RESUMO
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.
Assuntos
Elementos Facilitadores Genéticos , Processos de Determinação Sexual , Animais , Feminino , Humanos , Masculino , Camundongos , Sítios de Ligação/genética , Elementos Facilitadores Genéticos/genética , Deleção de Sequência , Processos de Determinação Sexual/genética , Proteína da Região Y Determinante do Sexo/genética , Proteína da Região Y Determinante do Sexo/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Testículo/crescimento & desenvolvimento , FenótipoRESUMO
The aim of this study was to assess the long-term effect of exposure to environmentally relevant doses of non-steroidal anti-inflammatory drugs (NSAIDs; ibuprofen, and diclofenac) and 17ß-ethinylestradiol (EE2) on the mouse uterus. NSAID-EE2 mixtures were administered in the drinking water from gestational day 8 until 8 weeks post-birth (i.e., during embryo development, lactation, puberty, and sexual maturity). The incidence of adenomyosis lesions (presence of endometrial glands in the inner myometrium) increased up to 60% in the uterus of 8-week-old exposed females (F1) and to 85% in F2 females (exposed father). Histological analysis revealed aberrant proliferation and apoptosis, vacuolization of epithelial cells, and increased incidence of abnormal glands in the luminal and glandular epithelium in F1 and F2 uteri. Moreover, myofibroblast proportion (alpha-smooth muscle actin (α-SMA) expression analysis) and collagen expression (Picrosirius red stain; a fibrosis hallmark) were increased in F1 and F2 endometrium. Connexin-43 was aberrantly distributed in the endometrial stroma and glands of F1 and F2 uteri. Conversely, uterine 17ß-estradiol and progesterone levels were not affected in F1 and F2 females. These findings demonstrated that in mice, chronic exposure to NSAID and EE2 mixtures at environmental doses intergenerationally affects uterine physiology, particularly the endometrium. It may serve as a model to study the pathophysiology of human adenomyosis.
Assuntos
Adenomiose , Feminino , Camundongos , Animais , Humanos , Adenomiose/patologia , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/metabolismo , Útero/metabolismo , Endométrio/metabolismo , Miométrio/metabolismoRESUMO
DMRT1 is the testis-determining factor in several species of vertebrates, but its involvement in mammalian testes differentiation, where SRY is the testis-determining gene, remains ambiguous. So far, DMRT1 loss-of-function has been described in two mammalian species and induces different phenotypes: Disorders of Sex Development (46, XY DSD) in men and male infertility in mice. We thus abolished DMRT1 expression by CRISPR/Cas9 in a third species of mammal, the rabbit. First, we observed that gonads from XY DMRT1-/- rabbit fetuses differentiated like ovaries, highlighting that DMRT1 is involved in testis determination. In addition to SRY, DMRT1 is required in the supporting cells to increase the expression of the SOX9 gene, which heads the testicular genetic cascade. Second, we highlighted another function of DMRT1 in the germline since XX and XY DMRT1-/- ovaries did not undergo meiosis and folliculogenesis. XX DMRT1-/- adult females were sterile, showing that DMRT1 is also crucial for female fertility. To conclude, these phenotypes indicate an evolutionary continuum between non-mammalian vertebrates such as birds and non-rodent mammals. Furthermore, our data support the potential involvement of DMRT1 mutations in different human pathologies, such as 46, XY DSD as well as male and female infertility.
