RESUMO
We used male BTBR mice carrying the Lepob mutation, which are subject to severe and progressive obesity and diabetes beginning at 6 wk of age, to examine the influence of one specific manifestation of sleep apnea, intermittent hypoxia (IH), on male urinary voiding physiology and genitourinary anatomy. A custom device was used to deliver continuous normoxia (control) or IH to wild-type and Lepob/ob (mutant) mice for 2 wk. IH was delivered during the 12-h inactive (light) period in the form of 90 s of 6% O2 followed by 90 s of room air. Continuous room air was delivered during the 12-h active (dark) period. We then evaluated genitourinary anatomy and physiology. As expected for the type 2 diabetes phenotype, mutant mice consumed more food and water, weighed more, and voided more frequently and in larger urine volumes. They also had larger bladder volumes but smaller prostates, seminal vesicles, and urethras than wild-type mice. IH decreased food consumption and increased bladder relative weight independent of genotype and increased urine glucose concentration in mutant mice. When evaluated based on genotype (normoxia + IH), the incidence of pathogenic bacteriuria was greater in mutant mice than in wild-type mice, and among mice exposed to IH, bacteriuria incidence was greater in mutant mice than in wild-type mice. We conclude that IH exposure and type 2 diabetes can act independently and together to modify male mouse urinary function. NEW & NOTEWORTHY Metabolic syndrome and obstructive sleep apnea are common in aging men, and both have been linked to urinary voiding dysfunction. Here, we show that metabolic syndrome and intermittent hypoxia (a manifestation of sleep apnea) have individual and combined influences on voiding function and urogenital anatomy in male mice.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Hipóxia/metabolismo , Síndrome Metabólica/metabolismo , Obesidade/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Modelos Animais de Doenças , Hipóxia/genética , Resistência à Insulina/fisiologia , Fígado/metabolismo , Masculino , Síndrome Metabólica/genética , Camundongos , Obesidade/genéticaRESUMO
The bladder's remarkable regenerative capacity had been thought to derive exclusively from its own progenitors. While examining consequences of DNA methyltransferase 1 (Dnmt1) inactivation in mouse embryonic bladder epithelium, we made the surprising discovery that Wolffian duct epithelial cells can support bladder regeneration. Conditional Dnmt1 inactivation in mouse urethral and bladder epithelium triggers widespread apoptosis, depletes basal and intermediate bladder cells, and disrupts uroplakin protein expression. These events coincide with Wolffian duct epithelial cell recruitment into Dnmt1 mutant urethra and bladder where they are reprogrammed to express bladder markers, including FOXA1, keratin 5, P63, and uroplakin. This is evidence that Wolffian duct epithelial cells are summoned in vivo to replace damaged bladder epithelium and function as a reservoir of cells for bladder regeneration.
Assuntos
Bexiga Urinária/fisiologia , Urotélio/fisiologia , Ductos Mesonéfricos/fisiologia , Animais , Animais Recém-Nascidos , Apoptose , Linhagem da Célula , DNA (Citosina-5-)-Metiltransferase 1/fisiologia , Dano ao DNA , Metilação de DNA , Células Epiteliais/fisiologia , Camundongos , RegeneraçãoRESUMO
DNA methyltransferase 1 (DNMT1) is required for embryogenesis but roles in late forming organ systems including the prostate, which emerges from the urethral epithelium, have not been fully examined. We used a targeted genetic approach involving a Shhcre recombinase to demonstrate requirement of epithelial DNA methyltransferase-1 (Dnmt1) in mouse prostate morphogenesis. Dnmt1 mutant urethral cells exhibit DNA hypomethylation, DNA damage, p53 accumulation and undergo cell cycle arrest and apoptosis. Urethral epithelial cells are disorganized in Dnmt1 mutants, leading to impaired prostate growth and maturation and failed glandular development. We evaluated oriented cell division as a mechanism of bud elongation and widening by demonstrating that mitotic spindle axes typically form parallel or perpendicular to prostatic bud elongation axes. We then deployed a ShhcreERT allele to delete Dnmt1 from a subset of urethral epithelial cells, creating mosaic mutants with which to interrogate the requirement for cell division in specific prostatic bud epithelial populations. DNMT1- cell distribution within prostatic buds is not random as would be expected in a process where DNMT1 was not required. Instead, replication competent DNMT1â¯+â¯cells primarily accumulate in prostatic bud margins and tips while replication impeded DNMT1- cells accumulate in prostatic bud cores. Together, these results highlight the role of DNMT1 in regulating epithelial bud formation by maintaining cell cycle progression and survival of rapidly dividing urethral epithelial cells, which can be extended to the study of other developing epithelial organs. In addition, our results show that prostatic buds consist of two epithelial cell populations with distinct molecular and functional characteristics that could potentially contribute to specialized lineages in the adult prostate.
