical spermatogenesis, the molecular and cellular mechanisms underlying the maintenance and decline of spermatogenesis are not yet understood. We used single-cell RNA sequencing to generate a developmental atlas of the aging zebrafish testis. All testes contained spermatogonia, but we observed a progressive decline in spermatogenesis that correlated with age. Testes from some older males only contained spermatogonia and a reduced population of spermatocytes. Spermatogonia in older males were transcriptionally distinct from spermatogonia in testes capable of robust spermatogenesis. Immune cells including macrophages and lymphocytes drastically increased in abundance in testes that could not complete spermatogenesis. Our developmental atlas reveals the cellular changes as the testis ages and defines a molecular roadmap for the regulation of spermatogenesis.

Complementary sex determination regulates female and male development in honeybees (Apis mellifera) via heterozygous versus homo-/hemizygous genotypes of the csd (complementary sex determiner) gene involving numerous naturally occurring alleles. This lineage-specific function offers a rare opportunity to understand an undescribed regulatory mechanism and the molecular evolutionary path leading to this mechanism. We reviewed recent advances in understanding how Csd recognizes different versus identical protein variants, how these variants regulate downstream pathways and sexual differentiation, and how this mechanism has evolved and been shaped by evolutionary forces. Finally, we highlighted the shared regulatory principles of sex determination despite the diversity of primary signals and demonstrated that lineage-specific mutations are very informative for characterizing newly evolved functions.

Mollusks are the second most diverse animal phylum, yet little is known about their endocrinology or how they respond to endocrine disrupting compound (EDC) pollution. Characteristic effects of endocrine disruption are reproductive impairment, skewed sex ratios, development of opposite sex characteristics, and population decline. However, whether classical vertebrate EDCs, such as steroid hormone-like chemicals and inhibitors of steroidogenesis, exert effects on mollusks is controversial. In the blue mussel, Mytilus edulis, EDC exposure is correlated with feminized sex ratios in wild and laboratory mussels, but sex reversal has not been confirmed. Here, we describe a non-destructive qPCR assay to identify the sex of M. edulis allowing identification of males and females prior to experimentation. We exposed male mussels to 17α-ethinylestradiol and female mussels to ketoconazole, EDCs that mimic vertebrate steroid hormones or inhibit their biosynthesis. Both chemicals changed the sex of individual mussels, interfered with gonadal development, and disrupted gene expression of the sex differentiation pathway. Impacts from ketoconazole treatment, including changes in steroid levels, confirmed a role for steroidogenesis and steroid-like hormones in mollusk endocrinology. The present study expands the possibilities for laboratory and field monitoring of mollusk species and provides key insights into endocrine disruption and sexual differentiation in bivalves.

In order to optimize the parameters for reversing masculinization and establish the techniques for sex induction of pseudo-males and creation of all-female fry in largemouth bass (Micropterus salmodies, LMB), 15-day-old LMB (1.00 ± 0.10 cm in length, 0.10 ± 0.01 g in weight) were fed a diet supplemented with either 17α-methyltestosterone (MT) or letrozole (LE) and their combination. The experimental groups were M20 (20 mg/kg MT), L20 (20 mg/kg LE) and M10L10 (10 mg/kg MT and 10 mg/kg LE). The control group, named C, was not feed MT or LE. After 60 days, exogenous hormone in the diets was stopped and the effects of MT and LE on growth, male ratio, and gonadal development in LMB were evaluated. At 12-month-old, blood and gonadal tissue samples were collected to measure sex steroid hormones levels, analyze expression levels of dmrt1 and cyp19a1a genes, as well as examine the gonads morphology. The results showed no significant differences in growth between the experimental groups and the control group after a 60-day feeding period with the formulated diet (p > 0.05). The sex reversal ratio of M20, L20, M10L10 were 95.00%, 80.00%, 76.47%, respectively. The gonadal tissue sections showed that the gonadal structure of masculinized fish morphologic resembled that of control male fish. At 12-month-old, the sex reversal ratio in M20, L20, M10L10 and C groups were 100%, 86.67%, 73.33% and 50.00%, respectively. The testicular of pseudo-male fish in the M20 group exhibited well-developed morphology similarities to that of the control group males. However, the testes of pseudo-male fish in the L20 and M10L10 groups were smaller size Estradiol (E2) levels in the experimental groups was significantly lower than those in the control group females (p < 0.05), while testosterone (T) levels were significantly higher than that of the control group (p < 0.05). Compared to the female fish in the control group, pseudo-male fish from all experimental groups showed significantly upregulated expression of dmrt1 (p < 0.05), and significantly downregulated expression of cyp19a1a (p < 0.05). Pseudo-males selected from group M20 exhibited a significantly higher proportion of female offspring (92.00%) compared to the control group (46.50%). In summary, 20 mg/kg MT was the optimal inducing concentration.