Animals that reproduce sexually have organs called gonads, the ovaries and testes, which produce eggs and sperm. These organs, which are different in males and females, originate from the same cells during the development of the embryo. As a general rule, the chromosomal sex of an embryo, which gets determined at fertilization, leads to the activation and repression of specific genes. This in turn, controls whether the cells that will form the gonads will differentiate to develop testes or ovaries. Disruption of the key genes involved in the differentiation of the gonads can lead to fertility problems, and in some cases, it can cause the gonads to develop in the 'opposite' direction, resulting in a sex reversal. Identifying these genes is therefore essential to know how to maintain or restore fertility. DMRT1 is a gene that drives the differentiation of gonadal cells into the testicular pathway in several species of animals with backbones, including species of fish, frogs and birds. However, its role in mammals where testis differentiation is driven by a different gene called SRY is not well understood. Indeed, when DMRT1 is disrupted in male humans it leads to disorders of sex development, while disrupting this gene in male mice causes infertility. To obtain more information about the roles of DMRT1 in mammalian species, Dujardin et al. disrupted the gene in a third species of mammal: the rabbit. Dujardin et al. observed that chromosomally-male rabbits lacking DMRT1 developed ovaries instead of testes, showing that in rabbits, both SRY and DMRT1 are both required to produce testes. Additionally, this effect is similar to what is seen in humans, suggesting that rabbits may be a better model for human gonadal differentiation than mice are. Additionally, Dujardin et al. were also able to show that in female rabbits, lack of DMRT1 led to infertility, an effect that had not been previously described in other species. The results of Dujardin et al. may lead to better models for gonadal development in humans, involving DMRT1 in the differentiation of testes. Interestingly, they also suggest the possibility that mutations in this gene may be responsible for some cases of infertility in women. Overall, these findings indicate that DMRT1 is a key fertility gene.
Assuntos
Transtorno 46,XY do Desenvolvimento Sexual , Testículo , Animais , Feminino , Masculino , Coelhos , Transtorno 46,XY do Desenvolvimento Sexual/genética , Transtorno 46,XY do Desenvolvimento Sexual/metabolismo , Fertilidade/genética , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/metabolismo , Mamíferos/genética , Processos de Determinação Sexual/genética , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Testículo/metabolismoRESUMO
Non-steroidal anti-inflammatory drugs (NSAIDs) and 17α-ethinylestradiol (EE2) are extensively used in human and veterinary medicine. Due to their partial removal by wastewater treatment plants, they are frequent environmental contaminants, particularly in drinking water. Here, we investigated the adverse outcomes of chronic exposure to mixtures of NSAIDs (ibuprofen, 2hydroxy-ibuprofen, diclofenac) and EE2 at two environmentally relevant doses in drinking water, on the reproductive organ development and fertility in F1-exposed male and female mice and in their F2 offspring. In male and female F1 mice, which were exposed to these mixtures, reproductive organ maturation, estrous cyclicity, and spermiogenesis were altered. These defects were observed also in F2 animals, in addition to some specific sperm parameter alterations in F2 males. Transcriptomic analysis revealed significant changes in gene expression patterns and associated pathways implicated in testis and ovarian physiology. Chronic exposure of mice to NSAID and EE2 mixtures at environmental doses intergenerationally affected male and female fertility (i.e. total number of pups and time between litters). Our study provides new insights into the adverse effects of these pharmaceuticals on the reproductive health and will facilitate the implementation of a future regulatory environmental risk assessment of NSAIDs and EE2 for human health.
Assuntos
Água Potável , Poluentes Químicos da Água , Humanos , Masculino , Animais , Camundongos , Etinilestradiol/toxicidade , Reprodução , Ibuprofeno/farmacologia , Sêmen , Fertilidade , Anti-Inflamatórios não Esteroides/toxicidade , Poluentes Químicos da Água/toxicidadeRESUMO
Non-steroidal anti-inflammatory drugs (NSAIDs) and 17α-ethinyl-estradiol (EE2) are among the most relevant endocrine-disrupting pharmaceuticals found in the environment, particularly in surface and drinking water due to their incomplete removal via wastewater treatment plants. Exposure of pregnant mice to NSAID therapeutic doses during the sex determination period has a negative impact on gonadal development and fertility in adults; however, the effects of their chronic exposure at lower doses are unknown. In this study, we investigated the impact of chronic exposure to a mixture containing ibuprofen, 2hydroxy-ibuprofen, diclofenac, and EE2 at two environmentally relevant doses (added to the drinking water from fetal life until puberty) on the reproductive tract in F1 exposed mice and their F2 offspring. In F1 animals, exposure delayed male puberty and accelerated female puberty. In post-pubertal F1 testes and ovaries, differentiation/maturation of the different gonad cell types was altered, and some of these modifications were observed also in the non-exposed F2 generation. Transcriptomic analysis of post-pubertal testes and ovaries of F1 (exposed) and F2 animals revealed significant changes in gene expression profiles and enriched pathways, particularly the inflammasome, metabolism and extracellular matrix pathways, compared with controls (non-exposed). This suggested that exposure to these drug cocktails has an intergenerational impact. The identified Adverse Outcome Pathway (AOP) networks for NSAIDs and EE2, at doses that are relevant to everyday human exposure, will improve the AOP network of the human reproductive system development concerning endocrine disruptor chemicals. It may serve to identify other putative endocrine disruptors for mammalian species based on the expression of biomarkers.