Assuntos
Ciclo Celular/fisiologia , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Células Epiteliais/enzimologia , Organogênese/fisiologia , Próstata/embriologia , Uretra/embriologia , Animais , Sobrevivência Celular , DNA (Citosina-5-)-Metiltransferase 1/genética , Células Epiteliais/citologia , Masculino , Camundongos , Camundongos Transgênicos , Próstata/citologia , Uretra/citologiaRESUMO
BACKGROUND: Castration-insensitive epithelial progenitors capable of regenerating the prostate have been proposed to be concentrated in the proximal region based on facultative assays. Functional characterization of prostate epithelial populations isolated with individual cell surface markers has failed to provide a consensus on the anatomical and transcriptional identity of proximal prostate progenitors. METHODS: Here, we use single-cell RNA sequencing to obtain a complete transcriptomic profile of all epithelial cells in the mouse prostate and urethra to objectively identify cellular subtypes. Pan-transcriptomic comparison to human prostate cell types identified a mouse equivalent of human urethral luminal cells, which highly expressed putative prostate progenitor markers. Validation of the urethral luminal cell cluster was performed using immunostaining and flow cytometry. RESULTS: Our data reveal that previously identified facultative progenitors marked by Trop2, Sca-1, KRT4, and PSCA are actually luminal epithelial cells of the urethra that extend into the proximal region of the prostate, and are resistant to castration-induced androgen deprivation. Mouse urethral luminal cells were identified to be the equivalent of previously identified human club and hillock cells that similarly extend into proximal prostate ducts. Benign prostatic hyperplasia (BPH) has long been considered an "embryonic reawakening," but the cellular origin of the hyperplastic growth concentrated in the periurethral region is unclear. We demonstrate an increase in urethral luminal cells within glandular nodules from BPH patients. Urethral luminal cells are further increased in patients treated with a 5-α reductase inhibitor. CONCLUSIONS: Our data demonstrate that cells of the proximal prostate that express putative progenitor markers, and are enriched by castration in the proximal prostate, are urethral luminal cells and that these cells may play an important role in the etiology of human BPH.
Assuntos
Próstata/citologia , Células-Tronco/citologia , Uretra/citologia , Adolescente , Adulto , Animais , Antígenos de Neoplasias/metabolismo , Moléculas de Adesão Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Próstata/metabolismo , Células-Tronco/metabolismo , Uretra/metabolismo , Adulto JovemRESUMO
Mouse urinary behavior is quantifiable and is used to pinpoint mechanisms of voiding dysfunction and evaluate potential human therapies. Approaches to evaluate mouse urinary function vary widely among laboratories, however, complicating cross-study comparisons. Here, we describe development and multi-institutional validation of a new tool for objective, consistent, and rapid analysis of mouse void spot assay (VSA) data. Void Whizzard is a freely available software plugin for FIJI (a distribution of ImageJ) that facilitates VSA image batch processing and data extraction. We describe its features, demonstrate them by evaluating how specific VSA method parameters influence voiding behavior, and establish Void Whizzard as an expedited method for VSA analysis. This study includes control and obese diabetic mice as models of urinary dysfunction to increase rigor and ensure relevance across distinct voiding patterns. In particular, we show that Void Whizzard is an effective tool for quantifying nonconcentric overlapping void spots, which commonly confound analyses. We also show that mouse genetics are consistently more influential than assay design parameters when it comes to VSA outcomes. None of the following procedural modifications to reduce overlapping spots masked these genetic-related differences: reduction of VSA testing duration, water access during the assay period, placement of a wire mesh cage bottom on top of or elevated over the filter paper, treatment of mesh with a hydrophobic spray, and size of wire mesh opening. The Void Whizzard software and rigorous validation of VSA methodological parameters described here advance the goal of standardizing mouse urinary phenotyping for comprehensive urinary phenome analyses.
