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Molecular dynamics (MD) simulations were conducted in order to investigate the effect of the naturally occurring point mutations of the transcription factor (TF) sex-determining region Y (SRY) on the structure and dynamics of the SRY-DNA complex. The normal SRY, along with the two mutants I13T and G40R, comprising point mutations on the SRY chain, which have been clinically identified in patients with sex developmental disorders, were modeled as DNA complexes. Our modeling work aims at elucidating atomic-level structural determinants of the aberrant SRY-DNA complexation by means of μs-long MD. The results suggest that the observed disorders brought about by the G40R-DNA and I13T-DNA may arise predominantly from the destabilization of the complex being in accord with

Sryon the Y chromosome is the master switch of sex determination in mammals. It has been well established thatSryencodes a transcription factor that is transiently expressed in somatic cells of the male gonad, leading to the formation of testes. In the testis of adult mice,Sryis expressed as a circular RNA (circRNA) transcript. However, the physiological function ofSrycircRNA (circSRY) remains unknown since its discovery in 1993. Here we show that circSRY is mainly expressed in the spermatocytes, but not in mature sperm or somatic cells of the testis. Loss of circSRY led to germ cell apoptosis and the reduction of sperm count in the epididymis. The level of γH2AX was decreased, and failure of XY body formation was noted in circSRY KO germ cells. Further study demonstrated that circSRY directly bound to miR-138-5p in spermatocytes, and in vitro assay suggested that circSRY regulates H2AX mRNA through sponging miR-138-5p. Our study demonstrates that, besides determining sex,Sryalso plays an important role in spermatogenesis as a circRNA.

Functional elucidation of bovine Y-chromosome genes requires available genome editing technologies. Meanwhile, it has yet to be proven whether the bovine Sry gene is the main or single factor involved in the development of the male phenotype in bovine. Here, we efficiently knocked out four Y-linked genes (Sry, ZFY, DDX3Y, and EIF2S3Y) in bovine fetal fibroblasts (BFFs) with transcription activator-like effector nucleases (TALENs) individually. Furthermore, we used TALEN-mediated gene knockin at the Sry gene and generated a sex-reversal bovine by somatic cell nuclear transfer (SCNT). The resulting bovine had only one ovary and was sterile. We demonstrate, for the first time, that the Sry gene is an important sex-determining gene in bovine. Our method lays a solid foundation for detecting the biology of the bovine Y chromosome, as it may provide an alternative biological model system for the study of mammalian sex determination, and new methods for the practical application in agricultural, especially for sex predetermination.

To summarize the clinical characteristics of Turner syndrome (TS) with a small supernumerary marker chromosome (sSMC) and discuss the clinical significance and management of TS patients with sSMC.A retrospective analysis was conducted on the clinical data of 244 patients with disorders of sexual development admitted to Peking Union Medical College Hospital from February 1984 to July 2020.Among the 244 patients with a disorder of sexual development, 69 cases of TS were identified in which 13 patients had sSMC. Their ages ranged from 3 to 28 years old with an average of 14.31 ± 6.40 years. All 13 sSMC-positive patients had typical clinical manifestations of TS except ambiguous genitalia in four cases. SRY gene testing was performed in 11sSMC-positive patients and 10 patients were positive for SRY and one was negative. Among the 10 SRY-positive patients, two cases had hirsutism and clitoral enlargement and two cases had clitoral enlargement only. Nine sSMC and SRY-positive patients underwent gonadectomy and one had left gonadal gonadoblastoma with seminomaAlthough the sSMC positive detection rate in DSD patients is uncommon (5.33% in our sample), the positive SRY detection rate in sSMC-positive TS patients was extremely high in our TS patients. And TS patients with sSMC and SRY positive had a significantly increased risk of gonadal germ cell tumors. Routine SRY screening should be performed in TS patients with sSMC, and a gonadectomy should be performed in TS patients with sSMC and SRY positive to prevent the occurrence of tumors.

lt;bgt;Background and Objective:lt;/bgt; The sex pre-selection for offspring before conception is desirable demand especially for the breeding program of farm animals. This study aimed to evaluate the preconception treatment of monovalent and divalent ions on the primary sex ratio, ovarian structures and serum minerals levels in New Zealand white rabbit does.lt;bgt;Materials and Methods:lt;/bgt; Nine New Zealand white rabbits doelt;igt;lt;/igt;(5.4±0.61 months of age and 2.4±0.35 kg of body weight) were used. Rabbits in the 1lt;supgt;stlt;/supgt; group were given drinking water only (control). While the 2lt;supgt;ndlt;/supgt; and 3lt;supgt;rdlt;/supgt; groups were given 1% of (calcium and magnesium) and (sodium and potassium) in daily drinking water, respectively for 15 days before mating. The embryos of each group were individually collected after three days of mating for primary sex detection using SRY (Sex Determining Region Y) Polymerase Chain Reaction (PCR) assay. Mineral analyses for all studied animals were weekly detected in serum before and after mating.lt;bgt;Results:lt;/bgt; The primary sex ratio for embryos of rabbits does receive (Na+K) produced more males (69.7%) while (Ca+Mg) administrated rabbits does produce more females (72.2%). The mineral treatment leads to a significant increase in the number of corpus luteum, total embryos, follicles bleeding and a significant decline in the count of large follicles. Also, there was no significant change in serum Na and Ca levels in the treated groups compared to the control.lt;bgt;Conclusion:lt;/bgt; The preconception administration of Ca+Mg could produce more females while Na+K could produce more males without adverse side effects on serum minerals concentration.