Assuntos
Água Potável , Disruptores Endócrinos , Poluentes Químicos da Água , Gravidez , Masculino , Humanos , Feminino , Camundongos , Animais , Etinilestradiol/efeitos adversos , Ibuprofeno , Maturidade Sexual , Anti-Inflamatórios não Esteroides , Poluentes Químicos da Água/toxicidade , Disruptores Endócrinos/toxicidade , MamíferosRESUMO
Gonadal sex determination in mice is a complex and dynamic process, which is crucial for the development of functional reproductive organs. The expression of genes involved in this process is regulated by a variety of genetic and epigenetic mechanisms. Recently, there has been increasing evidence that transposable elements (TEs), which are a class of mobile genetic elements, play a significant role in regulating gene expression during embryogenesis and organ development. In this study, we aimed to investigate the involvement of TEs in the regulation of gene expression during mouse embryonic gonadal development. Through bioinformatics analysis, we aimed to identify and characterize specific TEs that operate as regulatory elements for sex-specific genes, as well as their potential mechanisms of regulation. We identified TE loci expressed in a time- and sex-specific manner along fetal gonad development that correlate positively and negatively with nearby gene expression, suggesting that their expression is integrated to the gonadal regulatory network. Moreover, chromatin accessibility and histone post-transcriptional modification analyses in differentiating supporting cells revealed that TEs are acquiring a sex-specific signature for promoter-, enhancer-, and silencer-like elements, with some of them being proximal to critical sex-determining genes. Altogether, our study introduces TEs as the new potential players in the gene regulatory network that controls gonadal development in mammals.
RESUMO
Gonadal sexual fate in mammals is determined during embryonic development and must be actively maintained in adulthood. In the mouse ovary, oestrogen receptors and FOXL2 protect ovarian granulosa cells from transdifferentiation into Sertoli cells, their testicular counterpart. However, the mechanism underlying their protective effect is unknown. Here, we show that TRIM28 is required to prevent female-to-male sex reversal of the mouse ovary after birth. We found that upon loss of Trim28, ovarian granulosa cells transdifferentiate to Sertoli cells through an intermediate cell type, different from gonadal embryonic progenitors. TRIM28 is recruited on chromatin in the proximity of FOXL2 to maintain the ovarian pathway and to repress testicular-specific genes. The role of TRIM28 in ovarian maintenance depends on its E3-SUMO ligase activity that regulates the sex-specific SUMOylation profile of ovarian-specific genes. Our study identifies TRIM28 as a key factor in protecting the adult ovary from the testicular pathway.
