RESUMEN
The male urogenital system is composed of the reproductive system and the urinary tract; they have an interconnected embryonic development and share one of their anatomical components, the urethra. This system has a highly complex physiology deeply interconnected with the circulatory and nervous systems, as well as being capable of adapting to environmental variations; it also undergoes changes with aging and, in the case of the reproductive system, with seasonality. The stroma is an essential component in this physiological plasticity and its complexity has increased with the description in the last decade of a new cell type, the telocyte. Several studies have demonstrated the presence of telocytes in the organs of the male urogenital system and other systems; however, their exact function is not yet known. The present review addresses current knowledge about telocytes in the urogenital system in terms of their locations, interrelationships, possible functions and pathological implications. It has been found that telocytes in the urogenital system possibly have a leading role in stromal tissue organization/maintenance, in addition to participation in stem cell niches and an association with the immune system, as well as specific functions in the urogenital system, lipid synthesis in the testes, erythropoiesis in the kidneys and the micturition reflex in the bladder. There is also evidence that telocytes are involved in pathologies in the kidneys, urethra, bladder, prostate, and testes.
Asunto(s)
Telocitos/patología , Telocitos/fisiología , Sistema Urogenital/patología , Sistema Urogenital/fisiología , Animales , Enfermedades de los Genitales Masculinos/patología , Enfermedades de los Genitales Masculinos/fisiopatología , Humanos , Metabolismo de los Lípidos/fisiología , Masculino , Próstata/citología , Próstata/patología , Próstata/fisiología , Células Madre/patología , Células Madre/fisiología , Testículo/citología , Testículo/patología , Testículo/fisiología , Vejiga Urinaria/citología , Vejiga Urinaria/patología , Vejiga Urinaria/fisiología , Sistema Urogenital/citologíaRESUMEN
Technological advances in three-dimensional (3D) reconstruction techniques have previously enabled paradigm shifts in our understanding of human embryonic and fetal development. Light sheet fluorescence microscopy (LSFM) is a recently-developed technique that uses thin planes of light to optically section whole-mount cleared and immunolabeled biologic specimens. The advent of commercially-available light sheet microscopes has facilitated a new generation of research into protein localization and tissue dynamics at extremely high resolution. Our group has applied LSFM to study developing human fetal external genitalia, internal genitalia and kidneys. This review describes LSFM and presents our group's technique for preparing, clearing, immunostaining and imaging human fetal urogenital specimens. We then present light sheet images and videos of each element of the developing human urogenital system. To the extent of our knowledge, the work conducted by our laboratory represents the first description of a method for performing LSFM on the full human urogenital system during the embryonic and fetal periods.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Imagenología Tridimensional/métodos , Microscopía Fluorescente/métodos , Manejo de Especímenes/métodos , Sistema Urogenital/citología , HumanosRESUMEN
The evolutionarily conserved transcription factor, Tbx18, is expressed in a dynamic pattern throughout embryonic and early postnatal life and plays crucial roles in the development of multiple organ systems. Previous studies have indicated that this dynamic function is controlled by an expansive regulatory structure, extending far upstream and downstream of the gene. With the goal of identifying elements that interact with the Tbx18 promoter in developing prostate, we coupled chromatin conformation capture (4C) and ATAC-seq from embryonic day 18.5 (E18.5) mouse urogenital sinus (UGS), where Tbx18 is highly expressed. The data revealed dozens of active chromatin elements distributed throughout a 1.5 million base pair topologically associating domain (TAD). To identify cell types contributing to this chromatin signal, we used lineage tracing methods with a Tbx18 Cre "knock-in" allele; these data show clearly that Tbx18-expressing precursors differentiate into wide array of cell types in multiple tissue compartments, most of which have not been previously reported. We also used a 209â¯kb Cre-expressing Tbx18 transgene, to partition enhancers for specific precursor types into two rough spatial domains. Within this central 209â¯kb compartment, we identified ECR1, previously described to regulate Tbx18 expression in ureter, as an active regulator of UGS expression. Together these data define the diverse fates of Tbx18+ precursors in prostate-associated tissues for the first time, and identify a highly active TAD controlling the gene's essential function in this tissue.