Skipping events impose repeated binding attempts: profound kinetic implications of protein-DNA conformational changes.

The kinetics of protein-DNA recognition, along with its thermodynamic properties, including affinity and specificity, play a central role in shaping biological function. Protein-DNA recognition kinetics are characterized by two key elements: the time taken to locate the target site amid various nonspecific alternatives; and the kinetics involved in the recognition process, which may necessitate overcoming an energetic barrier. In this study, we developed a coarse-grained (CG) model to investigate interactions between a transcription factor called the sex-determining region Y (SRY) protein and DNA, in order to probe how DNA conformational changes affect SRY-DNA recognition and binding kinetics. We find that, not only does a requirement for such a conformational DNA transition correspond to a higher energetic barrier for binding and therefore slower kinetics, it may further impede the recognition kinetics by increasing unsuccessful binding events (skipping events) where the protein partially binds its DNA target site but fails to form the specific protein-DNA complex. Such skipping events impose the need for additional cycles protein search of nonspecific DNA sites, thus significantly extending the overall recognition time. Our results highlight a trade-off between the speed with which the protein scans nonspecific DNA and the rate at which the protein recognizes its specific target site. Finally, we examine molecular approaches potentially adopted by natural systems to enhance protein-DNA recognition despite its intrinsically slow kinetics.

Complete male-to-female sex reversal in XY mice lacking the miR-17~92 cluster.

Mammalian sex determination is controlled by antagonistic gene cascades operating in embryonic undifferentiated gonads. The expression of the Y-linked gene SRY is sufficient to trigger the testicular pathway, whereas its absence in XX embryos leads to ovarian differentiation. Yet, the potential involvement of non-coding regulation in this process remains unclear. Here we show that the deletion of a single microRNA cluster, miR-17~92, induces complete primary male-to-female sex reversal in XY mice. Sry expression is delayed in XY knockout gonads, which develop as ovaries. Sertoli cell differentiation is reduced, delayed and unable to sustain testicular development. Pre-supporting cells in mutant gonads undergo a transient state of sex ambiguity which is subsequently resolved towards the ovarian fate. The miR-17~92 predicted target genes are upregulated, affecting the fine regulation of gene networks controlling gonad development. Thus, microRNAs emerge as key components for mammalian sex determination, controlling Sry expression timing and Sertoli cell differentiation.

Two redundant transcription factor binding sites in a single enhancer are essential for mammalian sex determination.

Male development in mammals depends on the activity of the two SOX gene: Sry and Sox9, in the embryonic testis. As deletion of Enhancer 13 (Enh13) of the Sox9 gene results in XY male-to-female sex reversal, we explored the critical elements necessary for its function and hence, for testis and male development. Here, we demonstrate that while microdeletions of individual transcription factor binding sites (TFBS) in Enh13 lead to normal testicular development, combined microdeletions of just two SRY/SOX binding motifs can alone fully abolish Enh13 activity leading to XY male-to-female sex reversal. This suggests that for proper male development to occur, these few nucleotides of non-coding DNA must be intact. Interestingly, we show that depending on the nature of these TFBS mutations, dramatically different phenotypic outcomes can occur, providing a molecular explanation for the distinct clinical outcomes observed in patients harboring different variants in the same enhancer.

Sex-determining region Y gene promotes liver fibrosis and accounts for sexual dimorphism in its pathophysiology.

BACKGROUND & AIMS: Men are more prone to develop and die from liver fibrosis than women. In this study, we aim to investigate how sex-determining region Y gene (SRY) in hepatocytes promotes liver fibrosis. METHODS: Hepatocyte-specific Sry knock-in (KI), Sry knockout (KO), and Sry KI with platelet-derived growth factor receptor α (Pdgfrα) KO mice were generated. Liver fibrosis was induced in mice by bile duct ligation for 2 weeks or carbon tetrachloride treatment for 6 weeks. In addition, primary hepatocytes, hepatic stellate cells (HSCs), and immortalized cell lines were used for in vitro studies and mechanistic investigation. RESULTS: Compared to females, the severity of toxin- or cholestasis-induced liver fibrosis is similarly increased in castrated and uncastrated male mice. Among all Y chromosome-encoded genes, SRY was the most significantly upregulated and consistently increased gene in fibrotic/cirrhotic livers in male patients and in mouse models. Sry KI mice developed exacerbated liver fibrosis, whereas Sry KO mice had alleviated liver fibrosis, compared to age- and sex-matched control mice after bile duct ligation or administration of carbon tetrachloride. Mechanistically, both our in vivo and in vitro studies illustrated that SRY in hepatocytes can transcriptionally regulate Pdgfrα expression, and promote HMGB1 (high mobility group box 1) release and subsequent HSC activation. Pdgfrα KO or treatment with the SRY inhibitor DAX1 in Sry KI mice abolished SRY-induced HMGB1 secretion and liver fibrosis. CONCLUSIONS: SRY is a strong pro-fibrotic factor and accounts for the sex disparity observed in liver fibrosis, suggesting its critical role as a potentially sex-specific therapeutic target for prevention and treatment of the disease. IMPACT AND IMPLICATION: We identified that a male-specific gene, sex-determining region Y gene (SRY), is a strong pro-fibrotic gene that accounts for the sex disparity observed in liver fibrosis. As such, SRY might be an appropriate target for surveillance and treatment of liver fibrosis in a sex-specific manner. Additionally, SRY might be a key player in the sexual dimorphism observed in hepatic pathophysiology more generally.

