RESUMO
Heterozygous mutations in the human SOX9 gene cause the skeletal malformation syndrome campomelic dysplasia which in 75% of 46, XY individuals is associated with male-to-female sex reversal. Although studies in homozygous Sox9 knockout mouse models confirmed that SOX9 is critical for testis development, mice heterozygous for the Sox9-null allele were reported to develop normal testes. This led to the belief that the SOX9 dosage requirement for testis differentiation is different between humans, which often require both alleles, and mice, in which one allele is sufficient. However, in prior studies, gonadal phenotypes in heterozygous Sox9 XY mice were assessed only by either gross morphology, histological staining or analyzed on a mixed genetic background. In this study, we conditionally inactivated Sox9 in somatic cells of developing gonads using the Nr5a1-Cre mouse line on a pure C57BL/6 genetic background. Section and whole-mount immunofluorescence for testicular and ovarian markers showed that XY Sox9 heterozygous gonads developed as ovotestes. Quantitative droplet digital PCR confirmed a 50% reduction of Sox9 mRNA as well as partial sex reversal shown by an upregulation of ovarian genes. Our data show that haploinsufficiency of Sox9 can perturb testis development in mice, suggesting that mice may provide a more accurate model of human disorders/differences of sex development than previously thought.
Assuntos
Displasia Campomélica/patologia , Transtornos do Desenvolvimento Sexual/patologia , Gônadas/patologia , Heterozigoto , Fatores de Transcrição SOX9/fisiologia , Diferenciação Sexual , Fator Esteroidogênico 1/fisiologia , Animais , Displasia Campomélica/etiologia , Displasia Campomélica/metabolismo , Modelos Animais de Doenças , Transtornos do Desenvolvimento Sexual/etiologia , Transtornos do Desenvolvimento Sexual/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , FenótipoRESUMO
46,XY gonadal dysgenesis (GD) is a Disorder/Difference of Sex Development (DSD) that can present with phenotypes ranging from ambiguous genitalia to complete male-to-female sex reversal. Around 50% of 46,XY DSD cases receive a molecular diagnosis. In mice, Fibroblast growth factor 9 (FGF9) is an important component of the male sex-determining pathway. Two FGF9 variants reported to date disrupt testis development in mice, but not in humans. Here, we describe a female patient with 46,XY GD harbouring the rare FGF9 variant (missense mutation), NM_002010.2:c.583G > A;p.(Asp195Asn) (D195N). By biochemical and cell-based approaches, the D195N variant disrupts FGF9 protein homodimerisation and FGF9-heparin-binding, and reduces both Sertoli cell proliferation and Wnt4 repression. XY Fgf9D195N/D195N foetal mice show a transient disruption of testicular cord development, while XY Fgf9D195N/- foetal mice show partial male-to-female gonadal sex reversal. In the general population, the D195N variant occurs at an allele frequency of 2.4 × 10-5 , suggesting an oligogenic basis for the patient's DSD. Exome analysis of the patient reveals several known and novel variants in genes expressed in human foetal Sertoli cells at the time of sex determination. Taken together, our results indicate that disruption of FGF9 homodimerization impairs testis determination in mice and, potentially, also in humans in combination with other variants.
Assuntos
Fator 9 de Crescimento de Fibroblastos , Disgenesia Gonadal 46 XY , Humanos , Masculino , Feminino , Camundongos , Animais , Dimerização , Fator 9 de Crescimento de Fibroblastos/genética , Testículo , Gônadas , Disgenesia Gonadal 46 XY/genéticaRESUMO
The transcription factor SOX9 is essential for the development of multiple organs including bone, testis, heart, lung, pancreas, intestine and nervous system. Mutations in the human SOX9 gene led to campomelic dysplasia, a haploinsufficiency disorder with several skeletal malformations frequently accompanied by 46, XY sex reversal. The mechanisms underlying the diverse SOX9 functions during organ development including its post-translational modifications, the availability of binding partners, and tissue-specific accessibility to target gene chromatin. Here we summarize the expression, activities, and downstream target genes of SOX9 in molecular genetic pathways essential for organ development, maintenance, and function. We also provide an insight into understanding the mechanisms that regulate the versatile roles of SOX9 in different organs.
