RESUMEN
Sexual reproduction crucially depends on the production of sperm in males and oocytes in females. Both types of gamete arise from the same precursor, the germ cells. We review the events that characterize the development of germ cells during fetal life as they commit to, and prepare for, oogenesis or spermatogenesis. In females, fetal germ cells enter meiosis, whereas in males they delay meiosis and instead lose pluripotency, activate an irreversible program of prospermatogonial differentiation, and temporarily cease dividing. Both pathways involve sex-specific molecular signals from the somatic cells of the developing gonads and a suite of intrinsic receptors, signal transducers, transcription factors, RNA stability factors, and epigenetic modulators that act in complex, interconnected positive and negative regulatory networks. Understanding these networks is important in the contexts of the etiology, diagnosis, and treatment of infertility and gonadal cancers, and in efforts to augment human and animal fertility using stem cell approaches.
Asunto(s)
Infertilidad Femenina/genética , Infertilidad Masculina/genética , Oogénesis/genética , Procesos de Determinación del Sexo , Diferenciación Sexual/genética , Espermatogénesis/genética , Animales , Femenino , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Humanos , Infertilidad Femenina/metabolismo , Infertilidad Femenina/patología , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Meiosis , Oocitos/citología , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Óvulo/citología , Óvulo/crecimiento & desarrollo , Óvulo/metabolismo , Transducción de Señal , Espermatozoides/citología , Espermatozoides/crecimiento & desarrollo , Espermatozoides/metabolismoRESUMEN
The genetic regulation of ovarian development remains largely unclear. Indeed, in most cases of impaired ovarian development-such as 46,XX disorders of sex development (DSD) without SRY, and premature ovarian insufficiency (POI)-the genetic causes have not been identified, and the vast majority of disease-associated sequence variants could lie within non-coding regulatory sequences. In this study, we aimed to identify enhancers of five ovarian genes known to play key roles in early ovarian development, basing our analysis on the expression of enhancer derived transcripts (eRNAs), which are considered to characterize active enhancers. Temporal expression profile changes in mouse WT1-positive ovarian cells were obtained from cap analysis of gene expression at E13.5, E16.5 and P0. We compared the chronological expression profiles of ovarian-specific eRNA with expression profiles for each of the ovarian-specific genes, yielding two candidate sequences for enhancers of Wnt4 and Rspo1. Both sequences are conserved between mouse and human, and we confirmed their enhancer activities using transient expression assays in murine granulosa cells. Furthermore, by sequencing the region in patients with impaired ovarian development in 24 patients, such as POI, gonadal dysgenesis and 46,XX DSD, we identified rare single nucleotide variants in both sequences. Our results demonstrate that combined analysis of the temporal expression profiles of eRNA and mRNA of target genes presents a powerful tool for locating cis-element enhancers, and a means of identifying disease-associated sequence variants that lie within non-coding regulatory sequences, thus advancing an important unmet need in forward human genetics.