Fish sex is largely influenced by steroid hormones, especially sex hormones. Here, we established a steroid hormone-free genetic model by mutation of cyp11a1 in Nile tilapia, which was confirmed by EIA assay. Gonadal phenotype and transcriptome analyses showed that the XX mutants displayed sex reversal from female to male but with defective spermatogenesis. Despite the sex reversal, the aromatase encoding gene cyp19a1a was continuously expressed in the gonads of the XX mutants, which might be caused by androgen deficiency. Whole-mount fluorescence in situ hybridization and transcriptome analysis showed that the gonads of the XX mutants firstly developed towards ovary but shifted to testis between 10 to 15 days after hatching. Detailed expression analysis of key sex differentiation pathway genes foxl3 and dmrt1 combined with apoptosis analysis revealed transdifferentiation of germ cells from female to male during sex reversal. Rescue experiments showed that both P5 and E2 treatment rescued the sex reversal of cyp11a1 mutant XX fish. Overall, our results revealed a transient ovary-like stage and transdifferentiation of germ cells from female to male in the early gonads of the steroid hormone-deprived cyp11a1 mutant XX fish.

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.

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.

In adult gonads, disruption of somatic sexual identity leads to defective gametogenesis and infertility. However, the underlying mechanisms by which somatic signals regulate germline cells to achieve proper gametogenesis remain unclear. In our previous study, we introduced the chinmoSex Transformation (chinmoST ) mutant Drosophila testis phenotype as a valuable model for investigating the mechanisms underlying sex maintenance. In chinmoST testes, depletion of the Janus Kinase-Signal Transducer and Activator of Transcription downstream effector Chinmo from somatic cyst stem cells (CySCs) feminizes somatic cyst cells and arrests germline differentiation. Here, we use single-cell RNA sequencing to uncover chinmoST -specific cell populations and their transcriptomic changes during sex transformation. Comparative analysis of intercellular communication networks between wild-type and chinmoST testes revealed disruptions in several soma-germline signaling pathways in chinmoST testes. Notably, the insulin signaling pathway exhibited significant enhancement in germline stem cells (GSCs). Chinmo cleavage under targets and tagmentation (CUT&Tag) assay revealed that Chinmo directly regulates two male sex determination factors, doublesex (dsx) and fruitless (fru), as well as Ecdysone-inducible gene L2 (ImpL2), a negative regulator of the insulin signaling pathway. Further genetic manipulations confirmed that the impaired gametogenesis observed in chinmoST testes was partly contributed by dysregulation of the insulin signaling pathway. In summary, our study demonstrates that somatic sex maintenance promotes normal spermatogenesis through Chinmo-mediated conserved sex determination and the insulin signaling pathway. Our work offers new insights into the complex mechanisms of somatic stem cell sex maintenance and soma-germline communication at the single-cell level. Additionally, our discoveries highlight the potential significance of stem cell sex instability as a novel mechanism contributing to testicular tumorigenesis.

In vertebrate sexual development, two important steroid hormones, testosterone and estrogen, regulate the sex-specific development of many tissues. In contrast, invertebrates utilize a single steroid hormone, ecdysone, to regulate developmental timing in both sexes. However, here we show that in Drosophila melanogaster, sex-specific ecdysone (E) signaling controls important aspects of gonad sexual dimorphism. Rather than being regulated at the level of hormone production, hormone activity is regulated cell-autonomously through sex-specific hormone reception. Ecdysone receptor (EcR) expression is restricted to the developing ovary and is repressed in the testis at a time when ecdysone initiates ovary morphogenesis. Interestingly, EcR expression is regulated downstream of the sex determination factor Doublesex (Dsx), the founding member of the Dsx/Mab3 Related Transcription Factor (DMRT) family that regulates gonad development in all animals. E signaling is required for normal ovary development1,2, and ectopic activation of E signaling in the testis antagonized stem cell niche identity and feminized somatic support cells, which were transformed into follicle-like cells. This work demonstrates that invertebrates can also use steroid hormone signaling to control sex-specific development. Further, it may help explain recent work showing that vertebrate sexual development is surprisingly cell-autonomous. For example, chickens utilize testosterone and estrogen to control sex-specific development, but when they have a mixture of cells with male and female genotypes, the male cells develop as male and the female cells develop as female despite exposure to the same circulating hormones3. Sex-specific regulation of steroid hormone signaling may well underly such cell-autonomous sexual fate choices in vertebrates as it does in Drosophila.