Assuntos
Organogênese , Fatores de Transcrição SOX9/metabolismo , Displasia Campomélica , Cromatina , Transtornos do Desenvolvimento Sexual/genética , Humanos , Masculino , Mutação , Fatores de Transcrição SOX9/genéticaRESUMO
In mice, male sex determination depends on FGF9 signalling via FGFR2c in the bipotential gonads to maintain the expression of the key testis gene SOX9. In humans, however, while FGFR2 mutations have been linked to 46,XY disorders of sex development (DSD), the role of FGF9 is unresolved. The only reported pathogenic mutations in human FGF9, FGF9S99N and FGF9R62G, are dominant and result in craniosynostosis (fusion of cranial sutures) or multiple synostoses (fusion of limb joints). Whether these synostosis-causing FGF9 mutations impact upon gonadal development and DSD etiology has not been explored. We therefore examined embryonic gonads in the well-characterized Fgf9 missense mouse mutants, Fgf9S99N and Fgf9N143T, which phenocopy the skeletal defects of FGF9S99N and FGF9R62G variants, respectively. XY Fgf9S99N/S99N and XY Fgf9N143T/N143T fetal mouse gonads showed severely disorganized testis cords and partial XY sex reversal at 12.5 days post coitum (dpc), suggesting loss of FGF9 function. By 15.5 dpc, testis development in both mutants had partly recovered. Mitotic analysis in vivo and in vitro suggested that the testicular phenotypes in these mutants arise in part through reduced proliferation of the gonadal supporting cells. These data raise the possibility that human FGF9 mutations causative for dominant skeletal conditions can also lead to loss of FGF9 function in the developing testis, at least in mice. Our data suggest that, in humans, testis development is largely tolerant of deleterious FGF9 mutations which lead to skeletal defects, thus offering an explanation as to why XY DSDs are rare in patients with pathogenic FGF9 variants.
Assuntos
Fator 9 de Crescimento de Fibroblastos/genética , Transtornos Ovotesticulares do Desenvolvimento Sexual/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Sinostose/genética , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Gônadas/crescimento & desenvolvimento , Gônadas/patologia , Humanos , Masculino , Camundongos , Mutação de Sentido Incorreto/genética , Transtornos Ovotesticulares do Desenvolvimento Sexual/patologia , Fatores de Transcrição SOX9/genética , Processos de Determinação Sexual/genética , Desenvolvimento Sexual/genéticaRESUMO
Women and other mammalian females are born with a finite supply of oocytes that determine their reproductive lifespan. During fetal development, individual oocytes are enclosed by a protective layer of granulosa cells to form primordial follicles that will grow, mature, and eventually release the oocyte for potential fertilization. Despite the knowledge that follicles are dysfunctional and will die without granulosa cell-oocyte interactions, the mechanisms by which these cells establish communication is unknown. We previously identified that two members of the Iroquois homeobox transcription factor gene family, Irx3 and Irx5, are expressed within developing ovaries but not testes. Deletion of both factors (Irx3-Irx5EGFP/Irx3-Irx5EGFP) disrupted granulosa cell-oocyte contact during early follicle development leading to oocyte death. Thus, we hypothesized that Irx3 and Irx5 are required to develop cell-cell communication networks to maintain follicle integrity and female fertility. A series of Irx3 and Irx5 mutant mouse models were generated to assess roles for each factor. While both Irx3 and Irx5 single mutant females were subfertile, their breeding outcomes and ovary histology indicated distinct causes. Careful analysis of Irx3- and Irx5-reporter mice linked the cause of this disparity to dynamic spatio-temporal changes in their expression patterns. Both factors marked the progenitor pre-granulosa cell population in fetal ovaries. At the critical phase of germline nest breakdown and primordial follicle formation however, Irx3 and Irx5 transitioned to oocyte- and granulosa cell-specific expression respectively. Further investigation into the cause of follicle death in Irx3-Irx5EGFP/Irx3-Irx5EGFP ovaries uncovered specific defects in both granulosa cells and oocytes. Granulosa cell defects included poor contributions to basement membrane deposition and mis-localization of gap junction proteins. Granulosa cells and oocytes both presented fewer cell projections resulting in compromised cell-cell communication. Altogether, we conclude that Irx3 and Irx5 first work together to define the pregranulosa cell population of germline nests. During primordial follicle formation, they transition to oocyte- and granulosa cell-specific expression patterns where they cooperate in neighboring cells to build the foundation for follicle integrity. This foundation is left as their legacy of the essential oocyte-granulosa cell communication network that ensures and ultimately optimizes the integrity of the ovarian reserve and therefore, the female reproductive lifespan.