Mammalian male sex differentiation is triggered during embryogenesis by the activation of the Y-linked testis-determining gene SRY. Since insufficient or delayed expression of SRY results in XY gonadal sex reversal, accurate regulation of SRY is critical for male development in XY animals. In humans, dysregulation of SRY may cause disorders of sex development. Mouse Sry is the most intensively studied mammalian model of sex determination. Sry expression is controlled in a spatially and temporally stringent manner. Several transcription factors play a key role in sex determination as trans-acting factors for Sry expression. In addition, recent studies have shown that several epigenetic modifications of Sry are involved in sex determination as cis-acting factors for Sry expression. Herein, we review the current understanding of transcription factor- and epigenetic modifier-mediated regulation of SRY/Sry expression.

Male sex determination in mammals is initiated by SRY, a Y-encoded transcription factor. The protein contains a high-mobility-group (HMG) box mediating sequence-specific DNA bending. Mutations causing XY gonadal dysgenesis (Swyer syndrome) cluster in the box and ordinarily arisede novo. Rare inherited variants lead to male development in one genetic background (the father) but not another (his sterile XY daughter).De novoand inherited mutations occur at an invariant Tyr adjoining the motif’s basic tail (box position 72; Y127 in SRY). In SRY-responsive cell lines CH34 and LNCaP,de novomutations Y127H and Y127C reduced SRY activity (as assessed by transcriptional activation of principal target geneSox9) by 5- and 8-fold, respectively. Whereas Y127H impaired testis-specific enhancer assembly, Y127C caused accelerated proteasomal proteolysis; activity was in part rescued by proteasome inhibition. Inherited variant Y127F was better tolerated: its expression was unperturbed, and activity was reduced by only twofold, a threshold similar to other inherited variants. Biochemical studies of wild-type (WT) and variant HMG boxes demonstrated similar specific DNA affinities (within a twofold range), with only subtle differences in sharp DNA bending as probed by permutation gel electrophoresis and fluorescence resonance-energy transfer (FRET); thermodynamic stabilities of the free boxes were essentially identical. Such modest perturbations are within the range of species variation. Whereas our cell-based findings rationalize thede novogenotype-phenotype relationships, a molecular understanding of inherited mutation Y127F remains elusive. Our companion study uncovers cryptic biophysical perturbations suggesting that thepara-OH group of Y127 anchors a novel water-mediated DNA clamp.

Sex determination in mammals is controlled by the dominance of either pro-testis (SRY-SOX9-FGF9) or pro-ovary (RSPO1-WNT4-FOXL2) genetic pathways during early gonad development in XY and XX embryos, respectively. We have previously shown that early, robust expression of mouse Sry is dependent on the nuclear protein GADD45g. In the absence of GADD45g, XY gonadal sex reversal occurs, associated with a major reduction of Sry levels at 11.5 dpc. Here, we probe the relationship between Gadd45g and Sry further, using gain- and loss-of-function genetics. First, we show that transgenic Gadd45g overexpression can elevate Sry expression levels at 11.5 dpc in the B6.YPOS model of sex reversal, resulting in phenotypic rescue. We then show that the zygosity of pro-ovarian Rspo1 is critical for the degree of gonadal sex reversal observed in both B6.YPOS and Gadd45g-deficient XY gonads, in contrast to that of Foxl2. Phenotypic rescue of sex reversal is observed in XY gonads lacking both Gadd45g and Rspo1, but this is not associated with rescue of Sry expression levels at 11.5 dpc. Instead, Sox9 levels are rescued by around 12.5 dpc. We conclude that Gadd45g is absolutely required for timely expression of Sry in XY gonads, independently of RSPO1-mediated WNT signalling, and discuss these data in light of our understanding of antagonistic interactions between the pro-testis and pro-ovary pathways.

The detection of fetal cell-free DNA (cfDNA) from maternal plasma has enabled the development of essential techniques in prenatal diagnosis during recent years. Extracellular vesicles including exosomes were determined to carry fetal DNA fragments. Considering the known difficulties during isolation and stability of cfDNA, exosomes might provide a new opportunity for prenatal diagnosis and screening. In this study, comparison of cfDNA and exosome DNA (exoDNA) for predicting the fetal sex and Rhesus D (RHD) genotype was performed by using real-time polymerase chain reaction with simultaneous amplification of sequences of SRY and RHD genes. Fetal sex and RHD were determined in 100 and 81 RHD-negative pregnant women with cfDNA and exoDNA, respectively. The gestation ages of pregnant women were between 9 and 40 weeks. The results were compared with the neonatal phenotype for gender and a serological test for RHD. The cfDNA revealed 95.75% sensitivity and 100% specificity in RHD positivity and 100% sensitivity and 95.45% specificity in SRY positivity. Cohen’s agreement coefficient in the Kappa test ranged from 0.8 to 1.0 (P

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays critical roles in embryonic and organ developments and is involved in diverse physiological events. Loss of function of FGF9 exhibits male-to-female sex reversal in the transgenic mouse model and gain of FGF9 copy number was found in human 46, XX sex reversal patient with disorders of sex development. These results suggested that FGF9 plays a vital role in male sex development. Nevertheless, how FGF9/Fgf9 expression is regulated during testis determination remains unclear. In this study, we demonstrated that human and mouse SRY bind to −833 to −821 of human FGF9 and −1010 to −998 of mouse Fgf9, respectively, and control FGF9/Fgf9 mRNA expression. Interestingly, we showed that mouse SRY cooperates with SF1 to regulate Fgf9 expression, whereas human SRY-mediated FGF9 expression is SF1 independent. Furthermore, using an ex vivo gonadal culture system, we showed that FGF9 expression is sufficient to switch cell fate from female to male sex development in 12–16 tail somite XX mouse gonads. Taken together, our findings provide evidence to support the SRY-dependent, fate-determining role of FGF9 in male sex development.