Differences/disorders of sex development (DSDs) in individuals with a 46, XY karyotype are a group of congenital disorders that manifest as male gonadal hypoplasia or abnormalities of the external genitalia. Approximately 50% of patients with 46, XY DSDs cannot obtain a molecular diagnosis. The aims of this paper were to review the most common causative genes and rare genes in patients with 46, XY DSDs, analyze global molecular diagnostic cohorts for the prevalence and geographic distribution of causative genes, and identify the factors affecting cohort detection results. Although the spectrum of genetic variants varies across regions and the severity of the clinical phenotype varies across patients, next-generation sequencing (NGS), the most commonly used detection method, can still reveal genetic variants and aid in diagnosis. A comparison of the detection rates of various sequencing modalities revealed that whole-exome sequencing (WES) facilitates a greater rate of molecular diagnosis of the disease than panel sequencing. Whole-genome sequencing (WGS), third-generation sequencing, and algorithm advancements will contribute to the improvement of detection efficiency. The most commonly mutated genes associated with androgen synthesis and action are AR, SR5A2, and HSD17B3, and the most commonly mutated genes involved in gonadal formation are NR5A1 and MAP3K1. Detection results are affected by differences in enrollment criteria and sequencing technologies.

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.

The Chinese soft-shelled turtle, Pelodiscus sinensis, exhibits distinct sexual dimorphism, with the males growing faster and larger than the females. During breeding, all-male offspring can be obtained using 17β-estradiol (E2). However, the molecular mechanisms underlying E2-induced sexual reversal have not yet been elucidated. Previous studies have investigated the molecular sequence and expression characteristics of estrogen receptors (ERs).

In this study, primary liver cells and embryos of P. sinensis were treated with ER agonists or inhibitors. Cell incubation experiments revealed that nuclear ERs (nERs) were the main pathway for the transmission of estrogen signals. Our results showed that ERα agonist (ERα-ag) upregulated the expression of Rspo1, whereas ERα inhibitor (ERα-Inh) downregulated its expression. The expression of Dmrt1 was enhanced after ERα-Inh + G-ag treatment, indicating that the regulation of male genes may not act through a single estrogen receptor, but a combination of ERs. In embryos, only the ERα-ag remarkably promoted the expression levels of Rspo1, Wnt4, and β-catenin, whereas the ERα-Inh had a suppressive effect. Additionally, Dmrt1, Amh, and Sox9 expression levels were downregulated after ERβ inhibitor (ERβ-Inh) treatment. GPER agonist (G-ag) has a significant promotion effect on Rspo1, Wnt4, and β-catenin, while the inhibitor G-Inh does not affect male-related genes.

Overall, these results suggest that ERs play different roles during sexual reversal in P. sinensis and ERα may be the main carrier of estrogen-induced sexual reversal in P. sinensis. Further studies need to be performed to analyze the mechanism of ER action.

In mammals, the development of male or female gonads from fetal bipotential gonads depends on intricate genetic networks. Changes in dosage or temporal expression of sex-determining genes can lead to differences of gonadal development. Two rare conditions are associated with disruptions in ovarian determination, including 46,XX testicular differences in sex development (DSD), in which the 46,XX gonads differentiate into testes, and 46,XX ovotesticular DSD, characterized by the coexistence of ovarian and testicular tissue in the same individual. Several mechanisms have been identified that may contribute to the development of testicular tissue in XX gonads. This includes translocation of SRY to the X chromosome or an autosome. In the absence of SRY, other genes associated with testis development may be overexpressed or there may be a reduction in the activity of pro-ovarian/antitesticular factors. However, it is important to note that a significant number of patients with these DSD conditions have not yet recognized a genetic diagnosis. This finding suggests that there are additional genetic pathways or epigenetic mechanisms that have yet to be identified. The text will provide an overview of the current understanding of the genetic factors contributing to 46,XX DSD, specifically focusing on testicular and ovotesticular DSD conditions. It will summarize the existing knowledge regarding the genetic causes of these differences. Furthermore, it will explore the potential involvement of other factors, such as epigenetic mechanisms, in developing these conditions.