Assuntos
Células da Granulosa/fisiologia , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Animais , Comunicação Celular , Conexinas/genética , Conexinas/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas , Proteínas de Homeodomínio/genética , Camundongos , Camundongos Nus , Oócitos/fisiologia , Fatores de Transcrição/genéticaRESUMO
In mammalian embryonic gonads, SOX9 is required for the determination of Sertoli cells that orchestrate testis morphogenesis. To identify genetic networks directly regulated by SOX9, we combined analysis of SOX9-bound chromatin regions from murine and bovine foetal testes with sequencing of RNA samples from mouse testes lacking Sox9. We found that SOX9 controls a conserved genetic programme that involves most of the sex-determining genes. In foetal testes, SOX9 modulates both transcription and directly or indirectly sex-specific differential splicing of its target genes through binding to genomic regions with sequence motifs that are conserved among mammals and that we called 'Sertoli Cell Signature' (SCS). The SCS is characterized by a precise organization of binding motifs for the Sertoli cell reprogramming factors SOX9, GATA4 and DMRT1. As SOX9 biological role in mammalian gonads is to determine Sertoli cells, we correlated this genomic signature with the presence of SOX9 on chromatin in foetal testes, therefore equating this signature to a genomic bar code of the fate of foetal Sertoli cells. Starting from the hypothesis that nuclear factors that bind to genomic regions with SCS could functionally interact with SOX9, we identified TRIM28 as a new SOX9 partner in foetal testes.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Morfogênese/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Fatores de Transcrição SOX9/genética , Células de Sertoli/metabolismo , Transcriptoma , Animais , Bovinos , Cromatina/química , Cromatina/metabolismo , Embrião de Mamíferos , Feminino , Feto , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Redes Reguladoras de Genes , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas Repressoras/metabolismo , Fatores de Transcrição SOX9/metabolismo , Análise de Sequência de RNA , Células de Sertoli/citologia , Processos de Determinação Sexual , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 28 com Motivo TripartidoRESUMO
Patients with 46,XY gonadal dysgenesis (GD) exhibit genital anomalies, which range from hypospadias to complete male-to-female sex reversal. However, a molecular diagnosis is made in only 30% of cases. Heterozygous mutations in the human FGFR2 gene cause various craniosynostosis syndromes including Crouzon and Pfeiffer, but testicular defects were not reported. Here, we describe a patient whose features we would suggest represent a new FGFR2-related syndrome, craniosynostosis with XY male-to-female sex reversal or CSR. The craniosynostosis patient was chromosomally XY, but presented as a phenotypic female due to complete GD. DNA sequencing identified the FGFR2c heterozygous missense mutation, c.1025G>C (p.Cys342Ser). Substitution of Cys342 by Ser or other amino acids (Arg/Phe/Try/Tyr) has been previously reported in Crouzon and Pfeiffer syndrome. We show that the 'knock-in' Crouzon mouse model Fgfr2c(C342Y/C342Y) carrying a Cys342Tyr substitution displays XY gonadal sex reversal with variable expressivity. We also show that despite FGFR2c-Cys342Tyr being widely considered a gain-of-function mutation, Cys342Tyr substitution in the gonad leads to loss of function, as demonstrated by sex reversal in Fgfr2c(C342Y/-) mice carrying the knock-in allele on a null background. The rarity of our patient suggests the influence of modifier genes which exacerbated the testicular phenotype. Indeed, patient whole exome analysis revealed several potential modifiers expressed in Sertoli cells at the time of testis determination in mice. In summary, this study identifies the first FGFR2 mutation in a 46,XY GD patient. We conclude that, in certain rare genetic contexts, maintaining normal levels of FGFR2 signaling is important for human testis determination.