Escherichia coli is a widely-used cell factory for recombinant protein production, nevertheless, high amount of produced protein is seen in aggregated form. The purpose of this study was to improve the solubility of recombinant bovine sex-determining region Y protein (rbSRY) by exploring the effect of temperature, inducer, and water-arginine mixed solvent. Codon-optimized rbSRY expressed in Rosetta-gami B (DE3) pLysS and purified by NI–NTA His-select affinity chromatography in the native and denaturing conditions. A three-dimensional model of SRY was built and studied through molecular dynamics simulations in water and in the presence of l-arginine as co-solvent. Results indicated the significant effects of temperature and IPTG concentration (P

In colon cancer, downregulation of the transmembrane heparan sulfate proteoglycan syndecan-1 (Sdc-1) is associated with increased invasiveness, metastasis, and dedifferentiation. As Sdc-1 modulates signaling pathways relevant to stem cell function, we tested the hypothesis that it may regulate a tumor-initiating cell phenotype. Sdc-1 small-interfering RNA knockdown in the human colon cancer cell lines Caco2 and HT-29 resulted in an increased side population (SP), enhanced aldehyde dehydrogenase 1 activity, and higher expression of CD133, LGR5, EPCAM, NANOG, SRY (sex-determining region Y)-box 2, KLF2, and TCF4/TCF7L2. Sdc-1 knockdown enhanced sphere formation, cell viability, Matrigel invasiveness, and epithelial-to-mesenchymal transition-related gene expression. Sdc-1-depleted HT-29 xenograft growth was increased compared to controls. Decreased Sdc-1 expression was associated with an increased activation of β1-integrins, focal adhesion kinase (FAK), and wingless-type (Wnt) signaling. Pharmacological FAK and Wnt inhibition blocked the enhanced stem cell phenotype and invasive growth. Sequential flow cytometric SP enrichment substantially enhanced the stem cell phenotype of Sdc-1-depleted cells, which showed increased resistance to doxorubicin chemotherapy and irradiation. In conclusion, Sdc-1 depletion cooperatively enhances activation of integrins and FAK, which then generates signals for increased invasiveness and cancer stem cell properties. Our findings may provide a novel concept to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. DATABASES: The GEO accession number of the Affymetrix transcriptomic screening is GSE58751.

Objectives: The 46,XX disorders of sex development (DSD) is a rare genetic cause of male infertility and possible misdiagnosis of this condition has never been reported. We aim to investigate clinical characteristics and laboratory results of infertile males with possibly misdiagnosed 46,XX DSD. Methods: Between January 2008 and December 2017, a retrospective case series study was performed involving sixteen 46,XX DSD males without azoospermia factor (AZF) deletion. Demographics, clinical features, laboratory results and assisted reproductive technology (ART) outcomes of these patients were depicted, and the underlying accurate diagnosis was also discussed. Results: The mean age was 30.06 ± 5.40 years old. Thirteen patients (81.25%) merely obtained secondary school education. Gynaecomastia occurred in one case, and cryptorchidism appeared in two cases. Testicular volumes were equal to 15 mL on two sides in one patient who had severe asthenozoospermia. Thirteen patients (81.25%) had bilateral atrophic testes which were below 5 mL. The majority of patients were observed with elevated levels of gonadotropic hormones and decreased testosterone values. Neither AZF region nor sex-determining region Y gene was absent among all patients. Twelve patients had normal ejaculatory function, whereas four were diagnosed with ejaculatory dysfunction. Eleven patients (68.75%) were diagnosed with azoospermia. Testicular sperm aspiration was performed in six subjects (37.50%). The pathological results showed that Leydig cell hyperplasia with spermatic failure was found in each case, and no sperm was found in testicular tissue. ART with donor sperm was conducted in 15 patients. Live birth was achieved in three cases through artificial insemination by donor and in one case using in-vitro fertilization by donor. Conclusions: Chromosomal analysis rarely yields 46,XX karyotype combined with no deletion of AZF in infertile males. Under this condition, molecular analysis should be conducted to avoid potential misdiagnosis and false interpretation of other findings.