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Próstata/metabolismo , Elementos Reguladores de la Transcripción/genética , Proteínas de Dominio T Box/genética , Animales , Sitios de Unión/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Cromatina/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Masculino , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Regiones Promotoras Genéticas/genética , Próstata/citología , Próstata/embriología , Proteínas de Dominio T Box/metabolismo , Sistema Urogenital/citología , Sistema Urogenital/embriología , Sistema Urogenital/metabolismoRESUMEN
Extracellular vesicles (EVs) are released from almost all cells and tissues. They are able to transport substances (e.g. proteins, RNA or DNA) at higher concentrations than in their environment and may adhere in a receptor-controlled manner to specific cells or tissues in order to release their content into the respective target structure. Blood contains high concentrations of EVs mainly derived from platelets, and, at a smaller amount, from erythrocytes. The female and male reproductive tracts produce EVs which may be associated with fertility or infertility and are released into body fluids and mucosas of the urogenital organs. In this review, the currently relevant detection methods are presented and critically compared. During pregnancy, placenta-derived EVs are dynamically detectable in peripheral blood with changing profiles depending upon progress of pregnancy and different pregnancy-associated pathologies, such as preeclampsia. EVs offer novel non-invasive diagnostic tools which may reflect the situation of the placenta and the foetus. EVs in urine have the potential of reflecting urogenital diseases including cancers of the neighbouring organs. Several methods for detection, quantification and phenotyping of EVs have been established, which include electron microscopy, flow cytometry, ELISA-like methods, Western blotting and analyses based on Brownian motion. This review article summarises the current knowledge about EVs in blood and cord blood, in the different compartments of the male and female reproductive tracts, in trophoblast cells from normal and pre-eclamptic pregnancies, in placenta ex vivo perfusate, in the amniotic fluid, and in breast milk, as well as their potential effects on natural killer cells as possible targets.
Asunto(s)
Vesículas Extracelulares , Leche Humana/citología , Sistema Urogenital/citología , Células Sanguíneas/citología , Femenino , Sangre Fetal/citología , Humanos , Lactancia , Masculino , EmbarazoRESUMEN
Substances that mimic endogenous hormones may alter the cell signaling that govern prostate development and predispose it to developing lesions in adult and senile life. Bisphenol A is able to mimic estrogens, and studies have demonstrated that low levels of exposure to this compound have caused alterations during prostate development. The aim of this study was to describe the prostate development in both male and female neonatal gerbils in normal conditions and under exposure to BPA during intrauterine life, and also to analyze whether the effects of intrauterine exposure to BPA remain in adulthood. Morphological, stereological, three-dimensional reconstruction, and immunohistochemical methods were employed. The results demonstrated that in 1-day-old normal gerbils, the female paraurethral glands and the male ventral lobe are morphologically similar, although its tissue components-epithelial buds (EB), periurethral mesenchyme (PeM), paraurethral mesenchyme (PaM) or ventral mesenchymal pad (VMP), and smooth muscle (SM)-have presented different immunolabeling pattern for androgen receptor (AR), and for proliferating cell nuclear antigen (PCNA). Moreover, we observed a differential response of male and female prostate to intrauterine BPA exposure. In 1-day-old males, the intrauterine exposure to BPA caused a decrease of AR-positive cells in the PeM and SM, and a decrease of the proliferative status in the EB. In contrast, no morphological alterations were observed in ventral prostate of adult males. In 1-day-old females, BPA exposure promoted an increase of estrogen receptor alpha (ERα) positive cells in PeM and PaM, a decrease of AR-positive cells in EB and PeM, besides a reduction of cell proliferation in EB. Additionally, the adult female prostate of BPA-exposed animals presented an increase of AR- and PCNA-positive cells. These results suggest that the prostate of female gerbils were more susceptible to the intrauterine BPA effects, since they became more proliferative in adult life. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1740-1750, 2016.