Assuntos
Craniossinostoses/genética , Disgenesia Gonadal 46 XY/genética , Mutação de Sentido Incorreto , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Adolescente , Animais , Craniossinostoses/metabolismo , Análise Mutacional de DNA , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos Mutantes , SíndromeRESUMO
SOX9 is a key transcription factor for testis determination and development. Mutations in and around the SOX9 gene contribute to Differences/Disorders of Sex Development (DSD). However, a substantial proportion of DSD patients lack a definitive genetic diagnosis. SOX9 target genes are potentially DSD-causative genes, yet only a limited subset of these genes has been investigated during testis development. We hypothesize that SOX9 target genes play an integral role in testis development and could potentially be causative genes in DSD. In this study, we describe a novel testicular target gene of SOX9, Trpc3. Trpc3 exhibits high expression levels in the SOX9-expressing male Sertoli cells compared to female granulosa cells in mouse fetal gonads between embryonic day 11.5 (E11.5) and E13.5. In XY Sox9 knockout gonads, Trpc3 expression is markedly downregulated. Moreover, culture of E11.5 XY mouse gonads with TRPC3 inhibitor Pyr3 resulted in decreased germ cell numbers caused by reduced germ cell proliferation. Trpc3 is also expressed in endothelial cells and Pyr3-treated E11.5 XY mouse gonads showed a loss of the coelomic blood vessel due to increased apoptosis of endothelial cells. In the human testicular cell line NT2/D1, TRPC3 promotes cell proliferation and controls cell morphology, as observed by xCELLigence and HoloMonitor real-time analysis. In summary, our study suggests that SOX9 positively regulates Trpc3 in mouse testes and TRPC3 may mediate SOX9 function during Sertoli, germ and endothelial cell development.
RESUMO
ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome features genital and testicular abnormalities including atypical genitalia and small testes with few seminiferous tubules. Our mouse model recapitulated the testicular defects when Atrx was deleted in Sertoli cells (ScAtrxKO) which displayed G2/M arrest and apoptosis. Here, we investigated the mechanisms underlying these defects. In control mice, Sertoli cells contain a single novel "GATA4 PML nuclear body (NB)" that contained the transcription factor GATA4, ATRX, DAXX, HP1α, and PH3 and co-localized with the Y chromosome short arm (Yp). ScAtrxKO mice contain single giant GATA4 PML-NBs with frequent DNA double-strand breaks (DSBs) in G2/M-arrested apoptotic Sertoli cells. HP1α and PH3 were absent from giant GATA4 PML-NBs indicating a failure in heterochromatin formation and chromosome condensation. Our data suggest that ATRX protects a Yp region from DNA damage, thereby preventing Sertoli cell death. We discuss Y chromosome damage/decondensation as a mechanism for testicular failure.