Asunto(s)
Menopausia Prematura , Insuficiencia Ovárica Primaria , Animales , Elementos de Facilitación Genéticos/genética , Femenino , Variación Genética , Humanos , Menopausia Prematura/genética , Ratones , Insuficiencia Ovárica Primaria/genética , Insuficiencia Ovárica Primaria/metabolismo , ARN/genética , Factores de TiempoRESUMEN
In mice, the entry of germ cells into meiosis crucially depends on the expression of stimulated by retinoic acid gene 8 (Stra8). Stra8 is expressed specifically in pre-meiotic germ cells of females and males, at fetal and postnatal stages, respectively, but the mechanistic details of its spatiotemporal regulation are yet to be defined. In particular, there has been considerable debate regarding whether retinoic acid is required, in vivo, to initiate Stra8 expression in the mouse fetal ovary. We show that the distinctive anterior-to-posterior pattern of Stra8 initiation, characteristic of germ cells in the fetal ovary, is faithfully recapitulated when 2.9â kb of the Stra8 promoter is used to drive eGFP expression. Using in vitro transfection assays of cutdown and mutant constructs, we identified two functional retinoic acid responsive elements (RAREs) within this 2.9â kb regulatory element. We also show that the transcription factor DMRT1 enhances Stra8 expression, but only in the presence of RA and the most proximal RARE. Finally, we used CRISPR/Cas9-mediated targeted mutation studies to demonstrate that both RAREs are required for optimal Stra8 expression levels in vivo.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células Germinativas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Sitios de Unión , Sistemas CRISPR-Cas/genética , Femenino , Desarrollo Fetal/genética , Feto/citología , Feto/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células Germinativas/citología , Meiosis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutagénesis , Ovario/citología , Ovario/metabolismo , Regiones Promotoras Genéticas , Receptores X Retinoide/genética , Receptores X Retinoide/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/farmacología , Tretinoina/farmacologíaRESUMEN
Germline-specific Cre lines are useful for analyses of primordial germ cell, spermatogonial and oogonial development, but also for whole-body deletions when transmitted through subsequent generations. Several germ cell specific Cre mouse strains exist, with various degrees of specificity, efficiency, and temporal activation. Here, we describe the CRISPR/Cas9 targeted insertion of an improved Cre (iCre) sequence in-frame at the 3' end of the Ddx4 locus to generate the Ddx4-P2A-iCre allele. Our functional assessment of this new allele, designated Ddx4iCreJoBo , reveals that Cre activity begins in PGCs from at least E10.5, and that it achieves higher efficiency for early gonadal (E10.5-12.5) germline deletion when compared to the inducible Oct4CreERT2 line. We found the Ddx4iCreJoBo allele to be hypomorphic for Ddx4 expression and homozygous males, but not females, were infertile. Using two reporter lines (R26RLacZ and R26RtdTomato ) and a floxed gene of interest (Criptoflox ) we found ectopic activity in multiple organs; global recombination (a common feature of germline Cre alleles) varies from 10 to 100%, depending on the particular floxed allele. There is a strong maternal effect, and therefore it is preferable for Ddx4iCreJoBo to be inherited from the male parent if ubiquitous deletion is not desired. With these limitations considered, we describe the Ddx4iCreJoBo line as useful for germline studies in which early gonadal deletion is required.
Asunto(s)
Células Germinativas , Integrasas , Animales , Masculino , Ratones , Animales Modificados Genéticamente , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Células Germinativas/metabolismo , Integrasas/genética , Integrasas/metabolismo , Ratones TransgénicosRESUMEN
Members of the nuclear factor I (NFI) family are key regulators of stem cell biology during development, with well-documented roles for NFIA, NFIB, and NFIX in a variety of developing tissues, including brain, muscle, and lung. Given the central role these factors play in stem cell biology, we posited that they may be pivotal for spermatogonial stem cells or further developing spermatogonia during testicular development. Surprisingly, in stark contrast to other developing organ systems where NFI members are co-expressed, these NFI family members show discrete patterns of expression within the seminiferous tubules. Sertoli cells (spermatogenic supporting cells) express NFIA, spermatocytes express NFIX, round spermatids express NFIB, and peritubular myoid cells express each of these three family members. Further analysis of NFIX expression during the cycle of the seminiferous epithelium revealed expression not in spermatogonia, as we anticipated, but in spermatocytes. These data suggested a potential role for NFIX in spermatogenesis. To investigate, we analyzed mice with constitutive deletion of Nfix (Nfix-null). Assessment of germ cells in the postnatal day 20 (P20) testes of Nfix-null mice revealed that spermatocytes initiate meiosis, but zygotene stage spermatocytes display structural defects in the synaptonemal complex, and increased instances of unrepaired DNA double-strand breaks. Many developing spermatocytes in the Nfix-null testis exhibited multinucleation. As a result of these defects, spermatogenesis is blocked at early diplotene and very few round spermatids are produced. Collectively, these novel data establish the global requirement for NFIX in correct meiotic progression during the first wave of spermatogenesis.