Assuntos
Ovário , Sumoilação , Animais , Feminino , Masculino , Mamíferos/metabolismo , Camundongos , Ovário/metabolismo , Células de Sertoli/metabolismo , Testículo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína 28 com Motivo Tripartido/genética , Proteína 28 com Motivo Tripartido/metabolismoRESUMO
Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin (acetylsalicylic acid), diclofenac and ibuprofen (IBU), and analgesic drugs, such as acetaminophen (APAP, or paracetamol), are widely used to treat inflammation and pain. APAP and IBU are over-the-counter drugs and are among the most commonly taken drugs in the first trimester of pregnancy, even in combination. Furthermore, these drugs and their metabolites are released in the environment, and can be frequently detected in wastewater, surface water, and importantly in drinking water. Although their environmental concentrations are much lower than the therapeutics doses, this suggests an uncontrolled low-dose exposure of the general population, including pregnant women and young children, two particularly at risk populations. Epidemiological studies show that exposure to these molecules in the first and second trimester of gestation can favor genital malformations in new-born boys. To investigate the cellular, molecular and mechanistic effects of exposure to these molecules, ex vivo studies with human or rodent gonadal explants and in vivo experiments in rodents have been performed in the past years. This review recapitulates recent data obtained in rodent models after in utero or postnatal exposure to these drugs. The first part of this review discusses the mechanisms by which NSAIDs and analgesics may impair gonadal development and maturation, puberty development, sex hormone production, maturation and function of adult organs, and ultimately fertility in the exposed animals and their offspring. Like other endocrine disruptors, NSAIDs and APAP interfere with endocrine gland function and may have inter/transgenerational adverse effects. Particularly, they may target germ cells, resulting in reduced quality of male and female gametes, and decreased fertility of exposed individuals and their descendants. Then, this review discusses the effects of exposure to a single drug (APAP, aspirin, or IBU) or to combinations of drugs during early embryogenesis, and the consequences on postnatal gonadal development and adult reproductive health. Altogether, these data may increase medical and public awareness about these reproductive health concerns, particularly in women of childbearing age, pregnant women, and parents of young children.
RESUMO
In vertebrates, gonadal sex determination is the process by which transcription factors drive the choice between the testicular and ovarian identity of undifferentiated somatic progenitors through activation of 2 different transcriptional programs. Studies in animal models suggest that sex determination always involves sex-specific transcription factors that activate or repress sex-specific genes. These transcription factors control their target genes by recognizing their regulatory elements in the non-coding genome and their binding motifs within their DNA sequence. In the last 20 years, the development of genomic approaches that allow identifying all the genomic targets of a transcription factor in eukaryotic cells gave the opportunity to globally understand the function of the nuclear proteins that control complex genetic programs. Here, the major transcription factors involved in male and female vertebrate sex determination and the genomic profiling data of mouse gonads that contributed to deciphering their transcriptional regulation role will be reviewed.
Assuntos
Fatores de Transcrição SOX9 , Processos de Determinação Sexual , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Gônadas/metabolismo , Masculino , Camundongos , Ovário/metabolismo , Fatores de Transcrição SOX9/genética , Processos de Determinação Sexual/genética , Diferenciação Sexual/genética , Testículo/metabolismoRESUMO
Adenomyosis is characterised by epithelial gland and mesenchymal stroma invasion of the uterine myometrium. Adenomyosis is an oestrogen-dependent gynaecological disease in which a number of factors, such as inflammatory molecules, prostaglandins (PGs), angiogenic factors, cell proliferation and extracellular matrix remodelling proteins, also play a role as key disease mediators. In this study, we used mice lacking both lipocalin and hematopoietic-PG D synthase (L- and H-Pgds) genes in which PGD2 is not produced to elucidate PGD2 roles in the uterus. Gene expression studied by real-time PCR and hormone dosages performed by ELISA or liquid chromatography tandem mass spectroscopy in mouse uterus samples showed that components of the PGD2 signalling pathway, both PGDS and PGD2-receptors, are expressed in the mouse endometrium throughout the oestrus cycle with some differences among uterine compartments. We showed that PGE2 production and the steroidogenic pathway are dysregulated in the absence of PGD2. Histological analysis of L/H-Pgds-/- uteri, and immunohistochemistry and immunofluorescence analyses of proliferation (Ki67), endothelial cell (CD31), epithelial cell (pan-cytokeratin), myofibroblast (α-SMA) and mesenchymal cell (vimentin) markers, identify that 6-month-old L/H-Pgds-/- animals developed adenomyotic lesions, and that disease severity increased with age. In conclusion, this study suggests that the PGD2 pathway has major roles in the uterus by protecting the endometrium against adenomyosis development. Additional experiments, using for instance transcriptomic approaches, are necessary to fully determine the molecular mechanisms that lead to adenomyosis in L/H-Pgds-/- mice and to confirm whether this strain is an appropriate model for studying the human disease.