Asunto(s)
Compuestos de Bencidrilo/toxicidad , Disruptores Endocrinos/toxicidad , Fenoles/toxicidad , Sistema Urogenital/efectos de los fármacos , Factores de Edad , Animales , Animales Recién Nacidos , Proliferación Celular/efectos de los fármacos , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Receptor alfa de Estrógeno/metabolismo , Femenino , Gerbillinae , Masculino , Exposición Materna/efectos adversos , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Próstata/citología , Próstata/efectos de los fármacos , Próstata/embriología , Próstata/crecimiento & desarrollo , Receptores Androgénicos/metabolismo , Factores Sexuales , Sistema Urogenital/citología , Sistema Urogenital/embriología , Sistema Urogenital/crecimiento & desarrolloRESUMEN
The developing limb is a useful model for studying organogenesis and developmental processes. Although Cre alleles exist for conditional loss- or gain-of-function in limbs, Cre alleles targeting specific limb subdomains are desirable. Here we report on the generation of the Hoxa13:Cre line, in which the Cre gene is inserted in the endogenous Hoxa13 gene. We provide evidence that the Cre is active in embryonic tissues/regions where the endogenous Hoxa13 gene is expressed. Our results show that cells expressing Hoxa13 in developing limb buds contribute to the entire autopod (hand/feet) skeleton and validate Hoxa13 as a distal limb marker as far as the skeleton is concerned. In contrast, in the limb musculature, Cre-based fate mapping shows that almost all muscle masses of the zeugopod (forearm) and part of the triceps contain Hoxa13-expressing cells and/or their descendants. Besides the limb, the activity of the Cre is detectable in the urogenital system and the hindgut, primarily in the epithelium and smooth muscles. Together our data show that the Hoxa13:Cre allele is a useful tool for conditional gene manipulation in the urogenital system, posterior digestive tract, autopod and part of the limb musculature.
Asunto(s)
Tracto Gastrointestinal/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Esbozos de los Miembros/metabolismo , Sistema Urogenital/embriología , Sistema Urogenital/metabolismo , Animales , Femenino , Miembro Anterior/citología , Miembro Anterior/embriología , Miembro Anterior/metabolismo , Tracto Gastrointestinal/citología , Tracto Gastrointestinal/embriología , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Hibridación in Situ , Integrasas/genética , Integrasas/metabolismo , Esbozos de los Miembros/citología , Esbozos de los Miembros/embriología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Músculos/citología , Músculos/embriología , Músculos/metabolismo , Organogénesis/genética , Factores de Tiempo , Sistema Urogenital/citologíaRESUMEN
BACKGROUND: We generated a mouse model of prostate cancer based on the adult-prostate-specific inactivation of phosphatase and tensin homolog (PTEN) using the Cre-loxP system. The potential of our mice as a useful animal model was examined by evaluating the chemopreventive efficacy of the anti-androgen, chlormadinone acetate (CMA). MATERIALS AND METHODS: Six-week-old mice were treated subcutaneously with 50 µg/g of CMA three times a week for 9 or 14 weeks and sacrificed at weeks 15 and 20. Macroscopic change of the entire genitourinary tract (GUT) and histologically evident prostate gland tumor development were evaluated. Proliferation and apoptosis status in the prostate were examined by immunohistochemistry. RESULTS: CMA triggered significant shrinkage of not only the GUT but also prostate glands at 15 weeks compared to the control (p=0.017 and p=0.010, respectively), and the trend became more marked after a further five-weeks of treatment. The onset of prostate adenocarcinoma was not prevented but the proliferation of cancer cells was inhibited by CMA, which suggested the androgen axis is critical for cancer growth in these mice. CONCLUSIONS: Conditional PTEN-deficient mice are useful as a preclinical model for chemoprevention studies and serve as a valuable tool for the future screening of potential chemopreventive agents.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antagonistas de Andrógenos/uso terapéutico , Acetato de Clormadinona/uso terapéutico , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata/tratamiento farmacológico , Adenocarcinoma/patología , Adenocarcinoma/prevención & control , Animales , Anticarcinógenos/uso terapéutico , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimioprevención , Modelos Animales de Enfermedad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/prevención & control , Sistema Urogenital/citología , Sistema Urogenital/patologíaRESUMEN
Glial cell line-derived neurotrophic factor (GDNF) is a TGFß family member, and GDNF signals through a glycosyl-phosphatidylinositol-linked cell surface receptor (GFRα1) and RET receptor tyrosine kinase. GDNF signaling plays crucial roles in urogenital processes, ranging from cell fate decisions in germline progenitors to ureteric bud outgrowth and renal branching morphogenesis. Gene ablation studies in mice have revealed essential roles for GDNF signaling in urogenital development, although its role in prostate development is unclear. We investigated the functional role of GDNF signaling in the urogenital sinus (UGS) and the developing prostate of mice. GDNF, GFRα1, and RET show time-specific and cell-specific expression during prostate development in vivo. In the UGS, GDNF and GFRα1 are expressed in the urethral mesenchyme (UrM) and epithelium (UrE), whereas RET is restricted to the UrM. In each lobe of the developing prostate, GDNF and GFRα1 expression declines in the epithelium and becomes restricted to the stroma. Using a well-established organ culture system, we determined that exogenous GDNF increases proliferation of UrM and UrE cells, altering UGS morphology. With regard to mechanism, GDNF signaling in the UrM increased RET expression and phosphorylation of ERK1/2. Furthermore, inhibition of RET kinase activity or ERK kinases suppressed GDNF-induced proliferation of UrM cells but not UrE cells. We therefore propose that GDNF signaling in the UGS increases proliferation of UrM and UrE cells by different mechanisms, which are distinguished by the role of RET receptor tyrosine kinase and ERK kinase signaling, thus implicating GDNF signaling in prostate development and growth.