Assuntos
Diabetes Mellitus Experimental/fisiopatologia , Software , Micção/fisiologia , Urodinâmica/fisiologia , Animais , Objetivos , Masculino , Camundongos Transgênicos , Bexiga Urinária/fisiopatologiaRESUMO
AIMS: Mice are increasingly being used as models to investigate aspects of urinary dysfunction that humans with lower urinary tract symptoms (LUTS) experience. One method used to examine voiding function is the spontaneous void spot assay. The purpose of this study was to characterize and identify animal husbandry conditions that might confound results of the spontaneous void spot assay in male C57Bl/6J mice. METHODS: Mice were placed in cages lined with filter paper for 4 hr and urine was visualized with UV transillumination. Voiding parameters including urine spot number, spot size, total urine area, primary void area, corner and center voiding were quantified. RESULTS: Adult male mice void more frequently with advancing age and a subpopulation (5-10%) display a frequent spotting pattern at 6-9 weeks of age. Voiding was not significantly different in male mice weaned to group housing (4-6 per cage) versus single housing, and was not altered when they were used as breeders. Voiding was changed upon transferring group housed adult males to single density cages, which decreased total urine area. Repeated assays of male voiding behavior over three consecutive days increased primary void area by the third day of monitoring and revealed that voiding behavior is impacted by routine cage changes and time of day. CONCLUSIONS: Together these results identify housing and husbandry practices that influence male voiding behaviors in the spontaneous void spot assay and will inform voiding behavior analyses conducted with male C57Bl/6J mice.
Assuntos
Criação de Animais Domésticos/métodos , Técnicas de Diagnóstico Urológico , Abrigo para Animais , Micção , Urodinâmica , Fatores Etários , Animais , Comportamento Animal , Ritmo Circadiano , Manobra Psicológica , Masculino , Camundongos Endogâmicos C57BL , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
BACKGROUND: Epigenetic factors influence stem cell function and other developmental events but their role in prostate morphogenesis is not completely known. We tested the hypothesis that histone deacetylase (HDAC) activity is required for prostate morphogenesis. RESULTS: We identified the presence of class I nuclear HDACs in the mouse urogenital sinus (UGS) during prostate development and found that Hdac 2 mRNA abundance diminishes as development proceeds which is especially evident in prostatic epithelium. Blockade of HDACs with the inhibitor trichostatin A (TSA) decreased the number of prostatic buds formed in UGS explant cultures but not the number of buds undergoing branching morphogenesis. In the latter, TSA promoted an extensive branching phenotype that was reversed by exogenous NOGGIN protein, which functions as a bone morphogenetic protein (BMP) inhibitor. TSA also increased Bmp2 promoter H3K27ac abundance, Bmp2 and Bmp4 mRNA abundance, and the percentage of epithelial cells marked by BMP-responsive phosphorylated SMAD1/5/8 protein. TSA exposed UGS explants grafted under the kidney capsule of untreated host mice for continued development achieved a smaller size without an obvious difference in glandular histology compared with control treated grafts. CONCLUSIONS: These results are consistent with an active role for HDACs in shaping prostate morphogenesis by regulating Bmp abundance.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Histonas/metabolismo , Próstata/crescimento & desenvolvimento , Acetilação , Animais , Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 2/farmacologia , Proteína Morfogenética Óssea 4/metabolismo , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Histona Desacetilase 2/metabolismo , Humanos , Ácidos Hidroxâmicos/química , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de SinaisRESUMO