Assuntos
Adenomiose/metabolismo , Prostaglandina D2/fisiologia , Transdução de Sinais , Útero/metabolismo , Animais , Dinoprostona/metabolismo , Feminino , Oxirredutases Intramoleculares/metabolismo , Lipocalinas/metabolismo , Camundongos , Prostaglandina D2/genética , Prostaglandina D2/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Esteroides/biossíntese , Útero/fisiologiaRESUMO
Nonsteroidal anti-inflammatory drugs (NSAIDs) and analgesic drugs, such as acetaminophen (APAP), are frequently taken during pregnancy, even in combination. However, they can favour genital malformations in newborn boys and reproductive disorders in adults. Conversely, the consequences on postnatal ovarian development and female reproductive health after in utero exposure are unknown. Here, we found that in mice, in utero exposure to therapeutic doses of the APAP-ibuprofen combination during sex determination led to delayed meiosis entry and progression in female F1 embryonic germ cells. Consequently, follicular activation was reduced in postnatal ovaries through the AKT/FOXO3 pathway, leading in F2 animals to subfertility, accelerated ovarian aging with abnormal corpus luteum persistence, due to decreased apoptosis and increased AKT-mediated luteal cell survival. Our study suggests that administration of these drugs during the critical period of sex determination could lead in humans to adverse effects that might be passed to the offspring.
Assuntos
Acetaminofen/efeitos adversos , Envelhecimento/fisiologia , Ibuprofeno/efeitos adversos , Efeitos Tardios da Exposição Pré-Natal/patologia , Reprodução/fisiologia , Animais , Animais Recém-Nascidos , Proliferação de Células/efeitos dos fármacos , Feminino , Fertilidade , Proteína Forkhead Box O3/metabolismo , Células Germinativas/efeitos dos fármacos , Células Germinativas/patologia , Luteólise , Camundongos , Ovário/embriologia , Ovário/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Nonsteroidal antiinflammatory drugs and analgesic drugs, such as N-acetyl- p-aminophenol (APAP; acetaminophen, paracetamol), are widely used by pregnant women. Accumulating evidence has indicated that these molecules can favor genital malformations in newborn boys and reproductive disorders in adults. However, the consequences on postnatal testis development and adult reproductive health after exposure during early embryogenesis are still unknown. Using the mouse model, we show that in utero exposure to therapeutic doses of the widely used APAP-ibuprofen combination during the sex determination period leads to early differentiation and decreased proliferation of male embryonic germ cells, and early 5-methylcytosine and extracellular matrix protein deposition in 13.5 d postcoitum exposed testes. Consequently, in postnatal testes, Sertoli-cell maturation is delayed, the Leydig-cell compartment is hyperplasic, and the spermatogonia A pool is decreased. This results in a reduced production of testosterone and in epididymal sperm parameter defects. We observed a reduced sperm count (19%) in utero-exposed (F0) adult males and also a reduced sperm motility (40%) in their offspring (F1) when both parents were exposed, which leads to subfertility among the 6 mo old F1 animals. Our study suggests that the use of these drugs during the critical period of sex determination affects the germ-line development and leads to adverse effects that could be passed to the offspring.-Rossitto, M., Marchive, C., Pruvost, A., Sellem, E., Ghettas, A., Badiou, S., Sutra, T., Poulat, F., Philibert, P., Boizet-Bonhoure, B. Intergenerational effects on mouse sperm quality after in utero exposure to acetaminophen and ibuprofen.