Asunto(s)
Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Sistema Urogenital/citología , Animales , Proliferación Celular/efectos de los fármacos , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Femenino , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Ratones Endogámicos C57BL , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Técnicas de Cultivo de Órganos , Fosforilación/efectos de los fármacos , Próstata/efectos de los fármacos , Próstata/embriología , Próstata/metabolismo , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-ret/metabolismo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Uretra/efectos de los fármacos , Uretra/metabolismo , Sistema Urogenital/efectos de los fármacos , Sistema Urogenital/embriología , Sistema Urogenital/metabolismoRESUMEN
Although human umbilical cord mesenchymal stem cells (hUC-MSCs) have been identified as a new source of MSCs for potential application in regenerative medicine, their full potential of differentiation has not been determined. In particular, whether they have the capability to differentiate into epithelial cells of endodermal origin such as the prostate epithelial cells is unknown. Here we report that when hUC-MSCs were combined with rat urogenital sinus stromal cells (rUGSSs) and transplanted into the renal capsule in vivo, they could differentiate into prostate epithelial-like cells that could be verified by prostate epithelial cell-specific markers including the prostate specific antigen. The prostatic glandular structures formed in vivo displayed similar cellular architecture with lumens and branching features as seen for a normal prostate. In addition, the human origin of the hUC-MSCs was confirmed by immunocytochemistry for human nuclear antigen. These findings together indicate that hUC-MSCs have the capability to differentiate into epithelial-like cells that are normally derived from the endoderm, implicating their potential applications in tissue repair and regeneration of many endoderm-derived internal organs.
Asunto(s)
Diferenciación Celular , Células Epiteliales/citología , Células Madre Mesenquimatosas/citología , Próstata/citología , Cordón Umbilical/citología , Animales , Células Cultivadas , Células Epiteliales/química , Femenino , Humanos , Inmunohistoquímica , Queratina-8/análisis , Masculino , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/química , Ratones Endogámicos BALB C , Ratones Desnudos , Embarazo , Próstata/química , Ratas Sprague-Dawley , Receptores Androgénicos/análisis , Trasplante Heterólogo , Sistema Urogenital/citologíaRESUMEN
Autosomal recessive polycystic kidney disease, an inherited disorder characterized by the formation of cysts in renal collecting ducts and biliary dysgenesis, is caused by mutations of the polycystic kidney and hepatic disease 1 (PKHD1) gene. Expression of PKHD1 is tissue specific and developmentally regulated. Here, we show that a 2.0-kb genomic fragment containing the proximal promoter of mouse Pkhd1 directs tissue-specific expression of a lacZ reporter gene in transgenic mice. LacZ is expressed in renal collecting ducts beginning during embryonic development but is not expressed in extrarenal tissues. The Pkhd1 promoter contains a binding site for the transcription factor hepatocyte nuclear factor (HNF)-1ß, which is required for activity in transfected cells. Mutation of the HNF-1ß-binding site abolishes the expression of the lacZ reporter gene in renal collecting ducts. Transgenes containing the 2.0-kb promoter and 2.7 kb of additional genomic sequence extending downstream to the second exon are expressed in the kidney, intrahepatic bile ducts, and male reproductive tract. This pattern overlaps with the endogenous expression of Pkhd1 and coincides with sites of expression of HNF-1ß. We conclude that the proximal 2.0-kb promoter is sufficient for tissue-specific expression of Pkhd1 in renal collecting ducts in vivo and that HNF-1ß is required for Pkhd1 promoter activity in collecting ducts. Additional genomic sequences located from exons 1-2 or elsewhere in the gene locus are required for expression in extrarenal tissues.