In prostate and other epithelial cancers, E-cadherin (CDH1) is downregulated inappropriately by DNA methylation to promote an invasive phenotype. Though cancer frequently involves a reawakening of developmental signaling pathways, whether DNA methylation of Cdh1 occurs during organogenesis has not been determined. Here we show that DNA methylation of Cdh1 mediates outgrowth of developing prostate ducts. During the three-day gestational window leading up to and including prostate ductal initiation, Cdh1 promoter methylation increases and its mRNA and protein abundance decreases in epithelium giving rise to prostatic buds. DNA methylation is required for prostate specification, ductal outgrowth, and branching morphogenesis. All three endpoints are impaired by a DNA methylation inhibitor, which also decreases Cdh1 promoter methylation and increases Cdh1 mRNA and protein abundance. A CDH1 function-blocking antibody restores prostatic identity, bud outgrowth, and potentiates epithelial differentiation in the presence of the DNA methylation inhibitor. This is the first study to mechanistically link acquired changes in DNA methylation to the normal process of prostate organogenesis. We propose a novel mechanism whereby Cdh1 promoter methylation restricts Cdh1 abundance in developing prostate epithelium to create a permissive environment for prostatic bud outgrowth. Thus, DNA methylation primes the prostate primordium to respond to developmental cues mediating outgrowth, differentiation and maturation of the ductal network.
Assuntos
Caderinas/genética , Proteínas Cdh1/metabolismo , Metilação de DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Próstata/embriologia , Animais , Anticorpos Bloqueadores/imunologia , Proteínas Cdh1/genética , Proteínas Cdh1/imunologia , Diferenciação Celular/imunologia , Epitélio/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Morfogênese/genética , Regiões Promotoras Genéticas/genética , Próstata/metabolismo , RNA Mensageiro/metabolismoRESUMO
Androgen receptor (AR) signaling initiates mouse prostate development by stimulating prostate ductal bud formation and specifying bud patterns. Curiously, however, prostatic bud initiation lags behind the onset of gonadal testosterone synthesis by about three days. This study's objective was to test the hypothesis that DNA methylation controls the timing and scope of prostate ductal development by regulating Ar expression in the urogenital sinus (UGS) from which the prostate derives. We determined that Ar DNA methylation decreases in UGS mesenchyme during prostate bud formation in vivo and that this change correlates with decreased DNA methyltransferase expression in the same cell population during the same time period. To examine the role of DNA methylation in prostate development, fetal UGSs were grown in serum-free medium and 5 alpha dihydrotestosterone (DHT) and the DNA methylation inhibitor 5'-aza-2'-deoxycytidine (5AzadC) were introduced into the medium at specific times. As a measure of prostate development, in situ hybridization was used to visualize and count Nkx3-1 mRNA positive prostatic buds. We determined that inhibiting DNA methylation when prostatic buds are being specified, accelerates the onset of prostatic bud development, increases bud number, and sensitizes the budding response to androgens. Inhibition of DNA methylation also reduces Ar DNA methylation in UGS explants and increases Ar mRNA and protein in UGS mesenchyme and epithelium. Together, these results support a novel mechanism whereby Ar DNA methylation regulates UGS androgen sensitivity to control the rate and number of prostatic buds formed, thereby establishing a developmental checkpoint.