Asunto(s)
Factores de Transcripción NFI , Espermatogonias , Testículo , Animales , Masculino , Meiosis , Ratones , Ratones Noqueados , Factores de Transcripción NFI/genética , Factores de Transcripción NFI/metabolismo , Espermatocitos/metabolismo , Espermatogénesis/genética , Testículo/metabolismoRESUMEN
The nuclear receptor NR5A1 is equally expressed and required for development of the gonadal primordia of both sexes, but, after sex determination, it is upregulated in XY testes and downregulated in XX ovaries. We have recently demonstrated, in mice, that this downregulation is mediated by forkhead box L2 (FOXL2) and hypothesized that adequate suppression of Nr5a1 is essential for normal ovarian development. Further, analysis of human patients with disorders/differences of sex development suggests that overexpression of NR5A1 can result in XX (ovo)testicular development. Here, we tested the role of Nr5a1 by overexpression in fetal gonads using a Wt1-BAC (bacterial artificial chromosome) transgene system. Enforced Nr5a1 expression compromised ovarian development in 46,XX mice, resulting in late-onset infertility, but did not induce (ovo)testis differentiation. The phenotype was similar to that of XX mice lacking Notch signaling. The expression level of Notch2 was significantly reduced in Nr5a1 transgenic mice, and the ovarian phenotype was almost completely rescued by in utero treatment with a NOTCH2 agonist. We conclude that suppression of Nr5a1 during the fetal period optimizes ovarian development by fine-tuning Notch signaling.
Asunto(s)
Ovario/fisiología , Receptor Notch2/fisiología , Desarrollo Sexual , Factor Esteroidogénico 1/fisiología , Testículo/fisiología , Animales , Diferenciación Celular , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Transgénicos , Fenotipo , Receptor Notch2/genética , Transducción de Señal , Factor Esteroidogénico 1/genéticaRESUMEN
An adverse outcome pathway (AOP) is a simplified description of the sequence of mechanistic events that lead to a particular toxicological effect, from initial trigger to adverse outcome. Although designed to inform regulatory risk assessors, the AOP framework also provides a platform for innovative collaborations between experts from relevant research fields and the regulatory community. The underpinning for any AOP is basic knowledge about molecular and developmental processes; such knowledge can only be attained by solid bioscientific research. Starting with this fundamental knowledge, the objective is to devise novel testing strategies that focus on key events in a causative pathway. It is anticipated that such a knowledge-based approach will ultimately alleviate many of the burdens associated with classical chemical testing strategies that typically involve large-scale animal toxicity regimens. This hails from the notion that a solid understanding of the underlying mechanisms will allow the development and use of alternative test methods, including both in vitro and in silico approaches. This review is specifically targeted at professionals working in bioscientific fields, such as developmental and reproductive biology, and aims to (i) inform on the existence of the AOP framework and (ii) encourage new cross-disciplinary collaborations. It is hoped that fundamental biological knowledge can thus be better exploited for applied purposes: firstly, an improved understanding of how our perpetual exposure to environmental chemicals is causing human reproductive disease and, secondly, new approaches to screen for harmful chemicals more efficiently. This is not an instructional manual on how to create AOPs; rather, we discuss how to harness fundamental knowledge from the biosciences to assist regulatory toxicologists in their efforts to protect humans against chemicals that harm human reproductive development and function.