RESUMO
X-linked ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome in males is characterized by mental retardation, facial dysmorphism, alpha thalassemia and urogenital abnormalities, including small testes. It is unclear how mutations in the chromatin-remodeling protein ATRX cause these highly specific clinical features, since ATRX is widely expressed during organ development. To investigate the mechanisms underlying the testicular defects observed in ATR-X syndrome, we generated ScAtrxKO (Sertoli cell Atrx knockout) mice with Atrx specifically inactivated in the supporting cell lineage (Sertoli cells) of the mouse testis. ScAtrxKO mice developed small testes and discontinuous tubules, due to prolonged G2/M phase and apoptosis of proliferating Sertoli cells during fetal life. Apoptosis might be a consequence of the cell cycle defect. We also found that the onset of spermatogenesis was delayed in postnatal mice, with a range of spermatogenesis defects evident in adult ScAtrxKO mice. ATRX and the androgen receptor (AR) physically interact in the testis and in the Sertoli cell line TM4 and co-operatively activate the promoter of Rhox5, an important direct AR target. We also demonstrate that ATRX directly binds to the Rhox5 promoter in TM4 cells. Finally, gene expression of Rhox5 and of another AR-dependent gene, Spinlw1, was reduced in ScAtrxKO testes. These data suggest that ATRX can directly enhance the expression of androgen-dependent genes through physical interaction with AR. Recruitment of ATRX by DNA sequence-specific transcription factors could be a general mechanism by which ATRX achieves tissue-specific transcriptional regulation which could explain the highly specific clinical features of ATR-X syndrome when ATRX is mutated.
Assuntos
Proliferação de Células , DNA Helicases/genética , Proteínas Nucleares/genética , Receptores Androgênicos/metabolismo , Células de Sertoli/metabolismo , Animais , Apoptose , Ciclo Celular , Células Cultivadas , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imunoprecipitação , Masculino , Deficiência Intelectual Ligada ao Cromossomo X/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Testículo/anormalidades , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Nuclear Ligada ao X , Talassemia alfa/genéticaRESUMO
MicroRNAs are important regulators of developmental gene expression, but their contribution to fetal gonad development is not well understood. We have identified the evolutionarily conserved gonadal microRNAs miR-202-5p and miR-202-3p as having a potential role in regulating mouse embryonic gonad differentiation. These microRNAs are expressed in a sexually dimorphic pattern as the primordial XY gonad differentiates into a testis, with strong expression in Sertoli cells. In vivo, ectopic expression of pri-miR-202 in XX gonads did not result in molecular changes to the ovarian determination pathway. Expression of the primary transcript of miR-202-5p/3p remained low in XY gonads in a conditional Sox9-null mouse model, suggesting that pri-miR-202 transcription is downstream of SOX9, a transcription factor that is both necessary and sufficient for male sex determination. We identified the pri-miR-202 promoter that is sufficient to drive expression in XY but not XX fetal gonads ex vivo. Mutation of SOX9 and SF1 binding sites reduced ex vivo transactivation of the pri-miR-202 promoter, demonstrating that pri-miR-202 may be a direct transcriptional target of SOX9/SF1 during testis differentiation. Our findings indicate that expression of the conserved gonad microRNA, miR-202-5p/3p, is downstream of the testis-determining factor SOX9, suggesting an early role in testis development.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/metabolismo , Organogênese/genética , Fatores de Transcrição SOX9/metabolismo , Testículo/embriologia , Animais , Diferenciação Celular/genética , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética , Regiões Promotoras Genéticas , Fatores de Transcrição SOX9/genética , Células de Sertoli/citologia , Células de Sertoli/metabolismo , Diferenciação Sexual/genética , Testículo/citologia , Testículo/metabolismo , Transcrição GênicaRESUMO
In most mammals, the Y chromosomal Sry gene initiates testis formation within the bipotential gonad, resulting in male development. SRY is a transcription factor and together with SF1 it directly up-regulates the expression of the pivotal sex-determining gene Sox9 via a 1.3-kb cis-regulatory element (TESCO) which contains an evolutionarily conserved region (ECR) of 180 bp. Remarkably, several rodent species appear to determine sex in the absence of Sry and a Y chromosome, including the mole voles Ellobius lutescens and Ellobius tancrei, whereas Ellobius fuscocapillus of the same genus retained Sry. The sex-determining mechanisms in the Sry-negative species remain elusive. We have cloned and sequenced 1.1 kb of E. lutescens TESCO which shares 75% sequence identity with mouse TESCO indicating that testicular Sox9 expression in E. lutescens might still be regulated via TESCO. We have also cloned and sequenced the ECRs of E. tancrei and E. fuscocapillus. While the three Ellobius ECRs are highly similar (94-97% sequence identity), they all display a 14-bp deletion (Δ14) removing a highly conserved SOX/TCF site. Introducing Δ14 into mouse TESCO increased both basal activity and SF1-mediated activation of TESCO in HEK293T cells. We propose a model whereby Δ14 may have triggered up-regulation of Sox9 in XX gonads leading to destabilization of the XY/XX sex-determining mechanism in Ellobius. E. lutescens/E. tancrei and E. fuscocapillus could have independently stabilized their sex determination mechanisms by Sry-independent and Sry-dependent approaches, respectively.