Molecular and Functional Characterization of Human Sex-Determining Region on the Y Chromosome Variants Using Protamine 1 Promoter.

The male sex-determining gene, sex-determining region on the Y chromosome (SRY), is expressed in adult testicular germ cells; however, its role in regulating spermatogenesis remains unclear. The role of SRY in the postmeiotic gene expression was investigated by determining the effect of SRY on the promoter of the haploid-specific Protamine 1 (PRM1) gene, which harbors five distinct SRY-binding motifs. In a luciferase reporter assay system, SRY upregulates PRM1 promoter activity in vitro in a dose-dependent manner. Through a gel-shift assay involving a 31-bp DNA fragment encompassing the SRY element within the PRM1 promoter, the third SRY-binding site on the sense strand (-373/-367) was identified as crucial for PRM1 promoter activation. This assay was extended to analyze 9 SRY variants found in the testicular DNA of 44 azoospermia patients. The findings suggest that SRY regulates PRM1 promoter activity by directly binding to its specific motif within the PRM1 promoter.

The -KTS splice variant of WT1 is essential for ovarian determination in mice.

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified -KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of -KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify -KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.

CDYL reinforces male gonadal sex determination through epigenetically repressing Wnt4 transcription in mice.

In mammals, male and female gonads initially develop from bipotential progenitor cells, which can differentiate into either testicular or ovarian cells. The decision to adopt a testicular or ovarian fate relies on robust genetic forces, i.e., activation of the testis-determining gene Sry, as well as a delicate balance of expression levels for pro-testis and pro-ovary factors. Recently, epigenetic regulation has been found to be a key element in activation of Sry. Nevertheless, the mechanism by which epigenetic regulation controls the expression balance of pro-testis and pro-ovary factors remains unclear. Chromodomain Y-like protein (CDYL) is a reader protein for repressive histone H3 methylation marks. We found that a subpopulation of Cdyl-deficient mice exhibited XY sex reversal. Gene expression analysis revealed that the testis-promoting gene Sox9 was downregulated in XY Cdyl-deficient gonads during the sex determination period without affecting Sry expression. Instead, we found that the ovary-promoting gene Wnt4 was derepressed in XY Cdyl-deficient gonads prior to and during the sex-determination period. Wnt4 heterozygous deficiency restored SOX9 expression in Cdyl-deficient XY gonads, indicating that derepressed Wnt4 is a cause of the repression of Sox9. We found that CDYL directly bound to the Wnt4 promoter and maintained its H3K27me3 levels during the sex-determination period. These findings indicate that CDYL reinforces male gonadal sex determination by repressing the ovary-promoting pathway in mice.

Substrain differences in Sry expression at the stage of sex determination in C57BL/6 mouse strains.

The expression of sex determining region of the Y chromosome (Sry) in the fetal gonads is important for male development. In a mouse model of disorders of sex development (C57BL/6 (B6)-XYPOS), the gonadal phenotype and the timing of Sry expression differ due to differences among B6 substrains as the genetic background. Since differences in Sry expression among B6 substrains have been speculated, the present study examined Sry expression in B6J, B6JJmsSlc, and B6NCrl mice. These substrains differed in the number of Sry-expressing cells in the gonads of embryonic mice at each developmental stage, with B6NCrl having more than the other strains. The substrains differed also in the number of Sry-expressing cells between the left and right gonads, with B6J and B6NCrl, but not B6JJmsSlc, showing left gonad-dominant Sry expression. Substrain differences existed also in the distribution of Sry-expressing cells in the medial and lateral directions of gonads. In addition, in the left gonad-dominant Sry-expressing substrains B6J and B6NCrl, the medial and central regions of the left gonad had more Sry-expressing cells than those of the right gonad. Substrains of B6 mice have not always been considered in sex differentiation studies. In the present study, however, we observed substrain differences in the number of Sry-expressing cells, left-right distribution, and medial/lateral distribution during the early stages of gonadal development in B6 mice. Therefore, future studies on sex differentiation in B6 mice should consider substrain differences.

