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1.
Hum Mol Genet ; 29(23): 3781-3792, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33305798

RESUMEN

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.


Asunto(s)
Displasia Campomélica/patología , Trastornos del Desarrollo Sexual/patología , Gónadas/patología , Heterocigoto , Factor de Transcripción SOX9/fisiología , Diferenciación Sexual , Factor Esteroidogénico 1/fisiología , Animales , Displasia Campomélica/etiología , Displasia Campomélica/metabolismo , Modelos Animales de Enfermedad , Trastornos del Desarrollo Sexual/etiología , Trastornos del Desarrollo Sexual/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Gónadas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo
2.
Hum Reprod ; 37(9): 1994-2011, 2022 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-35856159

RESUMEN

STUDY QUESTION: What is the effect of the ketone ß-hydroxybutyrate (ßOHB) on preimplantation mouse embryo development, metabolism, epigenetics and post-transfer viability? SUMMARY ANSWER: In vitro ßOHB exposure at ketogenic diet (KD)-relevant serum concentrations significantly impaired preimplantation mouse embryo development, induced aberrant glycolytic metabolism and reduced post-transfer fetal viability in a sex-specific manner. WHAT IS KNOWN ALREADY: A maternal KD in humans elevates gamete and offspring ßOHB exposure during conception and gestation, and in rodents is associated with an increased time to pregnancy, and altered offspring organogenesis, post-natal growth and behaviour, suggesting a developmental programming effect. In vitro exposure to ßOHB at supraphysiological concentrations (8-80 mM) perturbs preimplantation mouse embryo development. STUDY DESIGN, SIZE, DURATION: A mouse model of embryo development and viability was utilized for this laboratory-based study. Embryo culture media were supplemented with ßOHB at KD-relevant concentrations, and the developmental competence, physiology, epigenetic state and post-transfer viability of in vitro cultured ßOHB-exposed embryos was assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS: Mouse embryos were cultured in vitro with or without ßOHB at concentrations representing serum levels during pregnancy (0.1 mM), standard diet consumption (0.25 mM), KD consumption (2 mM) and diabetic ketoacidosis (4 mM). The impact of ßOHB exposure on embryo development (blastocyst formation rate, morphokinetics and blastocyst total, inner cell mass and trophectoderm (TE) cell number), physiology (redox state, ßOHB metabolism, glycolytic metabolism), epigenetic state (histone 3 lysine 27 ß-hydroxybutyrylation, H3K27bhb) and post-transfer viability (implantation rate, fetal and placental development) was assessed. MAIN RESULTS AND THE ROLE OF CHANCE: All ßOHB concentrations tested slowed embryo development (P < 0.05), and ßOHB at KD-relevant serum levels (2 mM) delayed morphokinetic development, beginning at syngamy (P < 0.05). Compared with unexposed controls, ßOHB exposure reduced blastocyst total and TE cell number (≥0.25 mM; P < 0.05), reduced blastocyst glucose consumption (2 mM; P < 0.01) and increased lactate production (0.25 mM; P < 0.05) and glycolytic flux (0.25 and 2 mM; P < 0.01). Consumption of ßOHB by embryos, mediated via monocarboxylate transporters, was detected throughout preimplantation development. Supraphysiological (20 mM; P < 0.001), but not physiological (0.25-4 mM) ßOHB elevated H3K27bhb levels. Preimplantation ßOHB exposure at serum KD levels (2 mM) reduced post-transfer viability. Implantation and fetal development rates of ßOHB-treated embryos were 50% lower than controls (P < 0.05), and resultant fetuses had a shorter crown-rump length (P < 0.01) and placental diameter (P < 0.05). A strong sex-specific effect of ßOHB was detected, whereby female fetuses from ßOHB-treated embryos weighed less (P < 0.05), had a shorter crown-rump length (P < 0.05), and tended to have accelerated ear development (P < 0.08) compared with female control fetuses. LIMITATIONS, REASONS FOR CAUTION: This study only assessed embryo development, physiology and viability in a mouse model utilizing in vitro ßOHB exposure; the impact of in vivo exposure was not assessed. The concentrations of ßOHB utilized were modelled on blood/serum levels as the true oviduct and uterine concentrations are currently unknown. WIDER IMPLICATIONS OF THE FINDINGS: These findings indicate that the development, physiology and viability of mouse embryos is detrimentally impacted by preimplantation exposure to ßOHB within a physiological range. Maternal diets which increase ßOHB levels, such as a KD, may affect preimplantation embryo development and may therefore impair subsequent viability and long-term health. Consequently, our initial observations warrant follow-up studies in larger human populations. Furthermore, analysis of ßOHB concentrations within human and rodent oviduct and uterine fluid under different nutritional states is also required. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by the University of Melbourne and the Norma Hilda Schuster (nee Swift) Scholarship. The authors have no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.