Assuntos
Arvicolinae/genética , Regulação da Expressão Gênica , Fatores de Transcrição SOX9/metabolismo , Processos de Determinação Sexual , Cromossomo Y/metabolismo , Animais , Arvicolinae/metabolismo , Arvicolinae/fisiologia , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Evolução Molecular , Feminino , Variação Genética , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fatores de Transcrição SOX9/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Deleção de Sequência , Testículo/citologia , Testículo/metabolismo , Testículo/fisiologia , Cromossomo Y/genéticaRESUMO
During sex determination in the mouse, fibroblast growth factor 9 signals through the fibroblast growth factor receptor 2c isoform (FGFR2c) to trigger Sertoli cell and testis development from 11.5 days post coitum (dpc). In the XX gonad, the FOXL2 and WNT4/RSPO1 pathways drive granulosa cell and ovarian development. The function of FGFR2 in the developing ovary, and whether FGFR2 is required in the testis after sex determination, is not clear. In fetal mouse gonads from 12.5 dpc, FGFR2 shows sexually dimorphic expression. In XX gonads, FGFR2c is coexpressed with FOXL2 in pregranulosa cells, whereas XY gonads show FGFR2b expression in germ cells. Deletion of Fgfr2c in XX mice led to a marked decrease/absence of germ cells by 13.5 dpc in the ovary. This indicates that FGFR2c in the somatic pregranulosa cells is required for the maintenance of germ cells. Surprisingly, on the Fgfr2c-/- background, the germ cell phenotype could be rescued by ablation of Foxl2, suggesting a novel mechanism whereby FGFR2 and FOXL2 act antagonistically during germ cell development. Consistent with low/absent FGFR2 expression in the Sertoli cells of 12.5 and 13.5 dpc XY gonads, XY AMH:Cre; Fgfr2flox/flox mice showed normal testis morphology and structures during fetal development and in adulthood. Thus, FGFR2 is not essential for maintaining Sertoli cell fate after sex determination. Combined, these data show that FGFR2 is not necessary for Sertoli cell function after sex determination but does play an important role in the ovary.
Assuntos
Ovário , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Masculino , Feminino , Camundongos , Animais , Ovário/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Gônadas/metabolismo , Testículo/metabolismo , Células Germinativas/metabolismo , Processos de Determinação SexualRESUMO
Sex determination in fetal germ cells depends on a balance between exposure to retinoic acid (RA) and the degradation of RA achieved by the testis-specific expression of the catabolic cytochrome P450 enzyme, CYP26B1. Therefore, identification of factors regulating the expression of the Cyp26b1 gene is an important goal in reproductive biology. We used in situ hybridization to demonstrate that Cyp26b1 and transcription factor genes steroidogenic factor-1 (Sf1) and Sry-related HMG box 9 (Sox9) are coexpressed in Sertoli cells, whereas Cyp26b1 and Sf1 are coexpressed in Leydig cells in mouse fetal testes. In the mouse gonadal somatic cell line TM3, transfection of constructs expressing SOX9 and SF1 activated Cyp26b1 expression, independently of the positive regulator RA. In embryonic gonads deficient in SOX9 or SF1, Cyp26b1 expression was decreased relative to wild-type (WT) controls, as measured by quantitative RT-PCR (qRT-PCR). Furthermore, qRT-PCR showed that Cyp26b1 up-regulation by SOX9/SF1 was attenuated by the ovarian transcription factor Forkhead box L2 (FOXL2) in TM3 cells, whereas in Foxl2-null mice, Cyp26b1 expression in XX gonads was increased â¼20-fold relative to WT controls. These data support the hypothesis that SOX9 and SF1 ensure the male fate of germ cells by up-regulating Cyp26b1 and that FOXL2 acts to antagonize Cyp26b1 expression in ovaries.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Fatores de Transcrição SOX9/metabolismo , Testículo/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Ligação a DNA/genética , Proteína Forkhead Box L2 , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Masculino , Camundongos , Fatores de Processamento de RNA , Ácido Retinoico 4 Hidroxilase , Fatores de Transcrição SOX9/genética , Processos de Determinação Sexual/fisiologia , Testículo/metabolismo , Fatores de Transcrição/genética , Regulação para CimaRESUMO