Asunto(s)
Rutas de Resultados Adversos , Biología Evolutiva/métodos , Noxas/efectos adversos , Reproducción/efectos de los fármacos , Medicina Reproductiva/métodos , Toxicología/métodos , Canal Anal/embriología , Andrógenos/fisiología , Animales , Disruptores Endocrinos/toxicidad , Genitales/embriología , Humanos , Comunicación Interdisciplinaria , Internet , Modelos Animales , Pezones/embriología , Noxas/toxicidad , Reproducción/fisiología , Tretinoina/toxicidadRESUMEN
Spermatogenesis is a dynamic process involving self-renewal and differentiation of spermatogonial stem cells, meiosis, and ultimately, the differentiation of haploid spermatids into sperm. Centrosomal protein 55 kDa (CEP55) is necessary for somatic cell abscission during cytokinesis. It facilitates equal segregation of cytoplasmic contents between daughter cells by recruiting endosomal sorting complex required for transport machinery (ESCRT) at the midbody. In germ cells, CEP55, in partnership with testes expressed-14 (TEX14) protein, has also been shown to be an integral component of intercellular bridge before meiosis. Various in vitro studies have demonstrated a role for CEP55 in multiple cancers and other diseases. However, its oncogenic potential in vivo remains elusive. To investigate, we generated ubiquitously overexpressing Cep55 transgenic ( Cep55Tg/Tg) mice aiming to characterize its oncogenic role in cancer. Unexpectedly, we found that Cep55Tg/Tg male mice were sterile and had severe and progressive defects in spermatogenesis related to spermatogenic arrest and lack of spermatids in the testes. In this study, we characterized this male-specific phenotype and showed that excessively high levels of Cep55 results in hyperactivation of PI3K/protein kinase B (Akt) signaling in testis. In line with this finding, we observed increased phosphorylation of forkhead box protein O1 (FoxO1), and suppression of its nuclear retention, along with the relative enrichment of promyelocytic leukemia zinc finger (PLZF) -positive cells. Independently, we observed that Cep55 amplification favored upregulation of ret ( Ret) proto-oncogene and glial-derived neurotrophic factor family receptor α-1 ( Gfra1). Consistent with these data, we observed selective down-regulation of genes associated with germ cell differentiation in Cep55-overexpressing testes at postnatal day 10, including early growth response-4 ( Egr4) and spermatogenesis and oogenesis specific basic helix-loop-helix-1 ( Sohlh1). Thus, Cep55 amplification leads to a shift toward the initial maintenance of undifferentiated spermatogonia and ultimately results in progressive germ cell loss. Collectively, our findings demonstrate that Cep55 overexpression causes change in germ cell proportions and manifests as a Sertoli cell only tubule phenotype, similar to that seen in many azoospermic men.-Sinha, D., Kalimutho, M., Bowles, J., Chan, A.-L., Merriner, D. J., Bain, A. L., Simmons, J. L., Freire, R., Lopez, J. A., Hobbs, R. M., O'Bryan, M. K., Khanna, K. K. Cep55 overexpression causes male-specific sterility in mice by suppressing Foxo1 nuclear retention through sustained activation of PI3K/Akt signaling.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteína Forkhead Box O1/metabolismo , Infertilidad Masculina/metabolismo , Transducción de Señal , Espermatogonias/metabolismo , Animales , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores SexualesRESUMEN
KEY POINTS: In the present study, we investigated whether hypoxia during late pregnancy impairs kidney development in mouse offspring, and also whether this has long-lasting consequences affecting kidney function in adulthood. Hypoxia disrupted growth of the kidney, particularly the collecting duct network, in juvenile male offspring. By mid-late adulthood, these mice developed early signs of kidney disease, notably a compromised response to water deprivation. Female offspring showed no obvious signs of impaired kidney development and did not develop kidney disease, suggesting an underlying protection mechanism from the hypoxia insult. These results help us better understand the long-lasting impact of gestational hypoxia on kidney development and the increased risk of chronic kidney disease. ABSTRACT: Prenatal hypoxia is a common perturbation to arise during pregnancy, and can lead to adverse health outcomes in later life. The long-lasting impact of prenatal hypoxia on postnatal kidney development and maturation of the renal tubules, particularly the collecting duct system, is relatively unknown. In the present study, we used a model of moderate chronic maternal hypoxia throughout late gestation (12% O2 exposure from embryonic day 14.5 until birth). Histological analyses revealed marked changes in the tubular architecture of male hypoxia-exposed