RESUMEN
Epididymosomes (apocrine secreted epididymal vesicles) are assumed to play a crucial role in sperm maturation. Our aim has been to analyze the fusogenic properties of bovine epididymosomes and their involvement in the transfer of membrane components (lipids, proteins, plasma membrane Ca(2+)-ATPase 4 [PMCA4]) into bovine sperm. The fusogenic properties of epididymosomes with spermatozoa were investigated in vitro by using octadecyl rhodamine-B (R18)-labeled epididymosomes. Spermatozoa isolated from the epididymal caput showed a higher fusion rate than those taken from the cauda. The fusion rate was dependent on pH and time. Furthermore, the lipid and protein content in spermatozoa changed during epididymal transit and after in vitro fusion with epididymosomes. Following the in vitro fusion of caput spermatozoa with epididymosomes, the cholesterol/total phospholipid ratio of the sperm plasma membrane decreased. The effect was comparable with the cholesterol/total phospholipid ratio of native cauda spermatozoa. Co-incubation experiments of spermatozoa with biotinylated epididymosomes additionally revealed that proteins were transferred from epididymosomes to sperm. To examine the potential transfer of epididymis-derived PMCA4 to spermatozoa, immunofluorescence analysis and Ca(2+)-ATPase activity assays were performed. In caput spermatozoa, the PMCA4 fluorescence signal was slightly raised and Ca(2+)-ATPase activity increased after in vitro fusion. Thus, our experiments indicate significant changes in the lipid and protein composition of epididymal sperm following interaction with epididymosomes. Moreover, our results substantiate the presumption that PMCA4 is transferred to spermatozoa via epididymosomes.
Asunto(s)
Maduración del Esperma/fisiología , Espermatozoides/metabolismo , Animales , Transporte Biológico Activo/fisiología , Bovinos , Colesterol/metabolismo , Epidídimo/citología , Epidídimo/metabolismo , Metabolismo de los Lípidos/fisiología , Masculino , Fosfolípidos/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Espermatozoides/citologíaRESUMEN
The plasma membrane Ca(2+) -ATPase (PMCA) is the main restorer of Ca(2+) balance in sperm. Particularly, PMCA isoform 4 has an essential function in sperm fertility by its participation in gaining sperm hypermotility. PMCA activity is influenced by its lipid environment. Sperm membranes exhibit lipid raft microdomains or detergent-resistant membrane domains, enriched in sphingolipids and cholesterol, forming functional specialized areas. Lipid and protein composition of lipid rafts alters during the capacitation process, which is characterized by a cholesterol efflux. In this study, the localization of PMCA4 in lipid membrane fractions of the sperm plasma membrane was investigated. We identified PMCA4 in both the detergent-resistant membrane (DRM) and in the detergent-soluble (DS) fraction of caput and cauda sperm, respectively. Capacitation did not influence PMCA4 localization. In immunocytochemical studies PMCA4 was co-localized with the lipid raft/DRM marker caveolin in the mid piece of caput and cauda sperm. Functional studies with seminal vesicle major protein PDC-109 showed that the Ca(2+) -ATPase activity in DS fractions of cauda sperm and capacitated cauda sperm was stronger enhanced than in the DRMs. In both fractions the effect was statistically significant. In contrast, in lipid overlay experiments PDC-109 interacted stronger with the lipids extracted from DRMs than with lipids extracted from DS. Our results indicate a possible functional compartmentalization of PMCA in bull sperm membranes and point to a presumptive, yet unknown interaction partner of Ca(2+) -ATPase and PDC-109, mediating the PDC-109 action from DRMs to the DS fraction of sperm plasma membrane.
Asunto(s)
Membrana Celular/metabolismo , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Proteínas de Secreción de la Vesícula Seminal/fisiología , Espermatozoides/metabolismo , Animales , Bovinos , Inmunohistoquímica , Magnesio/farmacología , Masculino , Microdominios de Membrana/fisiología , Capacitación EspermáticaRESUMEN
BACKGROUND AND PURPOSE: Intraglandular injection of botulinum toxin (BoNT) leads to a transient denervation of the submandibular gland and this is associated with reduced salivary secretion. The purpose of the present study was to verify whether temporary acinar atrophy occurs simultaneously with chemical denervation of the glands. EXPERIMENTAL APPROACH: Tissue specimens of the right submandibular gland taken from 18 Wistar rats after intraglandular injection of BoNT A, BoNT B, or a combination of both were examined. As a sham control, an equivalent volume of saline was injected into the left submandibular gland. Morphometric measurements, immunohistochemistry, electron microscopy and western blot analysis were used to analyse the morphological and functional changes of the denervated glands. KEY RESULTS: Morphological and ultrastructural analyses of the cell organelles and secretory granula showed a clear atrophy of the acini, which was more prominent in glands injected with the combination of BoNT/A and B. Morphometric measurements of the glandular acini revealed a significant reduction of the area of the acinar cells after injection of BoNT (P=0.031). The expression of amylase was significantly reduced in BoNT treated glands. CONCLUSIONS AND IMPLICATIONS: Intraglandular application of BoNT induces structural and functional changes of the salivary glands indicated by glandular atrophy. These effects may be due to glandular denervation induced by the inhibition of the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) involved in acetylcholine release at the neuroglandular junction and also specially inhibition of those involved in exocytosis of the granula of the acinar cells.