Assuntos
Metilação de DNA/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Modelos Biológicos , Organogênese/fisiologia , Próstata/embriologia , Receptores Androgênicos/metabolismo , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Azacitidina/análogos & derivados , Sequência de Bases , Primers do DNA/genética , Decitabina , Di-Hidrotestosterona , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Imunoprecipitação , Hibridização In Situ , Técnicas In Vitro , Masculino , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Receptores Androgênicos/genética , Análise de Sequência de DNA , Fatores de Transcrição/metabolismoRESUMO
Aging men are susceptible to developing lower urinary tract symptoms, but the underlying etiology is unknown and the influence of dietary and environmental factors on them is unclear. We tested whether a folic acid-enriched diet changed urinary tract physiology and biology in control male mice and male mice with urinary dysfunction induced by exogenous testosterone and estradiol (T+E2), which mimics changing hormone levels in aging humans. T+E2 treatment increased mouse urine output, time between voiding events, and bladder capacity and compliance. Consumption of a folic acid-enriched diet moderated these changes without decreasing prostate wet weight or threshold voiding pressure. One potential mechanism for these changes involves water balance. T+E2 treatment increases plasma concentrations of anti-diuretic hormone, which is offset at least in part by a folic acid-enriched diet. Another potential mechanism involves neural control of micturition. The folic acid-enriched diet, fed to T+E2-treated mice, increased voiding frequency in response to intravesicular capsaicin infusion and increased mRNA abundance of the capsaicin-sensitive cation channel transient receptor potential vanilloid subfamily member 1 (Trpv1) in L6 and S1 dorsal root ganglia (DRG) neurons. T+E2 treatment and a folic acid-enriched diet also modified DNA methylation, which is capable of altering gene expression. We found the enriched diet increased global DNA methylation in dorsal and ventral prostate and L6 and S1 DRG. Our results are consistent with folic acid acting to slow or reverse T+E2-mediated alteration in urinary function in part by normalizing water balance and enhancing or preserving afferent neuronal function.
Assuntos
Estradiol/farmacologia , Ácido Fólico/farmacologia , Sintomas do Trato Urinário Inferior/tratamento farmacológico , Testosterona/farmacologia , Fenômenos Fisiológicos do Sistema Urinário/efeitos dos fármacos , Ração Animal , Animais , Capsaicina/administração & dosagem , Capsaicina/farmacologia , Dieta , Estradiol/administração & dosagem , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Testosterona/administração & dosagem , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismoRESUMO
Fetal prostate development is initiated by androgens and patterned by androgen dependent and independent signals. How these signals integrate to control epithelial cell differentiation and prostatic bud patterning is not fully understood. To test the role of beta-catenin (Ctnnb1) in this process, we used a genetic approach to conditionally delete or stabilize Ctnnb1 in urogenital sinus (UGS) epithelium from which the prostate derives. Two opposing mechanisms of action were revealed. By deleting Ctnnb1, we found it is required for separation of UGS from cloaca, emergence or maintenance of differentiated UGS basal epithelium and formation of prostatic buds. By genetically inducing a patchy subset of UGS epithelial cells to express excess CTNNB1, we found its excess abundance increases Bmp expression and leads to a global impairment of prostatic bud formation. Addition of NOGGIN partially restores prostatic budding in UGS explants with excess Ctnnb1. These results indicate a requirement for Ctnnb1 in UGS basal epithelial cell differentiation, prostatic bud initiation and bud spacing and suggest some of these actions are mediated in part through activation of BMP signaling.
Assuntos
Proteína Morfogenética Óssea 2/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , beta Catenina/biossíntese , Animais , Padronização Corporal , Diferenciação Celular , Cruzamentos Genéticos , Células Epiteliais/citologia , Deleção de Genes , Masculino , Camundongos , Microscopia Eletrônica de Varredura/métodos , Modelos Genéticos , Próstata/embriologia , Transdução de SinaisRESUMO
The purpose of this study was to validate a combined in situ hybridization (ISH)/immunohistochemistry (IHC) staining method for visualizing and quantifying mouse prostatic buds. To refine animal usage in prostate development studies, we also determined whether a comparable number of prostatic buds were formed in male and female mouse urogenital sinus (UGS) explants grown in vitro in the presence of androgen. We used IHC to label UGS epithelium and ISH to label prostatic buds with one of three different prostatic bud marking riboprobes: a previously identified prostatic bud marker, NK-3 transcription factor, locus 1 (Nkx3-1), and two newly identified prostatic bud markers, wingless-related MMTV integration site 10b (Wnt10b) and ectodysplasin-A receptor (Edar). We calculated total buds formed per UGS and the proportion marked by each mRNA after male UGS development in vivo and male and female UGS development in vitro. Nkx3-1 was first to mark the prostate field during UGS development in vivo but all three mRNAs marked prostatic buds during later developmental stages. The mRNAs localized to different domains: Nkx3-1 was present along about half the prostatic bud length while Edar and Wnt10b were restricted to distal bud tips. None of the mRNAs marked all buds formed in vitro and the proportion marked was developmental stage- and gender-dependent. Nkx3-1 marked the highest proportion of prostatic buds during in vitro UGS development. Together, our results reveal that ISH staining of mouse UGS can be used to quantify prostatic bud number, Nkx3-1 is currently the best suited riboprobe for this method, and female UGSs cannot be used interchangeably with male UGSs when conducting prostate development studies in vitro. We also found that Nkx3-1, Edar, and Wnt10b mark different prostatic bud regions and are likely to be useful in future studies of regional differences in prostatic bud gene expression.