Asunto(s)
Técnicas de Cultivo de Embriones , Placenta , Ácido 3-Hidroxibutírico/metabolismo , Ácido 3-Hidroxibutírico/farmacología , Animales , Blastocisto/metabolismo , Modelos Animales de Enfermedad , Técnicas de Cultivo de Embriones/métodos , Desarrollo Embrionario/fisiología , Femenino , Humanos , Masculino , Ratones , Embarazo
3.
PLoS Biol ; 17(4): e3000185, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30947255

RESUMEN

Dmrt1 is a highly conserved transcription factor, which is critically involved in regulation of gonad development of vertebrates. In medaka, a duplicate of dmrt1-acting as master sex-determining gene-has a tightly timely and spatially controlled gonadal expression pattern. In addition to transcriptional regulation, a sequence motif in the 3' UTR (D3U-box) mediates transcript stability of dmrt1 mRNAs from medaka and other vertebrates. We show here that in medaka, two RNA-binding proteins with antagonizing properties target this D3U-box, promoting either RNA stabilization in germ cells or degradation in the soma. The D3U-box is also conserved in other germ-cell transcripts, making them responsive to the same RNA binding proteins. The evolutionary conservation of the D3U-box motif within dmrt1 genes of metazoans-together with preserved expression patterns of the targeting RNA binding proteins in subsets of germ cells-suggest that this new mechanism for controlling RNA stability is not restricted to fishes but might also apply to other vertebrates.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Oryzias/genética , Procesos de Determinación del Sexo/genética , Regiones no Traducidas 3'/genética , Animales , Evolución Biológica , Femenino , Proteínas de Peces/genética , Células Germinativas/metabolismo , Masculino , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vertebrados/metabolismo
4.
PLoS Genet ; 14(8): e1007488, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-30071018

RESUMEN

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.


Asunto(s)
Células de la Granulosa/fisiología , Proteínas de Homeodominio/fisiología , Factores de Transcripción/fisiología , Animales , Comunicación Celular , Conexinas/genética , Conexinas/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Germinativas , Proteínas de Homeodominio/genética , Ratones , Ratones Desnudos , Oocitos/fisiología , Factores de Transcripción/genética
5.
BMC Biol ; 16(1): 16, 2018 01 29.
Artículo en Inglés | MEDLINE | ID: mdl-29378592

RESUMEN

BACKGROUND: Sex determination relies on a hierarchically structured network of genes, and is one of the most plastic processes in evolution. The evolution of sex-determining genes within a network, by neo- or sub-functionalization, also requires the regulatory landscape to be rewired to accommodate these novel gene functions. We previously showed that in medaka fish, the regulatory landscape of the master male-determining gene dmrt1bY underwent a profound rearrangement, concomitantly with acquiring a dominant position within the sex-determining network. This rewiring was brought about by the exaptation of a transposable element (TE) called Izanagi, which is co-opted to act as a silencer to turn off the dmrt1bY gene after it performed its function in sex determination. RESULTS: We now show that a second TE, Rex1, has been incorporated into Izanagi. The insertion of Rex1 brought in a preformed regulatory element for the transcription factor Sox5, which here functions in establishing the temporal and cell-type-specific expression pattern of dmrt1bY. Mutant analysis demonstrates the importance of Sox5 in the gonadal development of medaka, and possibly in mice, in a dmrt1bY-independent manner. Moreover, Sox5 medaka mutants have complete female-to-male sex reversal. CONCLUSIONS: Our work reveals an unexpected complexity in TE-mediated transcriptional rewiring, with the exaptation of a second TE into a network already rewired by a TE. We also show a dual role for Sox5 during sex determination: first, as an evolutionarily conserved regulator of germ-cell number in medaka, and second, by de novo regulation of dmrt1 transcriptional activity during primary sex determination due to exaptation of the Rex1 transposable element.