Assuntos
Acetaminofen/toxicidade , Ibuprofeno/toxicidade , Efeitos Tardios da Exposição Pré-Natal/patologia , Motilidade dos Espermatozoides/efeitos dos fármacos , Espermatozoides/patologia , Testículo/patologia , Analgésicos não Narcóticos/toxicidade , Animais , Anti-Inflamatórios não Esteroides/toxicidade , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/patologia , Células Germinativas Embrionárias/efeitos dos fármacos , Células Germinativas Embrionárias/patologia , Feminino , Masculino , Exposição Materna/efeitos adversos , Camundongos , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Reprodução , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Testículo/efeitos dos fármacos , Testículo/metabolismoRESUMO
Nuclear receptor subfamily 5 group A member 1/Steroidogenic factor 1 (NR5A1; SF-1; Ad4BP) mutations cause 46,XY disorders of sex development (DSD), with phenotypes ranging from developmentally mild (e.g., hypospadias) to severe (e.g., complete gonadal dysgenesis). The molecular mechanism underlying this spectrum is unclear. During sex determination, SF-1 regulates SOX9 (SRY [sex determining region Y]-box 9) expression. We hypothesized that SF-1 mutations in 46,XY DSD patients affect SOX9 expression via the Testis-specific Enhancer of Sox9 core element, TESCO. Our objective was to assess the ability of 20 SF-1 mutants found in 46,XY DSD patients to activate TESCO. Patient DNA was sequenced for SF-1 mutations and mutant SF-1 proteins were examined for transcriptional activity, protein expression, sub-cellular localization and in silico structural defects. Fifteen of the 20 mutants showed reduced SF-1 activation on TESCO, 11 with atypical sub-cellular localization. Fourteen SF-1 mutants were predicted in silico to alter DNA, ligand or cofactor interactions. Our study may implicate aberrant SF-1-mediated transcriptional regulation of SOX9 in 46,XY DSDs.
Assuntos
Transtorno 46,XY do Desenvolvimento Sexual/genética , Elementos Facilitadores Genéticos , Mutação , Fatores de Transcrição SOX9/genética , Fator Esteroidogênico 1/genética , Adolescente , Adulto , Criança , Pré-Escolar , Simulação por Computador , Regulação da Expressão Gênica , Células HEK293 , Humanos , Lactente , Recém-Nascido , Ligantes , Masculino , Ligação Proteica , Análise de Sequência de DNA/métodos , Fator Esteroidogênico 1/química , Fator Esteroidogênico 1/metabolismoRESUMO
Cell fate decisions require appropriate regulation of key genes. Sox9, a direct target of SRY, is pivotal in mammalian sex determination. In vivo high-throughput chromatin accessibility techniques, transgenic assays, and genome editing revealed several novel gonadal regulatory elements in the 2-megabase gene desert upstream of Sox9 Although others are redundant, enhancer 13 (Enh13), a 557-base pair element located 565 kilobases 5' from the transcriptional start site, is essential to initiate mouse testis development; its deletion results in XY females with Sox9 transcript levels equivalent to those in XX gonads. Our data are consistent with the time-sensitive activity of SRY and indicate a strict order of enhancer usage. Enh13 is conserved and embedded within a 32.5-kilobase region whose deletion in humans is associated with XY sex reversal, suggesting that it is also critical in humans.
Assuntos
Elementos Facilitadores Genéticos/genética , Disgenesia Gonadal 46 XY/genética , Fatores de Transcrição SOX9/genética , Processos de Determinação Sexual/genética , Proteína da Região Y Determinante do Sexo/metabolismo , Testículo/embriologia , Animais , Sequência Conservada , Feminino , Humanos , Masculino , Camundongos , Deleção de Sequência , Proteína da Região Y Determinante do Sexo/genética , Sítio de Iniciação de TranscriçãoRESUMO
Complete gonadal dysgenesis (CGD) is characterized by an incomplete differentiation of the genital organs in a patient with a 46,XY karyotype. It is induced by mutations in the sex-determining region Y (SRY) gene which plays a key role in testis-determining pathways. The aim of this study was to investigate the possible pathogenic nature of a novel SRY mutation (p.Y127H) identified in a 46,XY female patient. To determine the effect of this mutation on SRY function, we studied its impact on DNA interaction by electrophoretic mobility shift assays. Since tyrosine 127 is close to the C-terminal nuclear localization signal of SRY, we conducted HA-SRY protein expression to observe the impact of the mutation on the nuclear import function in transfected cells. Our results showed that the Y127H mutation nearly abolishes the DNA-binding capacity of SRY and strongly impairs the nuclear localization of the mutated protein. Together with a previously described mutation analyzed in parallel in this paper (p.Y127C), our results highlight this tyrosine residue as a crucial structural determinant of the high mobility group box domain. This is the first report to explain the molecular mechanism of the Y127H mutation causing sex reversal and gives new insights for clinical practice to benefit patients with disorders of sex development.