This dataset represents genes that are dysregulated in the postnatal day 12 (P12) mouse testis when ATRX is specifically inactivated in Sertoli cells (ScAtrxKO mice). The differentially expressed genes included in the dataset may play important roles in the testicular phenotypes observed in the ScAtrxKO mice, which were first reported in our previous work [1]. In fetal ScAtrxKO mice, Sertoli cells undergo apoptosis due to cell cycle defects, resulting in smaller testes with reduced tubule volume [1]. Adult ScAtrxKO mice show a wide range of spermatogenesis defects probably due to a failure of the dysfunctional ATRX protein to interact with the androgen receptor (AR) [1]. ATRX, a chromatin remodeling protein, is widely expressed in the human testis including Sertoli cells [2,3]. In XY individuals, the loss of ATRX leads to ATR-X (alpha thalassemia, mental retardation, X-linked) syndrome associated with a wide range of genital abnormalities such as hypospadias, ambiguous genitalia, and small testes with reduced tubule volume [4], [5], [6], [7], [8]. Our dataset contributes towards understanding the mechanism underlying ATRX regulation of testis development and spermatogenesis.
RESUMO
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.
Assuntos
Gônadas , Processos de Determinação Sexual , Humanos , Camundongos , Masculino , Animais , Processos de Determinação Sexual/genética , Proteína da Região Y Determinante do Sexo/genética , Proteína da Região Y Determinante do Sexo/metabolismo , Gônadas/metabolismo , Testículo/metabolismo , Diferenciação Sexual/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mamíferos/genética , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismoRESUMO
Sexual reproduction is essential for the propagation and the maintenance of fitness of our species, and is dependent on the correct development of the bipotential genital ridges into testes and ovaries. Although several transcription factors, secreted signaling molecules, and their receptors have been found to be important for testis determination and early gonad development, comparatively little research has been carried out into intracellular signal transduction pathways activated during these processes. Focal adhesion kinase (FAK) and protein tyrosine kinase 2 beta (PTK2B) form one group of cytosolic tyrosine kinases that are known to be important for processes such as cell proliferation, differentiation, and motility. Here, we describe the temporal and spatial expression patterns of Fak and Ptk2b mRNA and protein during sex determination and early gonadogenesis in mouse embryos. Ptk2b mRNA and PTK2B protein were expressed in testes from 11.5 days post coitum onward, predominantly in developing Sertoli cells, in a SOX9-dependent manner. Fak mRNA and FAK protein were expressed in gonads of both sexes at all stages examined. Our data suggest cell type- and stage-specific roles for PTK2B during early testis development.
Assuntos
Quinase 1 de Adesão Focal/metabolismo , Quinase 2 de Adesão Focal/genética , Gônadas/embriologia , Animais , Feminino , Quinase 1 de Adesão Focal/genética , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Células de Sertoli/metabolismo , Diferenciação Sexual/genética , Diferenciação Sexual/fisiologia , Testículo/embriologia , Testículo/metabolismoRESUMO
Sex determination occurs early during embryogenesis among vertebrates. It involves the differentiation of the bipotential gonad to ovaries or testes by a fascinating diversity of molecular switches. In most mammals, the switch is SRY (sex determining region Y); in other vertebrates it could be one of a variety of genes including Dmrt1 or dmy. Downstream of the switch gene, SOX9 upregulation is a central event in testes development, controlled by gonad-specific enhancers across the 2 Mb SOX9 locus. SOX9 is a 'hub' gene of gonadal development, regulated positively in males and negatively in females. Despite this diversity, SOX9 protein sequence and function among vertebrates remains highly conserved. This article explores the cellular, morphological, and genetic mechanisms initiated by SOX9 for male gonad differentiation.