neonates as early as postnatal day 7, with disrupted medullary development and altered expression of Ctnnb1 and Crabp2 (encoding a retinoic acid binding protein). Kidneys of the RARElacZ line offspring exposed to hypoxia showed reduced ß-galactosidase activity, indicating reduced retinoic acid-directed transcriptional activation. Wild-type male mice exposed to hypoxia had an early decline in urine concentrating capacity, evident at 4 months of age. At 12 months of age, hypoxia-exposed male mice displayed a compromised response to a water deprivation challenge, which was was correlated with an altered cellular composition of the collecting duct and diminished expression of aquaporin 2. There were no differences in the tubular structures or urine concentrating capacity between the control and hypoxia-exposed female offspring at any age. The findings of the present study suggest that prenatal hypoxia selectively disrupts collecting duct patterning through altered Wnt/ß-catenin and retinoic acid signalling and this results in impaired function in male mouse offspring in later life.
Asunto(s)
Hipoxia Fetal/fisiopatología , Túbulos Renales Colectores/fisiopatología , Animales , Animales Recién Nacidos , Femenino , Túbulos Renales Colectores/anatomía & histología , Túbulos Renales Colectores/crecimiento & desarrollo , Masculino , Ratones , Ratones Transgénicos , Embarazo , Factores SexualesRESUMEN
The mammalian sex-determining factor SRY comprises a conserved high-mobility group (HMG) box DNA-binding domain and poorly conserved regions outside the HMG box. Mouse Sry is unusual in that it includes a C-terminal polyglutamine (polyQ) tract that is absent in nonrodent SRY proteins, and yet, paradoxically, is essential for male sex determination. To dissect the molecular functions of this domain, we generated a series of Sry mutants, and studied their biochemical properties in cell lines and transgenic mouse embryos. Sry protein lacking the polyQ domain was unstable, due to proteasomal degradation. Replacing this domain with irrelevant sequences stabilized the protein but failed to restore Sry's ability to up-regulate its key target gene SRY-box 9 (Sox9) and its sex-determining function in vivo. These functions were restored only when a VP16 transactivation domain was substituted. We conclude that the polyQ domain has important roles in protein stabilization and transcriptional activation, both of which are essential for male sex determination in mice. Our data disprove the hypothesis that the conserved HMG box domain is the only functional domain of Sry, and highlight an evolutionary paradox whereby mouse Sry has evolved a novel bifunctional module to activate Sox9 directly, whereas SRY proteins in other taxa, including humans, seem to lack this ability, presumably making them dependent on partner proteins(s) to provide this function.
Asunto(s)
Genes sry , Procesos de Determinación del Sexo , Proteína de la Región Y Determinante del Sexo/genética , Proteína de la Región Y Determinante del Sexo/metabolismo , Animales , Evolución Molecular , Femenino , Genes Reporteros , Masculino , Ratones , Ratones Transgénicos , Mutagénesis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Péptidos/química , Embarazo , Complejo de la Endopetidasa Proteasomal/metabolismo , Desnaturalización Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Eliminación de Secuencia , Proteína de la Región Y Determinante del Sexo/química , Activación TranscripcionalRESUMEN
There are three established techniques described for ex vivo culture of the early embryonic organs: filter culture, agar block culture and hanging drop culture. Each of these protocols has advantages and disadvantages; here we assess the merits of each approach. Agar block culture has a long history and has been well described. This method results in good embryonic organ morphology. Filter culture has been used to culture a number of different embryonic organs and there are a variety of filter choices available. The key disadvantage of agar-block and filter based culture is that the large amount of media required can make the approach expensive, especially if biologicals such as growth factors are necessary; in addition, using these methods it can be difficult to track particular samples. Hanging drop culture is most commonly used to enable the aggregation of embryonic stem cells into embryoid bodies but it has also been employed for ex vivo organ culture. This method requires only 40µL of media per drop and isolates every organ to a trackable unit. We describe each of these methods and the use of different medias and provide the user with a matrix to help determine the optimal culture method for their needs. Glass-based culture methods required for live imaging are not discussed here.