Asunto(s)
Elementos Transponibles de ADN/fisiología , Células Germinativas/metabolismo , Factores de Transcripción SOXD/biosíntesis , Cromosomas Sexuales/metabolismo , Procesos de Determinación del Sexo/fisiología , Animales , Animales Modificados Genéticamente , Femenino , Masculino , Oryzias , Factores de Transcripción SOXD/genética , Cromosomas Sexuales/genética
6.
Dev Biol ; 412(2): 250-60, 2016 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-26939755

RESUMEN

Sexual development is initiated through differentiation of testicular Sertoli cells or ovarian granulosa cells. Although these supporting cells are considered to develop from common bipotential precursors, recent evidence suggests that distinct supporting cell populations are present in the ovary, with one providing granulosa cells of the medullary follicles and the other providing granulosa cells of the cortical follicles, the latter of which support lifelong fertility. Here, we demonstrate that XX fetal gonads contain GATA4 expressing supporting cells that either enter mitotic arrest, or remain proliferative. Blocking WNT signalling reduces XX supporting cell proliferation, while stabilising ß-catenin signalling promotes proliferation, indicating that the renewal of pre-granulosa cells is dependent on WNT/ß-catenin signalling in the proliferative supporting cell population. In contrast, XX supporting cells express p27 and FOXL2 and are maintained in mitotic arrest. Although FOXL2 is required for maintaining high levels of p27 expression, it is dispensable for entry and maintenance of mitotic arrest in XX supporting cells. Combined our data suggest that both medullary and cortical precursors arise from a common GATA4 expressing cell type. In addition, this work indicates that a balance between supporting cell self-renewal and differentiation is maintained in the developing ovary by relative WNT/ß-catenin and p27/FOXL2 activities. This study provides significant new insights into the origin and formation of ovarian follicles and evidence supporting a common fetal origin of medullary and cortical granulosa cells.


Asunto(s)
Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Factores de Transcripción Forkhead/metabolismo , Ovario/metabolismo , Proteína Wnt4/metabolismo , beta Catenina/metabolismo , Animales , Puntos de Control del Ciclo Celular , Diferenciación Celular , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteína Forkhead Box L2 , Factores de Transcripción Forkhead/genética , Células de la Granulosa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones de la Cepa 129 , Ratones Noqueados , Ratones Transgénicos , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Ovario/citología , Ovario/embriología
7.
PLoS Genet ; 10(5): e1004340, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24784881

RESUMEN

The Hedgehog (Hh) family of secreted proteins act as morphogens to control embryonic patterning and development in a variety of organ systems. Post-translational covalent attachment of cholesterol and palmitate to Hh proteins are critical for multimerization and long range signaling potency. However, the biological impact of lipid modifications on Hh ligand distribution and signal reception in humans remains unclear. In the present study, we report a unique case of autosomal recessive syndromic 46,XY Disorder of Sex Development (DSD) with testicular dysgenesis and chondrodysplasia resulting from a homozygous G287V missense mutation in the hedgehog acyl-transferase (HHAT) gene. This mutation occurred in the conserved membrane bound O-acyltransferase (MBOAT) domain and experimentally disrupted the ability of HHAT to palmitoylate Hh proteins such as DHH and SHH. Consistent with the patient phenotype, HHAT was found to be expressed in the somatic cells of both XX and XY gonads at the time of sex determination, and Hhat loss of function in mice recapitulates most of the testicular, skeletal, neuronal and growth defects observed in humans. In the developing testis, HHAT is not required for Sertoli cell commitment but plays a role in proper testis cord formation and the differentiation of fetal Leydig cells. Altogether, these results shed new light on the mechanisms of action of Hh proteins. Furthermore, they provide the first clinical evidence of the essential role played by lipid modification of Hh proteins in human testicular organogenesis and embryonic development.