Biased precursor ingression underlies the center-to-pole pattern of male sex determination in mouse.

During mammalian development, gonadal sex determination results from the commitment of bipotential supporting cells to Sertoli or granulosa cell fates. Typically, this decision is coordinated across the gonad to ensure commitment to a single organ fate. When unified commitment fails in an XY mouse, an ovotestis forms in which supporting cells in the center of the gonad typically develop as Sertoli cells, while supporting cells in the poles develop as granulosa cells. This central bias for Sertoli cell fate was thought to result from the initial expression of the drivers of Sertoli cell fate, SRY and/or SOX9, in the central domain, followed by paracrine expansion to the poles. However, we show here that the earliest cells expressing SRY and SOX9 are widely distributed across the gonad. In addition, Sertoli cell fate does not spread among supporting cells through paracrine relay. Instead, we uncover a center-biased pattern of supporting cell precursor ingression that occurs in both sexes and results in increased supporting cell density in the central domain. Our findings prompt a new model of gonad patterning in which a density-dependent organizing principle dominates Sertoli cell fate stabilization.

SOX9 and SRY binding sites on mouse mXYSRa/Enh13 enhancer redundantly regulate Sox9 expression to varying degrees.

Sox9 plays an essential role in mammalian testis formation. It has been reported that gene expression in the testes is regulated by enhancers. Among them, mXYSRa/Enh13-which is located at far upstream of the transcription start site-plays a critical role, wherein its deletion causes complete male-to-female sex reversal in mice. It has been proposed that the binding sites (BSs) of SOX9 and SRY, the latter of which is the sex determining gene on the Y chromosome, are associated with mXYSRa/Enh13. They function as an enhancer, whereby the sequences are evolutionarily conserved and in vivo binding of SOX9 and SRY to mXYSRa/Enh13 has been demonstrated previously. However, their precise in vivo functions have not been examined to date. To this end, this study generated mice with substitutions on the SOX9 and SRY BSs to reveal their in vivo functions. Homozygous mutants of SOX9 and SRY BS were indistinguishable from XY males, whereas double mutants had small testes, suggesting that these functions are redundant and that there is another functional sequence on mXYSRa/Enh13, since mXYSRa/Enh13 deletion mice are XY females. In addition, the majority of hemizygous mice with substitutions in SOX9 BS and SRY BS were female and male, respectively, suggesting that SOX9 BS contributes more to SRY BS for mXYSRa/Enh13 to function. The additive effect of SOX9 and SRY via these BSs was verified using an in vitro assay. In conclusion, SOX9 BS and SRY BS function redundantly in vivo, and at least one more functional sequence should exist in mXYSRa/Enh13.

Identification of a New Enhancer That Promotes Sox9 Expression by a Comparative Analysis of Mouse and Sry-Deficient Amami Spiny Rat.

INTRODUCTION: Testis differentiation is initiated by the SRY gene on the Y chromosome in mammalian species. However, the Amami spiny rat, Tokudaia osimensis, lacks both the Y chromosome and the Sry gene and acquired a unique Sox9 regulatory mechanism via a male-specific duplication upstream of Sox9, without Sry. In general mammalian species, the SRY protein binds to a testis-specific enhancer to promote SOX9 gene expression. Several enhancers located upstream of Sox9/SOX9 have been reported in mice and humans. In particular, the binding of SRY to the highly conserved enhancer Enh13 is thought to be a common mechanism underlying testis differentiation and sex determination in mammals. METHODS: Sequences of T. osimensis homologues of three Sox9 enhancers that were previously reported in mice, Enh8, Enh14, and Enh13, were determined. We performed in vitro assays to confirm enhancer activity involved in Sox9 regulation in T. osimensis. RESULTS: T. osimensis Enh13 showed enhancer activity when co-transfected with NR5A1 and SOX9. Mouse Enh13 was activated by NR5A1 and SRY; however, T. osimensis Enh13 did not respond to SRY, even though the binding sites of SRY and NR5A1 were conserved. To identify the key sequence that is present in mouse but absent from T. osimensis, we performed reporter gene assays using vectors in which partial sequences of T. osimensis Enh13 were replaced with mouse sequences. For T. osimensis Enh13 in which the second half (approximately 430 bp) was replaced with the corresponding mouse sequence, activity in response to NR5A1 and SRY was recovered. Further, reporter assays revealed that multiple regions in the second half of the mouse Enh13 sequence are required for the response to NR5A1 and SRY. The latter 49 bp was particularly important and contained four binding sites for three transcription factors, POU2F1, HOXA3, and GATA1. CONCLUSION: We showed that there are unknown sequences responsible for the interaction between NR5A1 and SRY and mEnh13 based on comparative analyses of Sry-dependent and Sry-independent species. Our comparative analyses revealed new molecular mechanisms underlying mammalian sex determination.