Assuntos
Hibridização In Situ/métodos , Próstata/embriologia , Animais , Receptor Edar/genética , Receptor Edar/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Próstata/metabolismo , RNA Mensageiro/biossíntese , Fatores Sexuais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismoRESUMO
Epithelial-stromal interactions in the lower urogenital tract (LUT) are integral to prostatic and seminal vesicle development in males, vaginal and uterine development in females, and urethral development in both sexes. Gene expression profiling of isolated LUT stroma and epithelium has unraveled mechanisms of LUT development, but such studies are confounded by heterogeneous and ill-defined cell sub-populations contained within each tissue compartment. We used in situ hybridization to synthesize a high-resolution molecular atlas of 17-day post-coitus fetal mouse LUT. We identified mRNAs that mark selective cell populations of the seminal vesicle, ejaculatory duct, prostate, urethra, and vagina, subdividing these tissues into 16 stromal and 8 epithelial sub-compartments. These results provide a powerful tool for mapping LUT gene expression patterns and also reveal previously uncharacterized sub-compartments that may play mechanistic roles in LUT development of which we were previously unaware.
Assuntos
Biomarcadores/metabolismo , Sistema Urogenital/anatomia & histologia , Sistema Urogenital/embriologia , Sistema Urogenital/metabolismo , Animais , Atlas como Assunto , Feminino , Perfilação da Expressão Gênica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismoRESUMO
Prostate development is influenced by ß-catenin signaling, but it is unclear which ß-catenin activators are involved, where they are synthesized, and whether their mRNA abundance is influenced by androgens. We identified WNT/ß-catenin-responsive ß-galactosidase activity in the lower urogenital tract (LUT) of transgenic reporter mice, but ß-galactosidase activity differed among the four mouse strains we examined. We used in situ hybridization to compare patterns of Wnts, r-spondins (Rspos, co-activators of ß-catenin signaling), ß-catenin-responsive mRNAs, and an androgen receptor-responsive mRNA in wild type fetal male, fetal female, and neonatal male LUT. Most Wnt and Rspo mRNAs were present in LUT during prostate development. Sexually dimorphic expression patterns were observed for WNT/ß-catenin-responsive genes, and for Wnt2b, Wnt4, Wnt7a, Wnt9b, Wnt10b, Wnt11, Wnt16, and Rspo3 mRNAs. These results reveal sexual differences in WNT/ß-catenin signaling in fetal LUT, supporting the idea that this pathway may be directly or indirectly responsive to androgens during prostate ductal development.