Asunto(s)
Aciltransferasas/genética , Trastorno del Desarrollo Sexual 46,XY/genética , Proteínas Hedgehog/metabolismo , Lipoilación/genética , Mutación Missense , Transducción de Señal/genética , Aciltransferasas/química , Aciltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Homocigoto , Humanos , Masculino , Ratones , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de Aminoácido , Testículo/embriología
8.
Dev Biol ; 404(2): 88-102, 2015 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-26116176

RESUMEN

ROBO2 plays a key role in regulating ureteric bud (UB) formation in the embryo, with mutations in humans and mice leading to supernumerary kidneys. Previous studies have established that the number and position of UB outgrowths is determined by the domain of metanephric mesenchymal Gdnf expression, which is expanded anteriorly in Robo2 mouse mutants. To clarify how this phenotype arises, we used high-resolution 3D imaging to reveal an increase in the number of nephrogenic cord cells, leading to extension of the metanephric mesenchyme field in Robo2-null mouse embryos. Ex vivo experiments suggested a dependence of this effect on proliferative signals from the Wolffian duct. Loss of Robo2 resulted in a failure of the normal separation of the mesenchyme from the Wolffian duct/ureteric epithelium, suggesting that aberrant juxtaposition of these two compartments in Robo2-null mice exposes the mesenchyme to abnormally high levels of proliferative stimuli. Our data suggest a new model in which SLIT-ROBO signalling acts not by attenuating Gdnf expression or activity, but instead by limiting epithelial/mesenchymal interactions in the nascent metanephros and restricting the extent of the nephrogenic field. These insights illuminate the aetiology of multiplex kidney formation in human individuals with ROBO2 mutations.


Asunto(s)
Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Proteínas de Homeodominio/genética , Riñón/embriología , Receptores Inmunológicos/genética , Factores de Transcripción/genética , Conductos Mesonéfricos/embriología , Animales , Línea Celular , Proliferación Celular , Células Epiteliales/citología , Factor Neurotrófico Derivado de la Línea Celular Glial/biosíntesis , Proteínas de Homeodominio/biosíntesis , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Mesodermo/citología , Mesodermo/embriología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas/genética , Receptores Inmunológicos/metabolismo , Factores de Transcripción/biosíntesis
9.
Adv Exp Med Biol ; 886: 13-32, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-26659485

RESUMEN

For many years the main role of RNA, it addition to the housekeeping functions of for example tRNAs and rRNAs, was believed to be a messenger between the genes encoded on the DNA and the functional units of the cell, the proteins. This changed drastically with the identification of the first small non-coding RNA, termed microRNA, some 20 years ago. This discovery opened the field of regulatory RNAs with no or little protein-coding potential. Since then many new classes of regulatory non-coding RNAs, including endogenous small interfering RNAs (endo-siRNAs), PIWI-associated RNAs (piRNAs), and long non-coding RNAs, have been identified and we have made amazing progress in elucidating their expression, biogenesis, mechanisms and mode of action, and function in many, if not all, biological processes. In this chapter we provide an introduction about the current knowledge of the main classes of non-coding RNAs, what is know about their biogenesis and mechanism of function.


Asunto(s)
MicroARNs/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Humanos , MicroARNs/genética , ARN Interferente Pequeño/genética
10.
PLoS Genet ; 9(1): e1003160, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23300479