Asunto(s)
Técnicas de Cultivo de Célula , Diferenciación Celular/genética , Células Madre Embrionarias/citología , Técnicas de Cultivo de Órganos/métodos , Animales , Desarrollo Embrionario/genética , RatonesRESUMEN
The technique described in this protocol allows the user to position small tissues in the optimal orientation for paraffin embedding and sectioning by first immobilising the tissue in an agarose/gelatin cube. This method is an adaptation of methods used for early embryos and can be used for any small tissues or embryo segments. Processing of larger tissue sections using molds to create agarose/gelatin blocks has been described previously; this detailed protocol provides a method for dealing with much smaller tissues or embryos (≤5mm). The tissue is briefly fixed then an agarose/gelatin drop is created to surround the tissue. The tissue can be orientated as per the user's preference in the drop before it sets as is carved into a cube with a domed top. The cube is then dehydrated and goes through the embedding and sectioning process. The domed cube is easy to orientate when embedding the tissue in a wax block giving the user assured orientation of the small tissue for sectioning. Additionally, the agarose/gelatin cube is easy to see in the unmolded wax once embedded, making the region of interest easy to identify.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Embrión de Mamíferos , Desarrollo Embrionario/genética , Adhesión en Parafina , Animales , Gelatina/química , Humanos , Ratones , Sefarosa/químicaRESUMEN
BACKGROUND: It is widely accepted that, during the development of testes in the mammalian embryo, male germ cells are influenced by signals from the surrounding somatic cells, but not vice versa, so that germ cells are dispensable for the formation of testes. RESULTS: We now demonstrate that development of the mouse fetal testis is compromised in the absence of germ cells. Using two- and three-dimensional imaging techniques, we reveal that W(e)/W(e) mutant testes devoid of germ cells have misshapen and poorly organized cords. We also found that mutant gonads have fewer Sertoli cells than normal and that the Leydig cells express key markers at higher than normal levels. CONCLUSIONS: These observations point to the existence of germ cell-derived signals that directly or indirectly affect the Sertoli and Leydig cell populations, and provide a new paradigm for the organogenesis of the mammalian testes.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Células Germinativas/metabolismo , Células Intersticiales del Testículo/metabolismo , Cordón Espermático/embriología , Animales , Masculino , Ratones , Ratones TransgénicosRESUMEN
During embryogenesis, lymphatic endothelial progenitor cells first arise from a subset of blood vascular endothelial cells in the dorsolateral aspects of the cardinal veins. The molecular cues responsible for defining the regionalisation of such a discrete pool of progenitors remain uncharacterised. Here we identify a novel function for CYP26B1, an enzyme known to play a role in tissue morphogenesis by fine-tuning retinoic acid (RA) concentration, in regulating lymphangiogenesis. Cyp26b1-null mice, in which RA levels are elevated, exhibited an increased number of lymphatic endothelial progenitor cells in the cardinal veins, together with hyperplastic, blood filled lymph sacs and hyperplastic dermal lymphatic vessels. Conversely, mice over-expressing Cyp26b1 had hypoplastic lymph sacs and lymphatic vessels. Our data suggest that RA clearance by CYP26B1 in the vicinity of lymphatic endothelial progenitor cells is important for determining the position and size of the progenitor pool specified. Our studies identify a genetic pathway that underpins the architecture of the developing lymphatics and define CYP26B1 as a novel modulator of lymphatic vascular patterning.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Regulación del Desarrollo de la Expresión Génica , Linfangiogénesis , Sistema Linfático/embriología , Vasos Linfáticos/metabolismo , Retinoides/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , Cruzamientos Genéticos , Células Endoteliales/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente , Fenotipo , Ácido Retinoico 4-Hidroxilasa , Transducción de Señal , Transgenes , Tretinoina/metabolismoRESUMEN