Asunto(s)
Túbulos Renales Colectores/fisiología , Riñón Poliquístico Autosómico Recesivo/fisiopatología , Regiones Promotoras Genéticas/fisiología , Receptores de Superficie Celular/fisiología , Animales , Sistema Biliar/citología , Sistema Biliar/fisiología , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/fisiología , Factor Nuclear 1-beta del Hepatocito/fisiología , Túbulos Renales Colectores/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Riñón Poliquístico Autosómico Recesivo/genética , Regiones Promotoras Genéticas/genética , Receptores de Superficie Celular/genética , Sistema Urogenital/citología , Sistema Urogenital/fisiologíaRESUMEN
Pericyte, also known as mural cell or Rouget cell, is one of the main cells that make up the wall of capillaries. Pericytes play important roles not only in the maturation, stability, and function maintenance of blood vessels, but also in the growth and development of tissues and organs, wound repair, and other physiological and pathological processes. Researches on the functions of pericytes are mainly concentrated on their multipotency, adjustment of vascular functions, and process of fibrosis, as well as on renal fibrosis, renal blood flow regulation, and glomerular filtration in urology, but are quite insufficient in andrology. This article reviews the location, origin, distribution, morphology, markers, and functions of pericytes, aiming to induce further studies of pericytes in andrology.
Asunto(s)
Pericitos/fisiología , Sistema Urogenital/citología , Fibrosis/patología , HumanosRESUMEN
Exposure to estrogen-mimicking chemicals during critical periods of development, such as infancy, may have adverse effects. However, these effects can be difficult to characterize in most epidemiologic studies. For example, growth of reproductive organs may be susceptible to estrogenic chemicals, but measuring it requires skilled ultrasound examination; timing of pubertal onset may be altered, but observing it requires long-term follow up. To address the need for a simple marker of response to estrogenic exposures in infants, we propose a novel application of a classic marker of estrogen response in adult women: cytological evaluation of urogenital epithelial cells. In this cross-sectional study of 34 female and 41 male infants, we demonstrate that epithelial cells can be obtained from swabs of the vaginal introitus (females) and urethral meatus (males), as well as from spun urine, and that these cells respond to differential estrogenic conditions, as indicated by the relative abundance of the superficial epithelial cell type. To model varying estrogen exposure, we sampled from infants who were either newborn (highly exposed to maternal estrogens), or 12 weeks old (12 W) (negligibly exposed to estrogen). Newborns had a higher percentage of superficial cells (%S), as compared to 12 W (mean ± standard error: 8.3 ± 1.8 vs. 0.9 ± 0.2) (p < 0.01), consistent with an estrogen response. This difference in %S from newborn to 12 W was observed similarly for swab (-7.6 ± 1.7) and urine (-7.3 ± 2.6) specimens and for males (-9.6 ± 2.9) and females (-5.2 ± 2.1). Examination of urogenital epithelial cells can successfully demonstrate estrogen response in both sexes, using cell specimens collected from either swab or urine sampling. In future studies, this simple, non-invasive method may be applied to assess whether estrogen-mimicking chemicals produce an estrogenic response in infants.