RESUMEN

Mouse sex determination provides an attractive model to study how regulatory genetic networks and signaling pathways control cell specification and cell fate decisions. This study characterizes in detail the essential role played by the insulin receptor (INSR) and the IGF type I receptor (IGF1R) in adrenogenital development and primary sex determination. Constitutive ablation of insulin/IGF signaling pathway led to reduced proliferation rate of somatic progenitor cells in both XX and XY gonads prior to sex determination together with the downregulation of hundreds of genes associated with the adrenal, testicular, and ovarian genetic programs. These findings indicate that prior to sex determination somatic progenitors in Insr;Igf1r mutant gonads are not lineage primed and thus incapable of upregulating/repressing the male and female genetic programs required for cell fate restriction. In consequence, embryos lacking functional insulin/IGF signaling exhibit (i) complete agenesis of the adrenal cortex, (ii) embryonic XY gonadal sex reversal, with a delay of Sry upregulation and the subsequent failure of the testicular genetic program, and (iii) a delay in ovarian differentiation so that Insr;Igf1r mutant gonads, irrespective of genetic sex, remained in an extended undifferentiated state, before the ovarian differentiation program ultimately is initiated at around E16.5.


Asunto(s)
Gónadas , Insulina , Receptor IGF Tipo 1 , Receptor de Insulina , Procesos de Determinación del Sexo/genética , Corteza Suprarrenal/crecimiento & desarrollo , Corteza Suprarrenal/patología , Glándulas Suprarrenales/crecimiento & desarrollo , Glándulas Suprarrenales/metabolismo , Animales , Diferenciación Celular/genética , Linaje de la Célula , Proliferación Celular , Trastornos del Desarrollo Sexual/genética , Femenino , Gónadas/crecimiento & desarrollo , Gónadas/metabolismo , Gónadas/patología , Humanos , Insulina/genética , Insulina/metabolismo , Masculino , Ratones , Ovario/crecimiento & desarrollo , Ovario/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Cromosomas Sexuales , Transducción de Señal , Testículo/crecimiento & desarrollo , Testículo/metabolismo
11.
Dev Biol ; 394(2): 242-52, 2014 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-25158167

RESUMEN

The two main functions of the ovary are the production of oocytes, which allows the continuation of the species, and secretion of female sex hormones, which control many aspects of female development and physiology. Normal development of the ovaries during embryogenesis is critical for their function and the health of the individual in later life. Although the adult ovary has been investigated in great detail, we are only starting to understand the cellular and molecular biology of early ovarian development. Here we show that the adult stem cell marker Lgr5 is expressed in the cortical region of the fetal ovary and this expression is mutually exclusive to FOXL2. Strikingly, a third somatic cell population can be identified, marked by the expression of NR2F2, which is expressed in LGR5- and FOXL2 double-negative ovarian somatic cells. Together, these three marker genes label distinct ovarian somatic cell types. Using lineage tracing in mice, we show that Lgr5-positive cells give rise to adult cortical granulosa cells, which form the follicles of the definitive reserve. Moreover, LGR5 is required for correct timing of germ cell differentiation as evidenced by a delay of entry into meiosis in Lgr5 loss-of-function mutants, demonstrating a key role for LGR5 in the differentiation of pre-granulosa cells, which ensure the differentiation of oogonia, the formation of the definitive follicle reserve, and long-term female fertility.


Asunto(s)
Feto/citología , Regulación del Desarrollo de la Expresión Génica/fisiología , Marcadores Genéticos/genética , Ovario/citología , Ovario/embriología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Factor de Transcripción COUP II/metabolismo , Linaje de la Célula/fisiología , Femenino , Técnica del Anticuerpo Fluorescente , Proteína Forkhead Box L2 , Factores de Transcripción Forkhead/metabolismo , Hibridación in Situ , Ratones , Ratones Endogámicos C57BL , Ovario/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa
12.
Reproduction ; 150(3): R93-107, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25995439

RESUMEN

A complex network of gene regulation and interaction drives male sex determination and differentiation. While many important protein-coding genes that are necessary for proper male development have been identified, many disorders in human sex development are still unexplained at the molecular level. This suggests that key factors and regulatory mechanisms are still unknown. In recent years, extensive data have shown that different classes of non-coding RNAs (ncRNAs) play a role in almost all developmental and physiological pathways. Here we review what is known about their role in male sex determination and differentiation not only in mammals, but also other species. While for some processes a key role for ncRNA has been identified, we are still far from having a complete picture.