Assuntos
Trombospondinas/genética , Sistema Urogenital/embriologia , Sistema Urogenital/metabolismo , Proteínas Wnt/genética , Anatomia Artística , Animais , Animais Recém-Nascidos , Atlas como Assunto , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Trombospondinas/metabolismo , Distribuição Tecidual , Sistema Urogenital/crescimento & desenvolvimento , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/genética , Via de Sinalização Wnt/fisiologiaRESUMO
Tubby-like protein 3 (TULP3) is required for proper embryonic development in mice. Disruption of mouse Tulp3 results in morphological defects in the embryonic craniofacial regions, the spinal neural tube and the limbs. Here, we show that TULP3 functions as a novel negative regulator of Sonic hedgehog (Shh) signaling in the mouse. In Tulp3 mutants, ventral cell types in the lumbar neural tube, which acquire their identities in response to Shh signaling, are ectopically specified at the expense of dorsal cell types. Genetic epistasis experiments show that this ventralized phenotype occurs independently of Shh and the transmembrane protein Smoothened, but it is dependent on the transcription factor Gli2. The ventralized phenotype is also dependent on the kinesin II subunit Kif3A, which is required for intraflagellar transport and ciliogenesis. In addition, TULP3 is required for proper Shh-dependent limb patterning and for maintaining the correct balance between differentiation and proliferation in the neural tube. Finally, the localization of TULP3 to the tips of primary cilia raises the possibility that it regulates the Hedgehog pathway within this structure.
Assuntos
Padronização Corporal , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Hedgehog/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Animais , Regulação para Baixo , Embrião de Mamíferos , Feminino , Proteínas Hedgehog/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Mutação , Tubo Neural/embriologia , Tubo Neural/metabolismo , Proteínas/genética , Medula Espinal/embriologia , Medula Espinal/metabolismoRESUMO
The ventral urogenital sinus (UGS) of control male mice has two rows of 3-4 prostatic buds at birth, but how androgens regulate ventral bud (VB) number and patterning is unclear. VBs in both sexes appeared to be a mixture of prostatic and urethral buds. UGSs from Tfm male and antiandrogen (flutamide)-exposed mice had small VBs, suggesting that initiation of some VBs is androgen independent. Tfm male mice are widely considered completely androgen insensitive yet their UGSs were 5alpha-dihydrotestosterone (DHT)- responsive. VBs (6-8) were generally distributed bimodally on the left-right axis at both minimal and normal male androgen signaling. Yet control females and DHT-exposed Tfm males had 13-14 VBs, whose left-right distribution was fairly uniform. These results suggest that VB number and distribution respond biphasically as androgen signaling increases from minimal, and that androgens regulate bud specification. Complete VB agenesis by the selective budding inhibitor 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) required high androgen signaling.
Assuntos
Androgênios/metabolismo , Padronização Corporal , Feto/metabolismo , Próstata/embriologia , Receptores Androgênicos/metabolismo , Antagonistas de Androgênios , Síndrome de Resistência a Andrógenos/metabolismo , Animais , Animais Recém-Nascidos , Di-Hidrotestosterona , Feminino , Flutamida , Proteínas de Homeodomínio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Técnicas de Cultura de Órgãos , Dibenzodioxinas Policloradas , Caracteres Sexuais , Transdução de Sinais , Teratogênicos , Terminologia como Assunto , Fatores de Transcrição/metabolismo , Uretra/embriologiaRESUMO
Urinary voiding dysfunction in aging men can cause bothersome symptoms and irreparable tissue damage. Underlying mechanisms are not fully known. We previously demonstrated that subcutaneous, slow-release testosterone and estradiol implants (T+E2) drive a pattern of urinary voiding dysfunction in male mice that resembles that of aging men. The initial goal of this study was to test the hypothesis that prostatic epithelial beta-catenin (Ctnnb1) is required for T+E2-mediated voiding dysfunction. Targeted Ctnnb1 deletion did not significantly change voiding function in control or T+E2 treated mice but led to the surprising discovery that the C57BL/6J × FVB/NJ × 129S1 mixed genetic background onto which Ctnnb1 loss of function alleles were maintained is profoundly susceptible to voiding dysfunction. The mixed background mice develop a more rapid T+E2-mediated increase in spontaneous urine spotting, are more impaired in ability to initiate bladder contraction, and develop larger and heavier bladders than T+E2 treated C57BL/6J pure bred mice. To better understand mechanisms, we separately evaluated contributions of T and E2 and found that E2 mediates voiding dysfunction. Our findings that genetic factors serve as modifiers of responsiveness to T and E2 demonstrate the need to control for genetic background in studies of male voiding dysfunction. We also show that genetic factors could control severity of voiding dysfunction. We demonstrate the importance of E2 as a key mediator of voiding impairment, and show that the concentration of E2 in subcutaneous implants determines the severity of voiding dysfunction in mice, demonstrating that the mouse model is tunable, a factor which is important for future pharmacological intervention studies.