Asunto(s)
Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Estrógenos/farmacología , Sistema Urogenital/citología , Biomarcadores , Femenino , Humanos , Lactante , Recién Nacido , MasculinoRESUMEN
In Drosophila melanogaster, much of our understanding of sexually dimorphic neuronal development and function comes from the study of male behavior, leaving female behavior less well understood. Here, we identify a post-embryonic population of Insulin-like peptide 7 (Ilp7)-expressing neurons in the posterior ventral nerve cord that innervate the reproductive tracts and exhibit a female bias in their function. They form two distinct dorsal and ventral subsets in females, but only a single dorsal subset in males, signifying a rare example of a female-specific neuronal subset. Female post-embryonic Ilp7 neurons are glutamatergic motoneurons innervating the oviduct and are required for female fertility. In males, they are serotonergic/glutamatergic neuromodulatory neurons innervating the seminal vesicle but are not required for male fertility. In both sexes, these neurons express the sex-differentially spliced fruitless-P1 transcript but not doublesex. The male fruitless-P1 isoform (fruM) was necessary and sufficient for serotonin expression in the shared dorsal Ilp7 subset, but although it was necessary for eliminating female-specific Ilp7 neurons in males, it was not sufficient for their elimination in females. By contrast, sex-specific RNA-splicing by female-specific transformer is necessary for female-type Ilp7 neurons in females and is sufficient for their induction in males. Thus, the emergence of female-biased post-embryonic Ilp7 neurons is mediated in a subset-specific manner by a tra- and fru-dependent mechanism in the shared dorsal subset, and a tra-dependent, fru-independent mechanism in the female-specific subset. These studies provide an important counterpoint to studies of the development and function of male-biased neuronal dimorphism in Drosophila.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Neuronas/metabolismo , Neuropéptidos/metabolismo , Caracteres Sexuales , Envejecimiento , Animales , Drosophila melanogaster/fisiología , Embrión no Mamífero/metabolismo , Femenino , Fertilidad , Masculino , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Neuronas/citología , Fenotipo , Vesículas Seminales/citología , Vesículas Seminales/metabolismo , Neuronas Serotoninérgicas/citología , Neuronas Serotoninérgicas/metabolismo , Sistema Urogenital/citología , Sistema Urogenital/inervaciónRESUMEN
Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-naïve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis. The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).
Asunto(s)
Células Madre Embrionarias/citología , Epitelio/crecimiento & desarrollo , Mesodermo/crecimiento & desarrollo , Próstata/crecimiento & desarrollo , Trasplante Heterólogo/métodos , Sistema Urogenital/crecimiento & desarrollo , Animales , Células Madre Embrionarias/trasplante , Femenino , Humanos , Masculino , Mesodermo/citología , Ratones , Ratones Desnudos , Embarazo , Próstata/citología , Sistema Urogenital/citologíaRESUMEN
Stem cell enrichment provides a tool to examine prostate stem cells obtained from benign and malignant tissue. Functional assays can enrich stem cells based on common stem cell phenotypes, such as high ATP binding cassette (ABC) transporter mediated efflux of Hoechst substrates (side population assay). This functional assay is based upon mechanisms that protect cells from environmental insult thus contributing to the survival and protection of the stem cell population. We have isolated and analyzed cells digested from twelve clinical prostate specimens based on the side population assay. Prostate stem cell properties of the isolated cells were tested by serial recombination with rat urogenital mesenchyme. Recombinants with side population cells demonstrate an increase in the frequency of human ductal growth and the number of glands per recombinant when compared to recombinants with non-side population cells. Isolated cells were capable of prostatic growth for up to three generations in the recombination assay with as little as 125 sorted prostate cells. The ability to reproducibly use cells isolated by fluorescence activated cell sorting from human prostate tissue is an essential step to a better understanding of human prostate stem cell biology. ABC transporter G2 (ABCG2) was expressed in recombinants from side population cells indicating the side population cells have self-renewal properties. Epithelial cell differentiation of recombinants was determined by immunohistochemical analysis for expression of the basal, luminal, and neuroendocrine markers, p63, androgen receptor, prostate specific antigen, and chromogranin A, respectively. Thus, the ABCG2 expressing side population demonstrates multipotency and self-renewal properties indicating stem cells are within this population.