Germ cells, the embryonic precursors of sperm or oocytes, respond to molecular cues that regulate their sex-specific development in the fetal gonads. In males in particular, the balance between continued proliferation and cell fate commitment is crucial: defects in proliferation result in insufficient spermatogonial stem cells for fertility, but escape from commitment and prolonged pluripotency can cause testicular germ cell tumors. However, the factors that regulate this balance remain unidentified. Here, we show that signaling by the TGFß morphogen Nodal and its co-receptor Cripto is active during a crucial window of male germ cell development. The Nodal pathway is triggered when somatic signals, including FGF9, induce testicular germ cells to upregulate Cripto. Germ cells of mutant mice with compromised Nodal signaling showed premature differentiation, reduced pluripotency marker expression and a reduced ability to form embryonic germ (EG) cell colonies in vitro. Conversely, human testicular tumors showed upregulation of NODAL and CRIPTO that was proportional to invasiveness and to the number of malignant cells. Thus, Nodal signaling provides a molecular control mechanism that regulates male germ cell potency in normal development and testicular cancer.
Asunto(s)
Factor de Crecimiento Epidérmico/metabolismo , Células Germinativas/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína Nodal/metabolismo , Transducción de Señal , Espermatogénesis/fisiología , Espermatogonias/metabolismo , Testículo/embriología , Animales , Diferenciación Celular , Proliferación Celular , Factor 9 de Crecimiento de Fibroblastos/metabolismo , Células Germinativas/citología , Humanos , Masculino , Ratones , Neoplasias de Células Germinales y Embrionarias/metabolismo , Células Madre Pluripotentes/citología , Espermatogonias/citología , Neoplasias Testiculares/metabolismo , Factor de Crecimiento Transformador betaRESUMEN
Male sex determination hinges on the development of testes in the embryo, beginning with the differentiation of Sertoli cells under the influence of the Y-linked gene SRY. Sertoli cells then orchestrate fetal testis formation including the specification of fetal Leydig cells (FLCs) that produce steroid hormones to direct virilization of the XY embryo. As the majority of XY disorders of sex development (DSDs) remain unexplained at the molecular genetic level, we reasoned that genes involved in FLC development might represent an unappreciated source of candidate XY DSD genes. To identify these genes, and to gain a more detailed understanding of the regulatory networks underpinning the specification and differentiation of the FLC population, we developed methods for isolating fetal Sertoli, Leydig, and interstitial cell-enriched subpopulations using an Sf1-eGFP transgenic mouse line. RNA sequencing followed by rigorous bioinformatic filtering identified 84 genes upregulated in FLCs, 704 genes upregulated in nonsteroidogenic interstitial cells, and 1217 genes upregulated in the Sertoli cells at 12.5 days postcoitum. The analysis revealed a trend for expression of components of neuroactive ligand interactions in FLCs and Sertoli cells and identified factors potentially involved in signaling between the Sertoli cells, FLCs, and interstitial cells. We identified 61 genes that were not known previously to be involved in specification or differentiation of FLCs. This dataset provides a platform for exploring the biology of FLCs and understanding the role of these cells in testicular development. In addition, it provides a basis for targeted studies designed to identify causes of idiopathic XY DSD.