Organ culture systems are useful for elucidating the process of testicular differentiation from mammalian undifferentiated genetically male gonads, as they permit various experiments, including experiments involving the control of gene expression. However, without addition of testicular differentiation-related factors, it is difficult to induce the formation of testis cord from immature gonads by a time point earlier 12 tail somites (ts) that corresponding to 11.0 days post coitum (dpc). In this study, we attempted to establish an organ culture system that induces testis formation from immature gonads (around 8 ts: 10.5 dpc) just before Sry (sex-determining region of the Y chromosome) expression. A paired gonad-mesonephros complex of around 8 ts was placed in the groove of an agarose gel block and put the semi-cylindrical piece of agarose gel to maintain the gonad morphology. The gonads were cultured in the gas phase for 96 hr. As a result, testis cord-like structures appeared in many genetically male gonads. Cells expressing the Sertoli cell markers Sox9 (SRY-box 9) and Amh (anti-Mullerian hormone) were observed, while granulosa cell marker Foxl2 (forkhead box L2) was not detected. In addition, Sox9- and Amh-expressing cells were observed throughout the entire gonad in many individuals. Amh mRNA expression was also upregulated. Surprisingly, formation of a partial testicular structure was observed from more immature gonads (6 ts). These results show that our gonadal organ culture system is useful for elucidating the regulation mechanism of Sry expression in undifferentiated bipotential gonads.

BackgroundSRY (sex determining region of Y) is one of the important genes involved in the process of human sex determination. The disturbed sex determination caused by an SRY mutation accounts for 10% to 15% of cases with 46, XY sex reversal. Recently, 3 distal enhancers were identified upstream of the SOX9 gene.ObjectivesThe purpose of this study was to investigate the molecular etiology of 46, XY sex reversal in 3 familial patients and a sporadic patient.DesignNext-generation sequencing was used to reveal the genotype and inherited pattern. Copy number variations and single nucleotide polymorphism haplotyping were analyzed to observe the alteration of enhancers of SOX9. Transcriptional activity of SRY mutation were assessed by a dual luciferase reporting system, and nuclear translocation was observed by confocal microscopy.ResultsTwo novel SRY gene mutations, p.Arg76Leu and p.Glu89flx15, were identified. In the pedigree with multiple patients, p.Arg76Leu mutation in SRY and p.Gly212Ser mutation in NR5A1 were identified in the proband. The heterozygous deletion far upstream of the SOX9 gene in chromosome 17 was identified in the 3 patients in this family, containing the distal enhancer eSR-A of SOX9 but not eSR-B and eALDI. The frameshift mutation p.Glu89flx15 was revealed to inhibit the transcriptional activity of the target gene, whereas the missense mutation p.Arg76Leu barely showed an effect.ConclusionIn contrast to sporadic cases, inherited single nucleotide variations of SRY are not the main cause of the severe phenotype of 46, XY sex reversal, and the enhancers of SOX9 should be investigated carefully in such patients.

Objective To investigate whether miR‐141 and the sex determination region of Y chromosome box 11 (SOX11) play roles in steroid‐induced avascular necrosis of the femoral head (SANFH), and to explore whether miR‐141 could target SOX11 to influence the proliferation of bone marrow mesenchymal stem cells (BMSC). Methods Bone marrow mesenchymal stem cells (BMSC) were isolated and cultured from 4‐week‐old Sprague Dawley rats. A flow cytometry assay was performed to identify BMSC. BMSC were divided into two groups: a control group and a dexamethasone (DEX) group. BMSC were transfected by miR‐141 mimic, miR‐141 inhibitor, and SOX11. Real‐time polymerase chain reaction (PCR) assay was performed to investigate the mRNA expression of miR‐141 and SOX11. The results were used to determine the effect of transfection and to verify the expression in each group and the association between miR‐141 and SOX11. Luciferase reporter assay revealed the targeted binding site between miR‐141 and the 3′‐untranslated region of SOX11 mRNA. MTT assays were performed to investigate the proliferation of BMSC in the miR‐141 mimic, miR‐141 inhibitor, and SOX11 groups. Result The results of the flow cytometry assay suggested that cells were positive for CD29 and CD90 while negative for CD45. This meant that the isolated and cultured cells were not hematopoietic stem cells. In addition, cell transfection was successful based on the expression of miR‐141 and SOX11. According to the results of real‐time PCR assay, the mRNA expression of miR‐141 in SANFH was upregulated (4.117 ± 0.042 vs 1 ± 0.027, P

The sex-determining region of the Y chromosome, Sry/SRY, is an initiation factor for testis development in both humans and mice. Although the functional compatibility between murine SRY and human SRY was previously examined in transgenic mice, their equivalency remains inconclusive. Because molecular interaction and timeline of mammalian sex determination were mostly described in murine experiments, we generated a mouse model in which Sry was substituted with human SRY to verify the compatibility. The mouse model had the human SRY open reading frame at the locus of murine Sry exon 1—Sry(SRY) mice—and was generated using the CRISPR/Cas9 system. The reproductive system of the mice was analyzed. The expression of human SRY in the fetal gonadal ridge of Sry(SRY) mice was detected. The external and internal genitalia of adult Sry(SRY) mice were similar to those of wild-type females, without any significant difference in anogenital distance. Sry(SRY) mice obtained gonads, which were morphologically considered as ovaries. Histological analysis revealed that the cortical regions of gonads from adult Sry(SRY) mice contained few follicles. We successfully replaced genes on the Y chromosome with targeted genome editing using the CRISPR/Cas9 system. Because the Sry(SRY) XY mice did not develop testis, we concluded that human SRY was insufficient to drive testis development in mouse embryos. The difference in response elements and lack of glutamine-rich domains may have invalidated human SRY function in mice. Signal transduction between Sry/SRY expression and Sox9/SOX9 activation is possibly organized in a species-specific manner.