In mammals, gonadal somatic cell lineage differentiation determines the development of the bipotential gonad into either the ovary or testis. Sertoli cells, the only somatic cells in the spermatogenic tubules, support spermatogenesis during gonadal development. During embryonic Sertoli cell lineage differentiation, relevant genes, including WT1, GATA4, SRY, SOX9, AMH, PTGDS, SF1, and DMRT1, are expressed at specific times and in specific locations to ensure the correct differentiation of the embryo toward the male phenotype. The dysregulated development of Sertoli cells leads to gonadal malformations and male fertility disorders. Nevertheless, the molecular pathways underlying the embryonic origin of Sertoli cells remain elusive. By reviewing recent advances in research on embryonic Sertoli cell genesis and its key regulators, this review provides novel insights into sex determination in male mammals as well as the molecular mechanisms underlying the genealogical differentiation of Sertoli cells in the male reproductive ridge.

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.

Stem cells are critical for replenishment of cells lost to death, damage or differentiation. Drosophila testes are a key model system for elucidating mechanisms regulating stem cell maintenance and differentiation. An intriguing gene identified through such studies is the transcription factor, chronologically inappropriate morphogenesis (Chinmo). Chinmo is a downstream effector of the Jak-STAT signaling pathway that acts in testis somatic stem cells to ensure maintenance of male stem cell fate and sexual identity. Defects in these processes can lead to infertility and the formation of germ cell tumors. While Chinmo's effect on testis stem cell behavior has been investigated in detail, there is still much to be learned about its structure, function, and interactions with other proteins. Using a two-hybrid screen, we find that Chinmo interacts with itself, the small ubiquitin-like modifier SUMO, the novel protein CG11180, and four other proteins (CG4318, Ova (ovaries absent), Taf3 (TBP-associated factor 3), and CG18269). Since both Chinmo and CG11180 contain sumoylation sites and SUMO-interacting motifs (SIMs), we analyzed their interaction in more detail. Using site-directed mutagenesis of a unique SIM in CG11180, we demonstrate that Chinmo's interaction with CG11180 is SUMO-dependent. Furthermore, to assess the functional relevance of both SUMO and CG11180, we performed RNAi-mediated knockdown of both proteins in somatic cells of the Drosophila testis. Using this approach, we find that CG11180 and SUMO are required in somatic cells of adult testes, and that reduction of either protein causes formation of germ cell tumors. Overall, our work suggests that SUMO may be involved in the interaction of Chinmo and CG11180 and that these genes are required in somatic cells of the adult Drosophila testis. Consistent with the CG11180 knockdown phenotype in male testes, and to underscore its connection to Chinmo, we propose the name Chigno (Childless Gambino) for CG11180.

Although sex determination is ubiquitous in vertebrates, mechanisms of sex determination vary from environmentally to genetically influenced. In vertebrates, genetic sex determination is typically accomplished with sex chromosomes. Groups like mammals maintain conserved sex chromosome systems, while sex chromosomes in most vertebrate clades are not conserved across similar evolutionary timescales. One group inferred to have an evolutionarily stable mode of sex determination is Anguimorpha, a clade of charismatic taxa including monitor lizards, Gila monsters, and crocodile lizards. The common ancestor of extant anguimorphs possessed a ZW system that has been retained across the clade. However, the sex chromosome system in the endangered, monotypic family of crocodile lizards (Shinisauridae) has remained elusive. Here, we analyze genomic data to demonstrate that Shinisaurus has replaced the ancestral anguimorph ZW system on LG7 with a novel ZW system on LG3. The linkage group, LG3, corresponds to chromosome 9 in chicken, and this is the first documented use of this syntenic block as a sex chromosome in amniotes. Additionally, this ~1 Mb region harbors approximately 10 genes, including a duplication of the sex-determining transcription factor, Foxl2, critical for the determination and maintenance of sexual differentiation in vertebrates, and thus a putative primary sex-determining gene for Shinisaurus.