Assuntos
Códon/genética , Disgenesia Gonadal 46 XY/genética , Disgenesia Gonadal/genética , Mutação/genética , Proteína da Região Y Determinante do Sexo/genética , Adolescente , Sequência de Bases , DNA/metabolismo , Análise Mutacional de DNA , Feminino , Células HEK293 , Humanos , Sinais de Localização Nuclear/genética , Tirosina/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
Cell lineages of the early human gonad commit to one of the two mutually antagonistic organogenetic fates, the testis or the ovary. Some individuals with a 46,XX karyotype develop testes or ovotestes (testicular or ovotesticular disorder of sex development; TDSD/OTDSD), due to the presence of the testis-determining gene, SRY Other rare complex syndromic forms of TDSD/OTDSD are associated with mutations in pro-ovarian genes that repress testis development (e.g. WNT4); however, the genetic cause of the more common non-syndromic forms is unknown. Steroidogenic factor-1 (known as NR5A1) is a key regulator of reproductive development and function. Loss-of-function changes in NR5A1 in 46,XY individuals are associated with a spectrum of phenotypes in humans ranging from a lack of testis formation to male infertility. Mutations in NR5A1 in 46,XX women are associated with primary ovarian insufficiency, which includes a lack of ovary formation, primary and secondary amenorrhoea as well as early menopause. Here, we show that a specific recurrent heterozygous missense mutation (p.Arg92Trp) in the accessory DNA-binding region of NR5A1 is associated with variable degree of testis development in 46,XX children and adults from four unrelated families. Remarkably, in one family a sibling raised as a girl and carrying this NR5A1 mutation was found to have a 46,XY karyotype with partial testicular dysgenesis. These unique findings highlight how a specific variant in a developmental transcription factor can switch organ fate from the ovary to testis in mammals and represents the first missense mutation causing isolated, non-syndromic 46,XX testicular/ovotesticular DSD in humans.
Assuntos
Proteínas de Ligação a DNA/genética , Transtorno 46,XY do Desenvolvimento Sexual/genética , Insuficiência Ovariana Primária/genética , Desenvolvimento Sexual/genética , Fator Esteroidogênico 1/genética , Adulto , Síndrome de Resistência a Andrógenos/genética , Síndrome de Resistência a Andrógenos/patologia , Linhagem da Célula/genética , Criança , Transtorno 46,XY do Desenvolvimento Sexual/patologia , Feminino , Gônadas/crescimento & desenvolvimento , Gônadas/patologia , Humanos , Cariótipo , Masculino , Mutação de Sentido Incorreto , Ovário/crescimento & desenvolvimento , Ovário/patologia , Linhagem , Insuficiência Ovariana Primária/patologia , Processos de Determinação Sexual , Testículo/crescimento & desenvolvimento , Testículo/patologiaRESUMO
Germ cells, the precursors of gametes, represent a unique cell lineage that is able to differentiate into spermatozoa or oocytes depending on the chromosomal sex of the organism. In the mammalian embryonic gonad, commitment to oogenesis involves pre-meiotic DNA replication and entry into the first meiotic division; whereas, commitment to spermatogenesis involves inhibition of meiotic initiation, suppression of pluripotency, mitotic arrest and expression of specific markers that will control the development of the male germ cells. The crucial decision made by the germ line to commit to either a male or a female fate has been partially explained by genetic and ex vivo studies in mice which have implicated a complex network of regulatory genes, numerous factors and pathways. Besides the reproductive failure that may follow a deregulation of this complex network, the germ cells may, in view of their proliferative and pluripotent nature, act as precursors of potential malignant transformation and as putative targets for exogenous environmental compounds. Our review summarizes and discusses recent developments that have improved our understanding on how germ cell precursors are committed to a male or a female cell fate in the mouse gonad.