SRY is the master regulator of male sex determination in eutherian mammals. In mice, Sry expression is transcriptionally and epigenetically controlled in a developmental stage-specific manner. The Sry promoter undergoes demethylation in embryonic gonadal somatic cells at the sex-determining period. However, its molecular mechanism and in vivo significance remain unclear. Here, we report that the Sry promoter is actively demethylated during gonadal development, and TET2 plays a fundamental role in Sry demethylation. Tet2-deficient mice showed absence of 5-hydroxymethylcytosine in the Sry promoter. Furthermore, Tet2 deficiency diminished Sry expression, indicating that TET2-mediated DNA demethylation regulates Sry expression positively. We previously showed that the deficiency of the H3K9 demethylase Jmjd1a compromises Sry expression and induces male-to-female sex reversal. Tet2 deficiency enhanced the sex reversal phenotype of Jmjd1a-deficient mice. Thus, TET2-mediated active DNA demethylation and JMJD1A-mediated H3K9 demethylation contribute synergistically to sex determination.

The SRY-related (SOX) transcription factor family pivotally contributes to determining cell fate and identity in many lineages. Since the original discovery that SRY deletions cause sex reversal, mutations in half of the twenty human SOX genes have been associated with rare congenital disorders, henceforward called SOXopathies. Mutations are generally de novo, heterozygous and inactivating, revealing gene haploinsufficiency, but other types, including duplications, have been reported too. Missense variants primarily target the HMG domain, the SOX hallmark that mediates DNA binding and bending, nuclear trafficking, and protein-protein interactions. We here review key clinical and molecular features of SOXopathies and discuss the prospect that the disease family likely involves more SOX genes and larger clinical and genetic spectrums than currently appreciated.

Parkinson’s disease (PD) is a debilitating neurodegenerative disorder caused by the loss of midbrain dopamine (DA) neurons. While the cause of DA cell loss in PD is unknown, male sex is a strong risk factor. Aside from the protective actions of sex hormones in females, emerging evidence suggests that sex-chromosome genes contribute to the male bias in PD. We previously showed that the Y-chromosome gene, SRY, directly regulates adult brain function in males independent of gonadal hormone influence. SRY protein colocalizes with DA neurons in the male substantia nigra, where it regulates DA biosynthesis and voluntary movement. Here we demonstrate that nigral SRY expression is highly and persistently up-regulated in animal and human cell culture models of PD. Remarkably, lowering nigral SRY expression with antisense oligonucleotides in male rats diminished motor deficits and nigral DA cell loss in 6-hydroxydopamine (6-OHDA)-induced and rotenone-induced rat models of PD. The protective effect of the SRY antisense oligonucleotides was associated with male-specific attenuation of DNA damage, mitochondrial degradation, and neuroinflammation in the toxin-induced rat models of PD. Moreover, reducing nigral SRY expression diminished or removed the male bias in nigrostriatal degeneration, mitochondrial degradation, DNA damage, and neuroinflammation in the 6-OHDA rat model of PD, suggesting that SRY directly contributes to the sex differences in PD. These findings demonstrate that SRY directs a previously unrecognized male-specific mechanism of DA cell death and suggests that suppressing nigral Sry synthesis represents a sex-specific strategy to slow or prevent DA cell loss in PD.

Cranial neural crest cells are multipotent cells that migrate into the pharyngeal arches of the vertebrate embryo and differentiate into various craniofacial organ derivatives. Therefore, migrating cranial neural crest cells are considered one of the most attractive candidate cell sources in regenerative medicine. We generated cranial neural crest like cell (cNCCs) using mouse-induced pluripotent stem cells cultured in neural crest-inducing medium for 14 days. Subsequently, we conducted RNA sequencing experiments to analyze gene expression profiles of cNCCs at different time points after induction. cNCCs expressed several neural crest specifier genes; however, some previously reported specifier genes such as paired box 3 and Forkhead box D3, which are essential for embryonic neural crest development, were not expressed. Moreover, ETS proto-oncogene 1, transcription factor and sex-determining region Y-box 10 were only expressed after 14 days of induction. Finally, cNCCs expressed multiple protocadherins and a disintegrin and metalloproteinase with thrombospondin motifs enzymes, which may be crucial for their migration. Electronic supplementary material The online version of this article (10.1007/s00795-019-00229-2) contains supplementary material, which is available to authorized users.

Men and women differ in circulating lipids and coronary artery disease (CAD). While sex hormones such as estrogens decrease CAD risk, hormone replacement therapy increases risk. Biological sex is determined by sex hormones and chromosomes, but effects of sex chromosomes on circulating lipids and atherosclerosis are unknown. Here, we use mouse models to separate effects of sex chromosomes and hormones on atherosclerosis, circulating lipids and intestinal fat metabolism. We assess atherosclerosis in multiple models and experimental paradigms that distinguish effects of sex chromosomes, and male or female gonads. Pro-atherogenic lipids and atherosclerosis are greater in XX than XY mice, indicating a primary effect of sex chromosomes. Small intestine expression of enzymes involved in lipid absorption and chylomicron assembly are greater in XX male and female mice with higher intestinal lipids. Together, our results show that an XX sex chromosome complement promotes the bioavailability of dietary fat to accelerate atherosclerosis., Men and women differ in their risk of developing coronary artery disease, in part due to differences in their levels of sex hormones. Here, AlSiraj et al. show that the XX sex genotype regulates lipid metabolism and promotes atherosclerosis independently of sex hormones in mice.