Assuntos
Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Vertebrados/crescimento & desenvolvimento , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Processos de Determinação Sexual , Diferenciação Sexual , Vertebrados/genéticaRESUMO
Disorders/differences of sex development (DSD) cause profound psychological and reproductive consequences for the affected individuals, however, most are still unexplained at the molecular level. Here, we present a novel gene, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMGCS2), encoding a metabolic enzyme in the liver important for energy production from fatty acids, that shows an unusual expression pattern in developing fetal mouse gonads. Shortly after gonadal sex determination it is up-regulated in the developing testes following a very similar spatial and temporal pattern as the male-determining gene Sry in Sertoli cells before switching to ovarian enriched expression. To test if Hmgcs2 is important for gonad development in mammals, we pursued two lines of investigations. Firstly, we generated Hmgcs2-null mice using CRISPR/Cas9 and found that these mice had gonads that developed normally even on a sensitized background. Secondly, we screened 46,XY DSD patients with gonadal dysgenesis and identified two unrelated patients with a deletion and a deleterious missense variant in HMGCS2 respectively. However, both variants were heterozygous, suggesting that HMGCS2 might not be the causative gene. Analysis of a larger number of patients in the future might shed more light into the possible association of HMGCS2 with human gonadal development.
Assuntos
Transtornos do Desenvolvimento Sexual/genética , Disgenesia Gonadal/genética , Gônadas/crescimento & desenvolvimento , Hidroximetilglutaril-CoA Sintase/genética , Adolescente , Animais , Transtornos do Desenvolvimento Sexual/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Disgenesia Gonadal/patologia , Gônadas/patologia , Heterozigoto , Humanos , Masculino , Camundongos , Mutação de Sentido Incorreto/genética , Ovário/crescimento & desenvolvimento , Ovário/patologia , Células de Sertoli/metabolismo , Proteína da Região Y Determinante do Sexo/genética , Testículo/crescimento & desenvolvimento , Testículo/patologiaRESUMO
In mammals, sex is determined in the bipotential embryonic gonad by a balanced network of gene actions which when altered causes disorders of sexual development (DSD, formerly known as intersex). In the XY gonad, presumptive Sertoli cells begin to differentiate when SRY up-regulates SOX9, which in turn activates FGF9 and PGDS to maintain its own expression. This study identifies a new and essential component of FGF signaling in sex determination. Fgfr2 mutant XY mice on a mixed 129/C57BL6 genetic background had either normal testes, or developed ovotestes, with predominantly testicular tissue. However, backcrossing to C57BL6 mice resulted in a wide range of gonadal phenotypes, from hypoplastic testes to ovotestes with predominantly ovarian tissue, similar to Fgf9 knockout mice. Since typical male-specific FGF9-binding to the coelomic epithelium was abolished in Fgfr2 mutant XY gonads, these results suggest that FGFR2 acts as the receptor for FGF9. Pgds and SOX9 remained expressed within the testicular portions of Fgfr2 mutant ovotestes, suggesting that the Prostaglandin pathway acts independently of FGFR2 to maintain SOX9 expression. We could further demonstrate that double-heterozygous Fgfr2/Sox9 knockout mice developed ovotestes, demonstrating that both Fgfr2 and Sox9 can act as modifier intersex genes in the heterozygous state. In summary, we provide evidence that FGFR2 is important for male sex determination in mice, thereby rendering human FGFR2 a candidate gene for unsolved DSD cases such as 10q26 deletions.