Asunto(s)
ARN no Traducido/metabolismo , Procesos de Determinación del Sexo/genética , Espermatogénesis/genética , Animales , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Genes sry , Humanos , Masculino , Morfogénesis , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN no Traducido/genética , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Espermatozoides/metabolismo , Testículo/embriología , Testículo/metabolismo , Cromosoma Y
13.
Nature ; 456(7222): 643-7, 2008 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-18931657

RESUMEN

The lymphatic system plays a key role in tissue fluid regulation and tumour metastasis, and lymphatic defects underlie many pathological states including lymphoedema, lymphangiectasia, lymphangioma and lymphatic dysplasia. However, the origins of the lymphatic system in the embryo, and the mechanisms that direct growth of the network of lymphatic vessels, remain unclear. Lymphatic vessels are thought to arise from endothelial precursor cells budding from the cardinal vein under the influence of the lymphatic hallmark gene Prox1 (prospero homeobox 1; ref. 4). Defects in the transcription factor gene SOX18 (SRY (sex determining region Y) box 18) cause lymphatic dysfunction in the human syndrome hypotrichosis-lymphoedema-telangiectasia, suggesting that Sox18 may also play a role in lymphatic development or function. Here we use molecular, cellular and genetic assays in mice to show that Sox18 acts as a molecular switch to induce differentiation of lymphatic endothelial cells. Sox18 is expressed in a subset of cardinal vein cells that later co-express Prox1 and migrate to form lymphatic vessels. Sox18 directly activates Prox1 transcription by binding to its proximal promoter. Overexpression of Sox18 in blood vascular endothelial cells induces them to express Prox1 and other lymphatic endothelial markers, while Sox18-null embryos show a complete blockade of lymphatic endothelial cell differentiation from the cardinal vein. Our findings demonstrate a critical role for Sox18 in developmental lymphangiogenesis, and suggest new avenues to investigate for therapeutic management of human lymphangiopathies.


Asunto(s)
Diferenciación Celular , Vasos Linfáticos/citología , Vasos Linfáticos/embriología , Factores de Transcripción SOXF/metabolismo , Animales , Biomarcadores/análisis , Movimiento Celular , Células Cultivadas , Edema/genética , Células Endoteliales/citología , Células Endoteliales/metabolismo , Efrina-B2/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Hipotricosis/genética , Linfangiogénesis , Vasos Linfáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Regiones Promotoras Genéticas/genética , Factores de Transcripción SOXF/deficiencia , Factores de Transcripción SOXF/genética , Telangiectasia/genética , Proteínas Supresoras de Tumor/genética , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genética , Venas/citología
14.
Genes (Basel) ; 15(3)2024 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-38540391

RESUMEN

Disruption of meiosis and DNA repair genes is associated with female fertility disorders like premature ovarian insufficiency (POI). In this study, we identified a homozygous missense variant in the HELQ gene (c.596 A>C; p.Gln199Pro) through whole exome sequencing in a POI patient, a condition associated with disrupted ovarian function and female infertility. HELQ, an enzyme involved in DNA repair, plays a crucial role in repairing DNA cross-links and has been linked to germ cell maintenance, fertility, and tumour suppression in mice. To explore the potential association of the HELQ variant with POI, we used CRISPR/Cas9 to create a knock-in mouse model harbouring the equivalent of the human HELQ variant identified in the POI patient. Surprisingly, Helq knock-in mice showed no discernible phenotype, with fertility levels, histological features, and follicle development similar to wild-type mice. Despite the lack of observable effects in mice, the potential role of HELQ in human fertility, especially in the context of POI, should not be dismissed. Larger studies encompassing diverse ethnic populations and alternative functional approaches will be necessary to further examine the role of HELQ in POI. Our results underscore the potential uncertainties associated with genomic variants and the limitations of in vivo animal modelling.