We have used the four core genotypes (FCG) mouse model, which allows a distinction between effects of gonadal secretions and chromosomal complement, to determine when sex differences in the immune system first appear and what influences their development. Using splenic T cell number as a measure that could be applied to neonates with as yet immature immune responses, we found no differences among the four genotypes at postnatal day 1, but by day 7, clear sex differences were observed. These sex differences were unexpectedly independent of chromosomal complement and similar in degree to gonadectomized FCG adults: both neonatal and gonadectomized adult females (XX and XY) showed 2-fold the number of CD4+ and 7-fold the number of CD8+ T cells versus their male (XX and XY) counterparts. Appearance of this long-lived sex difference between days 1 and 7 suggested a role for the male-specific perinatal surge of testicular testosterone. Interference with the testosterone surge significantly de-masculinized the male CD4+, but not CD8+ splenic profile. Treatment of neonates demonstrated elevated testosterone limited mature cell egress from the thymus, whereas estradiol reduced splenic T cell seeding in females. Neonatal male splenic epithelium/stroma expressed aromatase mRNA, suggesting capacity for splenic conversion of perinatal testosterone into estradiol in males, which, similar to administration of estradiol in females, would result in reduced splenic T cell seeding. These sex steroid effects affected both CD4+ and CD8+ cells and yet interference with the testosterone surge only significantly de-masculinized the splenic content of CD4+ cells. For CD8+ cells, male cells in the thymus were also found to express one third the density of sphingosine-1-phosphate thymic egress receptors per cell compared to female, a male characteristic most likely an indirect result of Sry expression. Interestingly, the data also support a previously unrecognized role for non-gonadal estradiol in the promotion of intra-thymic cell proliferation in neonates of both sexes. Microarray analysis suggested the thymic epithelium/stroma as the source of this hormone. We conclude that some immune sex differences appear long before puberty and more than one mechanism contributes to differential numbers and distribution of T cells.

The aims of the presented case report are to emphasize the importance of a proper diagnostics and treatment in the case of the coexistence of Klinefelter syndrome (KS, 47 XXY) and complete androgen insensitivity syndrome (CAIS). Since there is no causal treatment it is necessary to provide the patient with a good quality of life, including psychological and sexological support.The presented case report is the retrospective analysis of the patient's medical history over the 3 years.At the age of 15, the patient was directed to genetic testing due to primary amenorrhea. The results of the patient showed an incorrect male karyotype with the SRY gene present (47, XXY). A molecular diagnostics revealed a very rare variant of the androgen receptor (AR) mutation responsible for tissue insensitivity to androgens. The detected mutation has not been described in the available databases so far. Following a diagnosis of the presence of Klinefelter syndrome (KS, 47 XXY) together with complete androgen insensitivity syndrome (CAIS), the patient underwent a bilateral gonadectomy.In women with KS and CAIS physiological reproduction and maintenance of normal sex, hormone levels are not possible. A gonadectomy is performed due to the risk of malignant testicular tumors.

SRY is the Y-chromosomal gene that determines male sex development in humans and most other mammals. After three decades of study, we still lack a detailed understanding of which domains of the SRY protein are required to engage pathway of gene activity leading to testis development. Some insight has been gained from the study of genetic variations underlying differences/disorders of sex determination (DSD), but the lack of a system of experimentally generating SRY mutations and studying their consequences in vivo has limited progress in the field. To address this issue, we generated a mouse model carrying a humanSRYtransgene able to drive male sex determination in XX mice. Using CRISPR-Cas9 gene editing, we generated novel genetic modifications in each ofSRY’s three domains (N-terminal, HMG box, and C-terminal) and performed detailed analysis of their molecular and cellular effects on embryonic testis development. Our results provide new functional insights unique to humanSRYand the causes of DSD, and present a versatile and powerful system in which to demonstrate causality ofSRYvariations in DSD, to functionally study theSRYvariation database, and to characterize new pathogenicSRYvariations found in DSD.

The rare condition 46, XY disorders of sex development (DSDs) is characterized by the female phenotype and male karyotype. We aimed to describe the genetic basis of 46, XY DSDs in nine patients and the genotype���phenotype relationships of the genes involved. Targeted next-generation sequencing (NGS) was used to analyze the underlying hereditary etiology in nine female patients with 46, XY DSDs. In silico analyses were used to predict the effects of novel variants on the protein function of the identified genes. Primary amenorrhea with the absence of puberty, inguinal hernia, and clitoridauxe were common complaints. All enrolled patients had a differential etiology by genetic testing, and five novel genetic variants involved in four genes (SRY, AR, NR5A1, and LHCGR) were identified. A novel nonsense variant of SRY c.51C > G was found in XY patients without testicles. Two novel heterozygous variants, i.e. c.265A > T (Ile89Leu) and c.422T > C (Val141Ala), of the LHCGR gene were found in male pseudo-hermaphroditism. We expanded the genetic mutation spectrum and described in detail the genotype���phenotype relationships of 46, XY DSDs. DNA sequencing for SRY should be a priority in female patients with 46, XY DSDs. NGS is useful for clarifying genetic pathogenesis and could provide a basis for clinical diagnosis and treatments of patients with 46, XY DSDs.