A Simulation Study of the Effect of Naturally Occurring Point Mutations on the SRY-DNA Complex.

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 in vitro assays found elsewhere and from modifications of the DNA bending as revealed in this study. Comparative potential of mean force computations, over a sequence of short separation distances for the three complexes, verified a higher stability of the normal SRY-DNA. Examining the way the SRY mutations modulate the SRY-DNA complex dynamics at the microscopic level is important also toward elucidating molecular determinants of function for proteins capable of binding to DNA.

Tenuous Transcriptional Threshold of Human Sex Determination. I. SRY and Swyer Syndrome at the Edge of Ambiguity.

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 arise de novo. Rare inherited variants lead to male development in one genetic background (the father) but not another (his sterile XY daughter). De novo and 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 novo mutations Y127H and Y127C reduced SRY activity (as assessed by transcriptional activation of principal target gene Sox9) 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 the de novo genotype-phenotype relationships, a molecular understanding of inherited mutation Y127F remains elusive. Our companion study uncovers cryptic biophysical perturbations suggesting that the para-OH group of Y127 anchors a novel water-mediated DNA clamp.

Gadd45g is required for timely Sry expression independently of RSPO1 activity.

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.

Functional Analysis of Mmd2 and Related PAQR Genes During Sex Determination in Mice.

INTRODUCTION: Sex determination in eutherian mammals is controlled by the Y-linked gene Sry, which drives the formation of testes in male embryos. Despite extensive study, the genetic steps linking Sry action and male sex determination remain largely unknown. Here, we focused on Mmd2, a gene that encodes a member of the progestin and adipoQ receptor (PAQR) family. Mmd2 is expressed during the sex-determining period in XY but not XX gonads, suggesting a specific role in testis development. METHODS: We used CRISPR to generate mouse strains deficient in Mmd2 and its 2 closely related PAQR family members, Mmd and Paqr8, which are also expressed during testis development. Following characterization of Mmd2 expression in the developing testis, we studied sex determination in embryos from single knockout as well as Mmd2;Mmd and Mmd2;Paqr8 double knockout lines using quantitative RT-PCR and immunofluorescence. RESULTS: Analysis of knockout mice deficient in Sox9 and Nr5a1 revealed that Mmd2 operates downstream of these known sex-determining genes. However, fetal testis development progressed normally in Mmd2-null embryos. To determine if other genes might have compensated for the loss of Mmd2, we analyzed Paqr8 and Mmd-null embryos and confirmed that in both knockout lines, sex determination occurred normally. Finally, we generated Mmd2;Mmd and Mmd2;Paqr8 double-null embryos and again observed normal testis development. DISCUSSION: These results may reflect functional redundancy among PAQR factors, or their dispensability in gonadal development. Our findings highlight the difficulties involved in identifying genes with a functional role in sex determination and gonadal development through expression screening and loss-of-function analyses of individual candidate genes and may help to explain the paucity of genes in which variations have been found to cause human disorders/differences of sex development.

SRY is a Key Mediator of Sexual Dimorphism in Hepatic Ischemia/Reperfusion Injury.

OBJECTIVES: 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/reoxygen-ation 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-3ß (GSK-3ß) and ß-catenin, and promotes phosphorylation and degradation of ß-catenin, leading to suppression of the downstream FOXOs, and activation of NF-κBand TLR4 signaling. Furthermore, activation of ß-catenin almost completely reversed the SRYoverexpression-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/ß-catenin signaling, which accounts for the sex-based disparity in hepatic I/R injuries.

Transcriptional Regulation of the Y-Linked Mammalian Testis-Determining Gene SRY.

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.

Diallyl thiosulfinate enhanced the anti-cancer activity of dexamethasone in the side population cells of multiple myeloma by promoting miR-127-3p and deactivating the PI3K/AKT signaling pathway.

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.

Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs.

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.

Gene expression and functional abnormalities in XX/Sry Leydig cells.

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.