RESUMO
Beta-catenin (CTNNB1) directs ectodermal appendage spacing by activating ectodysplasin A receptor (EDAR) transcription, but whether CTNNB1 acts by a similar mechanism in the prostate, an endoderm-derived tissue, is unclear. Here we examined the expression, function, and CTNNB1 dependence of the EDAR pathway during prostate development. In situ hybridization studies reveal EDAR pathway components including Wnt10b in the developing prostate and localize these factors to prostatic bud epithelium where CTNNB1 target genes are co-expressed. We used a genetic approach to ectopically activate CTNNB1 in developing mouse prostate and observed focal increases in Edar and Wnt10b mRNAs. We also used a genetic approach to test the prostatic consequences of activating or inhibiting Edar expression. Edar overexpression does not visibly alter prostatic bud formation or branching morphogenesis, and Edar expression is not necessary for either of these events. However, Edar overexpression is associated with an abnormally thick and collagen-rich stroma in adult mouse prostates. These results support CTNNB1 as a transcriptional activator of Edar and Wnt10b in the developing prostate and demonstrate Edar is not only important for ectodermal appendage patterning but also influences collagen organization in adult prostates.This article has an associated First Person interview with the first author of the paper.
RESUMO
Bothersome lower urinary tract symptoms (LUTS) manifest as urinary frequency, urgency, incontinence and incomplete bladder emptying. Existing treatments ameliorate but do not eliminate most symptoms, leading to financial and personal burdens attributable to sustained medical therapies that may last a lifetime. The purpose of this review is to highlight evidence of causal associations between LUTS and several common comorbidities, including intermittent hypoxia (IH) concomitant with obstructive sleep apnea (OSA), obesity, metabolic syndrome and type 2 diabetes. Links between these conditions, including therapies targeted to co-occurring complications that have demonstrated benefits for LUTS, suggest compelling avenues of research and also underscore critical gaps in understanding the mechanisms underlying urinary dysfunction. These gaps are prominent in the IH field, where an acknowledged link between OSA and LUTS has gone largely uninvestigated. New tools, models, or reappropriation of existing ones, especially rodent models, is required to parse the associations between IH/OSA, LUTS and obesity/diabetes and to elucidate their underlying, and potentially shared, etiologies.
Assuntos
Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/terapia , Hipóxia , Sintomas do Trato Urinário Inferior/complicações , Sintomas do Trato Urinário Inferior/terapia , Animais , HumanosRESUMO
Benign prostatic hyperplasia, prostate cancer, and changes in the ratio of circulating testosterone and estradiol often occur concurrently in aging men and can lead to lower urinary tract (LUT) dysfunction. To explore the possibility of a fetal basis for the development of LUT dysfunction in adulthood, Tg(CMV-cre);Nkx3-1(+/-);Pten(fl/+) mice, which are genetically predisposed to prostate neoplasia, were exposedin uteroand during lactation to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 1 µg/kg po) or corn oil vehicle (5 ml/kg) after a single maternal dose on 13 days post coitus, and subsequently were aged without further manipulation, or at 8 weeks of age were exposed to exogenous 17 ß-estradiol (2.5 mg) and testosterone (25 mg) (T+E2) via slow release subcutaneous implants.In uteroand lactational (IUL) TCDD exposure in the absence of exogenous hormone treatment reduced voiding pressure in adult mice, but otherwise had little effect on mouse LUT anatomy or function. By comparison, IUL TCDD exposure followed by exogenous hormone treatment increased relative kidney, bladder, dorsolateral prostate, and seminal vesicle weights, hydronephrosis incidence, and prostate epithelial cell proliferation, thickened prostate periductal smooth muscle, and altered prostate and bladder collagen fiber distribution. We propose a 2-hit model whereby IUL TCDD exposure sensitizes mice to exogenous-hormone-induced urinary tract dysfunction later in life.