Introduction: Disorders of sex development (DSD) constitutes a group of congenital conditions that affect uro-genital differentiation and are associated with chromosomal, gonadal and phenotypic sex abnormalities. Objective: To evaluate the clinical and genetic features of childhood DSD cases. Materials and methods: DSD patients followed up between the years of 2002 & ndash;2018 were evaluated in terms of their complaints, demographic, clinical features and genetic diagnoses. Results: Out of 289 patients, 143(49.5%) were classified as 46XY DSD, 62(21.5%) as 46XX DSD and 84(29%) as sex chromosomal DSD. Genetic diagnosis was achieved in 150 patients (51.9%). The distribution of the mo-lecular diagnosis of the 46XY DSD patients were; 12 (26.6%) SRD5A2, 10 (22.2%) AR, 7 (15.5%) HSD17B3, 3 (6.6%) WT-1, 2 (4.4%) AMHR2, 2 (4.4%) AMH, 2 (4.4%) LHCGR, 2 (4.4%) HSD3B2, 1 (2.2%) NR5A1, 1 (2.2%) CYP17A1 and 1 (2.2%) SRY mutation. Fifty (80.6%) of the 46XX DSD patients received a diagnosis with clinical and laboratory findings. Twenty-four (38.7%) of them were 21-hydroxylase deficiency, 9(14.5%) Rokitansky-K & uuml;ster-Hauser Syndrome, 4 (6.5%) 11-beta hydroxylase deficiency, 3 (4.8%) gonadal dysgenesis and 2 (3.2%) aromatase deficiency. in 46XX group pathogenic mutations were detected in 21(33.8%) of the patients. Eighty-four (29%) patients were diagnosed as sex chromosomal disorder. of these 66 (78.5%) were Turner Syndrome, 6 (7.2%) Klinefelter Syndrome and 10 (11.9%) mix gonadal dysgenesis. Gender re-assignment was decided in 11 patients. Malignant and pre-invasive lesions was diagnosed in 8 (2.7%) patients. Conclusion: Many of DSD & rsquo;s are clinically similar and etiology of numerous of them still cannot be established. A multi-disciplinary approach and new rapid genetic diagnostic methods are needed in the process from diagnosis to gender assignment and follow-up.

The SRY gene induces testis development even in XX individuals. However, XX/Sry testes fail to produce mature sperm, due to the absence of Y chromosome carrying genes essential for spermatogenesis. XX/Sry Sertoli cells show abnormalities in the production of lactate and cholesterol required for germ cell development. Leydig cells are essential for male functions through testosterone production. However, whether XX/Sry adult Leydig cells (XX/Sry ALCs) function normally remains unclear. In this study, the transcriptomes from XY and XX/Sry ALCs demonstrated that immediate early and cholesterogenic gene expressions differed between these cells. Interestingly, cholesterogenic genes were upregulated in XX/Sry ALCs, although downregulated in XX/Sry Sertoli cells. Among the steroidogenic enzymes, CYP17A1 mediates steroid 17α-hydroxylation and 17,20-lyase reaction, necessary for testosterone production. In XX/Sry ALCs, the latter reaction was selectively decreased. The defects in XX/Sry ALCs, together with those in the germ and Sertoli cells, might explain the infertility of XX/Sry testes.

Significance The present work characterizes the porcine sex-determining region on the Y chromosome (SRY) gene and demonstrates its pivotal role in sex determination. We provide evidence that genetically male pigs with a knockout of the SRY gene undergo sex reversal of the external and internal genitalia. This discovery of SRY as the main switch for sex determination in pigs may provide an alternative for surgical castration in pig production, preventing boar taint. As the pig shares many genetic, physiological, and anatomical similarities with humans, it also provides a suitable large animal model for human sex reversal syndromes, allowing for the development of new interventions for human sex disorders., The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located “high mobility group” (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.

To explore the pathogenesis for a SRY-negative male with 46,XX disorder of sex development (DSD).Peripheral blood samples of the patient and his family members were subjected to chromosomal karyotyping, routine PCR, real-time fluorescence quantitative PCR, whole exome sequencing and whole genome sequencing. The data was analyzed with NextGENe software.Both the proband and his brother presented a 46,XX karyotype with negative SRY gene, while their father presented normal phenotype and karyotype with positive SRY gene. No pathogenic variant associated with sex development was detected by whole exome sequencing, while a 243 kb duplication was detected by whole genome sequencing in the 5' upstream region of the SOX9 gene in the proband, his brother and father. The same duplication was not found in his sister and mother.The 243 kb duplication at the 5' upstream of the SOX9 gene may predispose to the 46,XX DSD in this family. It is speculated that there exist an unknown core regulatory element in the upstream of the SOX9, and its duplication may trigger expression of SOX9 and initiate testicular differentiation in the absence of SRY gene.

The risk of a gonadal tumor is high in testicular disorder of sexual development (DSD) with the Y chromosome, but cases of DSD without the Y chromosome are extremely rare. We reported a gonadal tumor in a phenotypically male individual with 46, XX testicular DSD. A testicular tumor was incidentally found in a 32-year-old phenotypic male who was presented to the hospital with male infertility. A diagnosis of 46, XX testicular DSD was made by the presentation of karyotype analysis of 46, XX with the sex-determining region of the Y chromosome (SRY) positive and gonadal tissue without female gonads. Surgery was performed due to a gradually growing tumor. The partial orchidectomy was performed with the diagnosis of a benign Leydig cell tumor in frozen biopsy.