The Nile tilapia (Oreochromis niloticus), with a system of XX/XY sex determination, is a worldwide farmed fish with a shorter sexual maturation time than that of most cultured fish. Tilapia show a spawning cycle of approximately 14 days and can be artificially propagated in the laboratory all year round to obtain genetically all female (XX) and all male (XY) fry. Its genome sequence has been opened, and a perfect gene editing platform has been established. With a moderate body size, it is convenient for taking enough blood to measure hormone level. In recent years, using tilapia as animal model, we have confirmed that estrogen is crucial for female development because 1) mutation of star2, cyp17a1 or cyp19a1a (encoding aromatase, the key enzyme for estrogen synthesis) results in sex reversal (SR) due to estrogen deficiency in XX tilapia, while mutation of star1, cyp11a1, cyp17a2, cyp19a1b or cyp11c1 affects fertility due to abnormal androgen, cortisol and DHP levels in XY tilapia; 2) when the estrogen receptors (esr2a/esr2b) are mutated, the sex is reversed from female to male, while when the androgen receptors are mutated, the sex cannot be reversed; 3) the differentiated ovary can be transdifferentiated into functional testis by inhibition of estrogen synthesis, and the differentiated testis can be transdifferentiated into ovary by simultaneous addition of exogenous estrogen and androgen synthase inhibitor; 4) loss of male pathway genes amhy, dmrt1, gsdf causes SR with upregulation of cyp19a1a in XY tilapia. Disruption of estrogen synthesis rescues the male to female SR of amhy and gsdf but not dmrt1 mutants; 5) mutation of female pathway genes foxl2 and sf-1 causes SR with downregulation of cyp19a1a in XX tilapia; 6) the germ cell SR of foxl3 mutants fails to be rescued by estrogen treatment, indicating that estrogen determines female germ cell fate through foxl3. This review also summarized the effects of deficiency of other steroid hormones, such as androgen, DHP and cortisol, on fish reproduction. Overall, these studies demonstrate that tilapia is an excellent animal model for studying reproductive endocrinology of fish.

A systematic analysis of genes related to reproduction is crucial for obtaining a comprehensive understanding of the molecular mechanisms that underlie male reproductive traits in mammals. Here, we utilized 435 goat transcriptome datasets to unveil the testicular tissue-specific genes (TSGs), allele-specific expression (ASE) genes and their uncharacterized transcriptional features related to male goat reproduction. Results showed a total of 1790 TSGs were identified in goat testis, which was the most among all tissues. GO enrichment analyses suggested that testicular TSGs were mainly involved in spermatogenesis, multicellular organism development, spermatid development, and flagellated sperm motility. Subsequently, a total of 95 highly conserved TSGs (HCTSGs), 508 middle conserved TSGs (MCTSGs) and 42 no conserved TSGs (NCTSGs) were identified in goat testis. GO enrichment analyses suggested that the HCTSGs and MCTSGs has a more important association with male reproduction than NCTSGs. Additionally, we identified 644 ASE genes, including 88 tissue-specific ASE (TS-ASE) genes (e.g., FSIP2, TDRD9). GO enrichment analyses indicated that both ASE genes and TS-ASE genes were associated with goat male reproduction. Overall, this study revealed an extensive gene set involved in the regulation of male goat reproduction and their dynamic transcription patterns. Data reported here provide valuable insights for a further improvement of the economic benefits of goats as well as future treatments for male infertility.

To investigate the role of the nuclear receptor NR5A1 in the testis after sex determination, we analyzed mice lacking NR5A1 in Sertoli cells (SCs) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impaired the expression of genes characteristic of SC identity (e.g. Sox9 and Amh), caused SC death from E14.5 onwards through a Trp53-independent mechanism related to anoikis, and induced disorganization of the testis cords. Together, these effects caused germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SCs changed their molecular identity: some acquired a 'pre-granulosa-like' cell identity, whereas other reverted to a 'supporting progenitor-like' cell identity, most of them being 'intersex' because they expressed both testicular and ovarian genes. Fetal Leydig cells (LCs) did not display significant changes, indicating that SCs are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LCs were absent from postnatal testes. In addition, adult mutant males displayed persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which could be explained by the loss of AMH and testosterone synthesis due to SC failure.