Asunto(s)
Infertilidad Femenina , Insuficiencia Ovárica Primaria , Animales , Femenino , Humanos , Ratones , ADN Helicasas/genética , Homocigoto , Infertilidad Femenina/genética , Mutación Missense , Insuficiencia Ovárica Primaria/genética
15.
Biol Reprod ; 89(2): 34, 2013 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-23843232

RESUMEN

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.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , MicroARNs/metabolismo , Organogénesis/genética , Factor de Transcripción SOX9/metabolismo , Testículo/embriología , Animales , Diferenciación Celular/genética , Masculino , Ratones , Ratones Noqueados , MicroARNs/genética , Regiones Promotoras Genéticas , Factor de Transcripción SOX9/genética , Células de Sertoli/citología , Células de Sertoli/metabolismo , Diferenciación Sexual/genética , Testículo/citología , Testículo/metabolismo , Transcripción Genética
16.
Biol Reprod ; 88(6): 143, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23616593

RESUMEN

MicroRNAs (miRNAs) have been shown to play key regulatory roles in a range of biological processes, including cell differentiation and development. To identify miRNAs that participate in gonad differentiation, a fundamental and tightly regulated developmental process, we examined miRNA expression profiles at the time of sex determination and during the early fetal differentiation of mouse testes and ovaries using high-throughput sequencing. We identified several miRNAs that were expressed in a sexually dimorphic pattern, including several members of the let-7 family, miR-378, and miR-140-3p. We focused our analysis on the most highly expressed, sexually dimorphic miRNA, miR-140-3p, and found that both miR-140-3p and its more lowly expressed counterpart, the previously annotated guide strand, miR-140-5p, are testis enriched and expressed in testis cords. Analysis of the miR-140-5p/miR-140-3p-null mouse revealed a significant increase in the number of Leydig cells in the developing XY gonad, strongly suggesting an important role for miR-140-5p/miR-140-3p in testis differentiation in mouse.


Asunto(s)
Diferenciación Celular/genética , Células Intersticiales del Testículo/citología , MicroARNs/metabolismo , Testículo/citología , Animales , Recuento de Células , Células Intersticiales del Testículo/metabolismo , Masculino , Ratones , Ratones Noqueados , MicroARNs/genética , Testículo/embriología , Testículo/metabolismo
17.
Bioinformatics ; 28(23): 3042-50, 2012 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-23044541

RESUMEN

MOTIVATION: Comparing transcriptomic data with proteomic data to identify protein-coding sequences is a long-standing challenge in molecular biology, one that is exacerbated by the increasing size of high-throughput datasets. To address this challenge, and thereby to improve the quality of genome annotation and understanding of genome biology, we have developed an integrated suite of programs, called Pinstripe. We demonstrate its application, utility and discovery power using transcriptomic and proteomic data from publicly available datasets. RESULTS: To demonstrate the efficacy of Pinstripe for large-scale analysis, we applied Pinstripe's reverse peptide mapping pipeline to a transcript library including de novo assembled transcriptomes from the human Illumina Body Atlas (IBA2) and GENCODE v10 gene annotations, and the EBI Proteomics Identifications Database (PRIDE) peptide database. This analysis identified 736 canonical open reading frames (ORFs) supported by three or more PRIDE peptide fragments that are positioned outside any known coding DNA sequence (CDS). Because of the unfiltered nature of the PRIDE database and high probability of false discovery, we further refined this list using independent evidence for translation, including the presence of a Kozak sequence or functional domains, synonymous/non-synonymous substitution ratios and ORF length. Using this integrative approach, we observed evidence of translation from a previously unknown let7e primary transcript, the archetypical lncRNA H19, and a homolog of RD3. Reciprocally, by exclusion of transcripts with mapped peptides or significant ORFs (>80 codon), we identify 32 187 loci with RNAs longer than 2000 nt that are unlikely to encode proteins. AVAILABILITY AND IMPLEMENTATION: Pinstripe (pinstripe.matticklab.com) is freely available as source code or a Mono binary. Pinstripe is written in C# and runs under the Mono framework on Linux or Mac OS X, and both under Mono and .Net under Windows. CONTACT: m.dinger@garvan.org.au or j.mattick@garvan.org.au SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Genómica/métodos , Proteómica/métodos , Programas Informáticos , Biología Computacional/métodos , Bases de Datos de Proteínas , Exones , Biblioteca de Genes , Genoma , Humanos , Anotación de Secuencia Molecular , Sistemas de Lectura Abierta , Proteínas/genética , ARN Largo no Codificante/genética , ARN Mensajero/genética , Análisis de Secuencia de ARN
18.
Nucleic Acids Res ; 39(6): 2393-403, 2011 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21075793