Objective To identify the role and mechanism of a male specific gene, SRY, in I/R-induced hepatic injury. Background Males are more vulnerable to I/R injury than females. However, the mechanism of these sex-based differences remains poorly defined. Methods Clinicopathologic data of patients who underwent hepatic resection were identified from an international multi-institutional database. Liver specific SRY TG mice were generated, and subjected to I/R insult with their littermate WT controls in vivo. In vitro experiments were performed by treating primary hepatocytes from TG and WT mice with hypoxia/reoxygenation stimulation. Results Clinical data showed that postoperative aminotransferase level, incidence of overall morbidity and liver failure were markedly higher among 1267 male versus 508 female patients who underwent hepatic resection. SRY was dramatically upregulated during hepatic I/R injury. Overexpression of SRY in male TG mice and ectopic expression of SRY in female TG mice exacerbated liver I/R injury compared with WTs as manifested by increased inflammatory reaction, oxidative stress and cell death in vivo and in vitro. Mechanistically, SRY interacts with Glycogen synthase kinase-3β (GSK-3β) and β-catenin, and promotes phosphorylation and degradation of β-catenin, leading to suppression of the downstream FOXOs, and activation of NF-κB and TLR4 signaling. Furthermore, activation of β-catenin almost completely reversed the SRY overexpression-mediated exacerbation of hepatic I/R damage. Conclusions SRY is a novel hepatic I/R mediator that promotes hepatic inflammatory reaction, oxidative stress and cell necrosis via inhibiting Wnt/β-catenin signaling, which accounts for the sex-based disparity in hepatic I/R injuries.

Background Disorders of sex development (DSD) is a chronic autoimmune disease characterized by systemic inflammation, pathological osteogenesis, and endocrine abnormality. However, its genetic etiology remains mostly unknown. In addition, little research focuses on the regulation mechanism from the view of transcriptomics in the hypothalamic-pituitary-gonadal axis (HPGA). The hypothalamus is the integrated center of the HPGA mediating neural, hormonal, and environmental stimulus to sex development. Methods Three XX-DSD (SRY-negative) pig (DSD) and three NF pigs (five months old, 40 kg ± 5 kg) were selected by external genitalia observation and sex determining region Y gene (SRY) detection. The hypothalamus were sampled for RNA isolation, and the mRNA, lncRNA and miRNA expression profiles were analyzed by sequencing. Results A total of 1,258 lncRNAs, 1,086 mRNAs, and 61 microRNAs were found to differentially express in XX-DSD pigs compared with normal female pigs. Many genes in hormone biosynthesis and secretion pathway are significantly up-regulated, and the up-regulation of GNRH1, KISS1 and AVP may be the candidate genes leading the abnormal secretion of GnRH. Next, we predicted the lncRNA-miRNA-mRNA co-expression triplets and constructed three competing endogenous RNA (ceRNA) potentially associated with DSD. Functional enrichment suggested TCONS_00340886, TCONS_00000204 and miR-181a were related to GnRH secretion. Conclusions Our research revealed the first transcriptomic profile in the hypothalamus of XX-DSD pigs and provided new insight in coding and non-coding RNAs that may be associated with DSD in pigs.

Background Side population (SP) cells, which have similar features to those of cancer stem cells, show resistance to dexamethasone (Dex) treatment. Thus, new drugs that can be used in combination with Dex to reduce the population of SP cells in multiple myeloma (MM) are required. Diallyl thiosulfinate (DATS, allicin), a natural organosulfur compound derived from garlic, has been shown to inhibit the proliferation of SP cells in MM cell lines. Therefore, we investigated the effect of a combination of DATS and Dex (DAT + Dex) on MM SP cells. Methods SP cells were sorted from MM RPMI-8226 and NCI-H929 cell lines using Hoechst 33342-labeled fluorescence-activated cell sorting. The growth of SP cells was evaluated using the cell counting kit-8 assay. Cell cycle and apoptosis assays were conducted using a BD Calibur flow cytometer. miRNA expression was measured using quantitative reverse transcription-polymerase chain reaction. Phosphoinositide 3-kinase (PI3K), phosphorylated AKT (p-AKT), AKT, p-mechanistic target of rapamycin (mTOR), and mTOR levels were measured using western blot analysis. Results Our results showed that the combination of DATS+Dex inhibited sphere formation, colony formation, and proliferation of MM SP cells by inducing apoptosis and cell cycle arrest in the G1/S phase. In addition, the combination of DATS+Dex promoted miR-127-3p expression and inhibited PI3K, p-AKT, and p-mTOR expression in SP cells. Knockdown of miR-127-3p expression weakened the effect of DATS+Dex on cell proliferation, colony formation, apoptosis, and cell cycle of MM SP cells. Additionally, knockdown of miR-127-3p activated the PI3K/AKT/mTOR signaling pathway in MM SP cells cotreated with DATS+Dex. Conclusion We demonstrated that cotreatment with DATS+Dex reduced cell proliferation, promoted apoptosis, and caused cell cycle arrest of MM SP cells by promoting miR-127-3p expression and deactivating the PI3K/AKT/mTOR signaling pathway.