Asunto(s)
Mesodermo/citología , Próstata/citología , Células de Población Lateral/citología , Sistema Urogenital/citología , Animales , Diferenciación Celular , Separación Celular , Células Epiteliales/citología , Humanos , Masculino , RatasRESUMEN
During development neural crest (NC)-derived neuronal progenitors migrate away from the neural tube to form autonomic ganglia in visceral organs like the intestine and lower urinary tract. Both during development and in mature tissues these cells are often widely dispersed throughout tissues so that isolation of discrete populations using methods like laser capture micro-dissection is difficult. They can however be directly visualized by expression of fluorescent reporters driven from regulatory regions of neuron-specific genes like Tyrosine hydroxylase (TH). We describe a method optimized for high yields of viable TH+ neuronal progenitors from fetal mouse visceral tissues, including intestine and lower urogenital tract (LUT), based on dissociation and fluorescence-activated cell sorting (FACS). The Th gene encodes the rate-limiting enzyme for production of catecholamines. Enteric neuronal progenitors begin to express TH during their migration in the fetal intestine and TH is also present in a subset of adult pelvic ganglia neurons . The first appearance of this lineage and the distribution of these neurons in other aspects of the LUT, and their isolation has not been described. Neuronal progenitors expressing TH can be readily visualized by expression of EGFP in mice carrying the transgene construct Tg(Th-EGFP)DJ76Gsat/Mmnc. We imaged expression of this transgene in fetal mice to document the distribution of TH+ cells in the developing LUT at 15.5 days post coitus (dpc), designating the morning of plug detection as 0.5 dpc, and observed that a subset of neuronal progenitors in the coalescing pelvic ganglia express EGFP. To isolate LUT TH+ neuronal progenitors, we optimized methods that were initially used to purify neural crest stem cells from fetal mouse intestine. Prior efforts to isolate NC-derived populations relied upon digestion with a cocktail of collagenase and trypsin to obtain cell suspensions for flow cytometry. In our hands these methods produced cell suspensions from the LUT with relatively low viability. Given the already low incidence of neuronal progenitors in fetal LUT tissues, we set out to optimize dissociation methods such that cell survival in the final dissociates would be increased. We determined that gentle dissociation in Accumax (Innovative Cell Technologies, Inc), manual filtering, and flow sorting at low pressures allowed us to achieve consistently greater survival (>70% of total cells) with subsequent yields of neuronal progenitors sufficient for downstream analysis. The method we describe can be broadly applied to isolate a variety of neuronal populations from either fetal or adult murine tissues.
Asunto(s)
Citometría de Flujo/métodos , Células-Madre Neurales/citología , Animales , Embrión de Mamíferos , Femenino , Intestinos/citología , Intestinos/embriología , Ratones , Embarazo , Sistema Urogenital/citología , Sistema Urogenital/embriologíaRESUMEN
Gangliosides have been suggested to play important roles in various functions such as adhesion, cell differentiation, growth control, and signaling. Mouse follicular development, ovulation, and luteinization during the estrous cycle are regulated by several hormones and cell-cell interactions. In addition, spermatogenesis in seminiferous tubules of adult testes is also regulated by several hormones, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and cell-cell interactions. The regulation of these processes by hormones and cell-cell interactions provides evidence for the importance of surface membrane components, including gangliosides. During preimplantation embryo development, a mammalian embryo undergoes a series of cleavage divisions whereby a zygote is converted into a blastocyst that is sufficiently competent to be implanted in the ma ternal uterus and continue its development. Mouse embryonic stem (mES) cells are pluripotent cells derived from mouse embryo, specifically, from the inner cell mass of blastocysts. Differentiated neuronal cells are derived from mES cells through the formation of embryonic bodies (EBs). EBs recapitulate many aspects of lineage-specific differentiation and temporal and spatial gene expression patterns during early embryogenesis. Previous studies on ganglioside expression during mouse embryonic development (including during in vitro fertilization, ovulation, spermatogenesis, and embryogenesis) reported that gangliosides were expressed in both undifferentiated and differentiated (or differentiating) mES cells. In this review, we summarize some of the advances in our understanding of the functional roles of gangliosides during the stages of mouse embryonic development, including ovulation, spermatogenesis, and embryogenesis, focusing on undifferentiated and differentiated mES cells (neuronal cells).
Asunto(s)
Diferenciación Celular , Desarrollo Embrionario , Células Madre Embrionarias/citología , Gangliósidos/metabolismo , Animales , Células Madre Embrionarias/metabolismo , Gametogénesis , Ratones , Sistema Urogenital/citología , Sistema Urogenital/embriología , Sistema Urogenital/metabolismoRESUMEN
Gangliosides have been suggested to play important roles in various functions such as adhesion, cell differentiation, growth control, and signaling. Mouse follicular development, ovulation, and luteinization during the estrous cycle are regulated by several hormones and cell-cell interactions. In addition, spermatogenesis in seminiferous tubules of adult testes is also regulated by several hormones, including follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and cell-cell interactions. The regulation of these processes by hormones and cell-cell interactions provides evidence for the importance of surface membrane components, including gangliosides. During preimplantation embryo development, a mammalian embryo undergoes a series of cleavage divisions whereby a zygote is converted into a blastocyst that is sufficiently competent to be implanted in the maternal uterus and continue its development. Mouse embryonic stem (mES) cells are pluripotent cells derived from mouse embryo, specifically, from the inner cell mass of blastocysts. Differentiated neuronal cells are derived from mES cells through the formation of embryonic bodies (EBs). EBs recapitulate many aspects of lineage-specific differentiation and temporal and spatial gene expression patterns during early embryogenesis. Previous studies on ganglioside expression during mouse embryonic development (including during in vitro fertilization, ovulation, spermatogenesis, and embryogenesis) reported that gangliosides were expressed in both undifferentiated and differentiated (or differentiating) mES cells. In this review, we summarize some of the advances in our understanding of the functional roles of gangliosides during the stages of mouse embryonic development, including ovulation, spermatogenesis, and embryogenesis, focusing on undifferentiated and differentiated mES cells (neuronal cells).