RESUMEN

The 3' untranslated regions (3'UTRs) of eukaryotic genes regulate mRNA stability, localization and translation. Here, we present evidence that large numbers of 3'UTRs in human, mouse and fly are also expressed separately from the associated protein-coding sequences to which they are normally linked, likely by post-transcriptional cleavage. Analysis of CAGE (capped analysis of gene expression), SAGE (serial analysis of gene expression) and cDNA libraries, as well as microarray expression profiles, demonstrate that the independent expression of 3'UTRs is a regulated and conserved genome-wide phenomenon. We characterize the expression of several 3'UTR-derived RNAs (uaRNAs) in detail in mouse embryos, showing by in situ hybridization that these transcripts are expressed in a cell- and subcellular-specific manner. Our results suggest that 3'UTR sequences can function not only in cis to regulate protein expression, but also intrinsically and independently in trans, likely as noncoding RNAs, a conclusion supported by a number of previous genetic studies. Our findings suggest novel functions for 3'UTRs, as well as caution in the use of 3'UTR sequence probes to analyze gene expression.


Asunto(s)
Regiones no Traducidas 3' , ARN no Traducido/metabolismo , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Desarrollo Embrionario/genética , Exones , Perfilación de la Expresión Génica , Humanos , Ratones , Procesamiento Postranscripcional del ARN
19.
Hum Mol Genet ; 19(3): 506-16, 2010 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-19933217

RESUMEN

Male development in mammals is normally initiated by the Y-linked gene Sry, which activates expression of Sox9, leading to a cascade of gene activity required for testis formation. Although defects in this genetic cascade lead to human disorders of sex development (DSD), only a dozen DSD genes have been identified, and causes of 46,XX DSD (XX maleness) other than SRY translocation are almost completely unknown. Here, we show that transgenic expression of Sox10, a close relative of Sox9, in gonads of XX mice resulted in development of testes and male physiology. The degree of sex reversal correlated with levels of Sox10 expression in different transgenic lines. Sox10 was expressed at low levels in primordial gonads of both sexes during normal mouse development, becoming male-specific during testis differentiation. SOX10 protein was able to activate transcriptional targets of SOX9, explaining at a mechanistic level its ability to direct male development. Because over-expression of SOX10 alone is able to mimic the XX DSD phenotypes associated with duplication of human chromosome 22q13, and given that human SOX10 maps to 22q13.1, our results functionally implicate SOX10 in the etiology of these DSDs.


Asunto(s)
Cromosomas Humanos Par 22/genética , Trastornos del Desarrollo Sexual , Trastornos del Desarrollo Sexual/genética , Trastornos del Desarrollo Sexual/metabolismo , Factores de Transcripción SOXE/metabolismo , Animales , Diferenciación Celular , Modelos Animales de Enfermedad , Trastornos del Desarrollo Sexual/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Masculino , Ratones , Ratones Transgénicos , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Factores de Transcripción SOXE/genética , Testículo/embriología , Testículo/metabolismo
20.
PLoS Biol ; 7(9): e1000196, 2009 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-19753101

RESUMEN

Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY) gonad, sex-determining region of the Y (SRY) protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK) signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg) mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4), a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas). These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and create a novel entry point into the molecular and cellular mechanisms underlying sex determination in mice and disorders of sexual development in humans.


Asunto(s)
MAP Quinasa Quinasa Quinasa 4/deficiencia , Sistema de Señalización de MAP Quinasas , Procesos de Determinación del Sexo , Animales , Trastornos del Desarrollo Sexual , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , MAP Quinasa Quinasa Quinasa 4/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ovario/citología , Ovario/embriología , Mutación Puntual , Proteína de la Región Y Determinante del Sexo/genética , Proteína de la Región Y Determinante del Sexo/metabolismo , Testículo/citología , Testículo/embriología
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