Asunto(s)
Diferenciación Celular/genética , Células Intersticiales del Testículo/metabolismo , Células de Sertoli/metabolismo , Procesos de Determinación del Sexo/genética , Testículo/metabolismo , Transcriptoma , Animales , Feto/metabolismo , Células Intersticiales del Testículo/citología , Masculino , Ratones , Ratones Transgénicos , Células de Sertoli/citología , Testículo/citologíaRESUMEN
In addition to their role as endocrine organs, the gonads nurture and protect germ cells, and regulate the formation of gametes competent to convey the genome to the following generation. After sex determination, gonadal somatic cells use several known signalling pathways to direct germ cell development. However, the extent to which germ cells communicate back to the soma, the molecular signals they use to do so and the significance of any such signalling remain as open questions. Herein, we review findings arising from the study of gonadal development and function in the absence of germ cells in a range of organisms. Most published studies support the view that germ cells are unimportant for foetal gonadal development in mammals, but later become critical for stabilisation of gonadal function and somatic cell phenotype. However, the lack of consistency in the data, and clear differences between mammals and other vertebrates and invertebrates, suggests that the story may not be so simple and would benefit from more careful analysis using contemporary molecular, cell biology and imaging tools.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Células Germinativas/citología , Gónadas/crecimiento & desarrollo , Mamíferos/crecimiento & desarrollo , Transducción de Señal , Animales , Diferenciación Celular , Células Germinativas/fisiología , Gónadas/metabolismo , Humanos , Mamíferos/metabolismoRESUMEN
ER71, also known as ETV2, is an ETS transcription factor that is expressed during embryogenesis and in adult testes. We show that Er71 transcription can be up-regulated by SRY, the key determinant of male differentiation. Accordingly, SRY bound to and activated the Er71 promoter, and mutation of a putative SRY binding site abolished this promoter activation. In turn, ER71 was able to bind to the promoter of Sox9, the primary target of SRY and a critical transcription factor for maintenance of the Sertoli cell phenotype. Mutation of the ER71 binding site in the Sox9 promoter suppressed ER71-dependent up-regulation of Sox9 transcription, and a dominant-negative ER71 molecule severely reduced Sox9 transcription in a Sertoli cell line. Conversely, SOX9 bound the Er71 promoter in vivo and Sox9 down-regulation reduced Er71 transcript levels. Together, these data suggest a mechanism by which SRY induces Sox9 and Er71 transcription early in testis differentiation, whereas ER71 and SOX9 participate in an autoregulatory loop to sustain each other's expression after Sry expression has subsided in mice. Thereby, ER71 and SOX9 may affect late testis development as well as the function of the adult male gonad.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Factor de Transcripción SOX9/genética , Testículo/metabolismo , Factores de Transcripción/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Línea Celular Tumoral , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Retroalimentación Fisiológica , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Hibridación in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas/genética , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción SOX9/metabolismo , Células de Sertoli/metabolismo , Proteína de la Región Y Determinante del Sexo/genética , Proteína de la Región Y Determinante del Sexo/metabolismo , Testículo/embriología , Testículo/crecimiento & desarrollo , Factores de Transcripción/metabolismoRESUMEN
The fetal gonad contains a great variety of differentiating cell populations, of which germ cells make up a relatively small percentage. In order to study germ cell-specific gene and protein expression, as well as determine direct effects of signaling molecules, it is necessary to prepare enriched populations of germ cells and maintain them in culture for several hours to multiple days. The protocols in this chapter are designed to provide a guide for the isolation or enrichment of primordial germ cells (from 9.5 days post coitum (dpc) to 18.5 dpc) by flow cytometry (Subheading 3.1) or magnetic sorting (Subheading 3.2), followed by feeder-free primary germ cell culture (Subheading 3.3).
Asunto(s)
Feto , Células Germinativas , Ratones , Animales , Células Germinativas/metabolismo , Técnicas de Cultivo , Citometría de Flujo , GónadasRESUMEN
Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Here, we identify recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which we tentatively propose be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XY GONadal dysgenesis). Our findings will enable additional diagnoses and improved outcomes for individuals born with this condition.