Asunto(s)
Animales , Ratones , Diferenciación Celular , Desarrollo Embrionario , Células Madre Embrionarias/citología , Gametogénesis , Gangliósidos/metabolismo , Sistema Urogenital/citologíaRESUMEN
The transcription factor OCT4 plays a crucial role in the earliest differentiation of the mammalian embryo and in self-renewal of embryonic stem cells. However, it remains controversial whether this gene is also expressed in somatic tissues. Here, we use a combination of RT-PCR on whole and microdissected tissues, in situ hybridization, immunohistochemistry and western blotting to show that OCT4 and SOX2 together with downstream targets, UTF1 and REX1/ZFP42, are expressed in the human male urogenital tract. We further support these results by the analysis of DNA methylation of a region in the OCT4 promoter. In culture, human primary epididymal cells formed spheres that continued to express the investigated genes for at least 20 days. Transcriptomic analysis of cultured cells showed up-regulation of CD29, CD44 and CD133 that are normally associated with sphere-forming cancer stem cells. Furthermore, stimulation with retinoic acid resulted in down-regulation of OCT4 expression, however, without multilineage differentiation. Our results show that OCT4 and associated genes are expressed in somatic epithelial cells from the urogenital tract and that these cells can form spheres, a general marker of stem cell behaviour.
Asunto(s)
Epidídimo/citología , Células Epiteliales/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Sistema Urogenital/citología , Células Cultivadas , Células Madre Embrionarias/citología , Epidídimo/efectos de los fármacos , Epidídimo/metabolismo , Células Epiteliales/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Queratolíticos/farmacología , Masculino , Análisis por Micromatrices , Próstata/citología , Próstata/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tretinoina/farmacologíaRESUMEN
The Julia Creek dunnart (Sminthopsis douglasi) is an endangered carnivorous marsupial belonging to the family Dasyuridae. This study investigated the oestrous cycle of this species in terms of its reproductive physiology and behaviour to explore more efficient methods of oestrus detection. Ten sexually mature captive female dunnarts were monitored daily at David Fleay Wildlife Park, Burleigh Heads, Australia, from mid September to late December 2006 for changes in urogenital cytology within the urine (0, 1+, 2+ and 3+), running wheel activity, body weight, uneaten food, faecal steroid metabolites (progesterone and oestradiol) and pouch development. Periods of increased running wheel activity were associated (p=0.004) with an increase in the proportion of cornified urogenital epithelial cells found in the urine; periods of decreasing weight (p<0.001) and uneaten food (p<0.001) were also associated with changes in urogenital cytology but not to the point where they would be useful for oestrus detection. Between 60.3% and 92.0% of peak distances (confidence interval 95%) occurred when the epithelial cell index was 2+ or 3+. Only 15.5-37.5% of peak weights (CI: 95%) and 28.1-49.9% of incidences of uneaten food (CI: 95%) occurred when the epithelial cell index was 2+ or 3+. There was no significant difference in the mean length of the oestrous cycle when measured by urogenital cytology (mean+/-SD: 25.0+/-5.7 days; n=20) or peak distance travelled (mean+/-SD: 25.4+/-5.7 days; n=20). Changes in the concentration of oestradiol metabolites in Julia Creek Dunnart faeces were not useful in characterising the oestrous cycle. Wheel running activity declined markedly with increased faecal progestagen concentration. The majority of the pouch variables examined showed maximum development during the inter-oestrus period but as there was considerable variation between animals, the pouch was not considered a useful index of oestrus.