Asunto(s)
Toxinas Botulínicas Tipo A/toxicidad , Toxinas Botulínicas/toxicidad , Parasimpatectomía , Sistema Nervioso Parasimpático/efectos de los fármacos , Glándula Submandibular/efectos de los fármacos , Amilasas/análisis , Animales , Atrofia , Tamaño de la Célula , Masculino , Microscopía Electrónica , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Wistar , Glándula Submandibular/enzimología , Glándula Submandibular/inervación , Glándula Submandibular/ultraestructuraRESUMEN
A complete documentation of German anatomical science and its representatives during the period of national socialism has not been published as yet--contrary to the situation in other medical disciplines. Instead of German anatomists, American anatomists have occasionally addressed this issue during their meetings and have reported on special aspects, such as the use of Nazi symbols in anatomical textbooks and atlases (Pernkopf 1952) and the use of corpses of justice victims for anatomical research and student education. Also, the genesis of the atrocious collection of "racial" skulls, initiated along with the SS-institution of the "Ahnenerbe" by the anatomist August Hirt of Strasbourg (who ordered more than 90 inmates from concentration camps to be murdered in the gas chamber built in the concentration camp of Natzweiler-Struthof close to Strasbourg, Alsace) has been described by Frederic Kasten and others. A broader view of the patterns of behaviour and political actions and fates of contemporary scientists, ranging from dismissal to clandestine opportunism, affirmative cooperation and fanatic activism can be obtained by the analysis of the activities in research, medical education and academic positions of the following members of the Institute of Anatomy at the Philipp-University in Marburg: Ernst Göppert, Eduard Jacobshagen, Ernst-Theodor Nauck, Adolf Dabelow, Helmut Becher and Alfred Benninghoff, whose activities and fates differ in several respects and allow more general deductions. Also, the individual fates of a number of prosecuted Jewish anatomists (Wassermann, München; Poll, Hamburg), of devoted and active members of the Nazi party (Clara, Leipzig; Blotevogel, Breslau) and of criminal fanatics (Hirt, Strasbourg; Kremer, Münster) are briefly discussed. The present contribution is an attempt to initiate a more detailed study of all German departments of anatomy during the Hitler regime and to generate a public discussion among the younger generation of German anatomists.
Asunto(s)
Anatomía/historia , Nacionalsocialismo/historia , Alemania , Historia del Siglo XX , Humanos , Judíos/historia , Universidades/historiaRESUMEN
Glucocorticoids are anti inflammatory stress hormones and have been suggested to be involved in a large number of pathological processes. To test the effects of glucocorticoids on stromal prostatic cell growth and proliferation in vitro, the influence of a synthetic glucocorticoid (dexamethasone, dex) on recently established human primary cells from prostatic stroma (hPCPs) was analysed. The localization and distribution of the glucocorticoid receptor (GR) was investigated by immunohistochemistry. In addition, expression of the active isoform of the receptor (alpha-GR) was examined by reverse transcription PCR, and the effect of different doses of dex on proliferation of the stromal cells evaluated using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and amido black assays. alpha-GR mRNA was expressed by the hPCPs, and the GR protein was detected in the cytoplasm and nucleus of these cells. Incubating the cells with dex resulted in an enhanced cell proliferation that was mainly restricted to the fibroblasts. Moreover, fibronectin (FN) gene expression and secretion of the protein was increased by high doses of dex (> or = 10(-8) M), whereas low doses of dex (10(-10)M) showed no effect. Human prostatic stromal cells show sensitivity to dex in vitro, resulting in an increase in cell proliferation and FN synthesis. The authors assume that locally accumulating glucocorticoids can also influence the regulation of cell growth and extracellular matrix synthesis in the human prostate in vivo and may play a role in the pathologically altered prostate.
Asunto(s)
División Celular/efectos de los fármacos , Dexametasona/farmacología , Fibronectinas/biosíntesis , Glucocorticoides/farmacología , Próstata/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Células Cultivadas , Colorimetría , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/citología , Fibronectinas/genética , Técnica del Anticuerpo Fluorescente , Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Músculo Liso/citología , Próstata/citología , Próstata/metabolismo , ARN Mensajero/análisis , Receptores de Glucocorticoides/análisis , Receptores de Glucocorticoides/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
The control of testicular development and differentiation depends on hormones and a variety of cell-cell interactions mediated mainly by paracrine factors. In the second and third weeks of post-natal development important changes take place in the rat testis, e.g. the tubular lumen starts to open on post-natal day 10, the blood-testis barrier starts to form on day 15, and Sertoli cell proliferation ceases on day 15. In the present study the expression in different testicular compartments of the androgen receptor (AR), progesterone receptor (PR), and extracellular matrix proteins such as laminin, entactin-1 (nidogen-1) and fibronectin, during post-natal development was examined using immunohistochemistry and semiquantitative image analysis. An intratubular AR peak on days 14-17, an increase in intratubular PR expression on days 14-16, and an increase in peritubular entactin-1 expression during the second and third weeks post-partum are demonstrated. These results suggest that a variety of changes occur at the cellular level during this period when certain milestones of testicular development occur, substantiating the hypothesis of a particular role for paracrine interactions during the development of the rat testis.
Asunto(s)
Proteínas de la Matriz Extracelular/análisis , Receptores Androgénicos/análisis , Receptores de Progesterona/análisis , Testículo/crecimiento & desarrollo , Envejecimiento , Animales , Vasos Sanguíneos/química , Núcleo Celular/química , Femenino , Fibronectinas/análisis , Inmunohistoquímica , Laminina/análisis , Células Intersticiales del Testículo , Masculino , Glicoproteínas de Membrana/análisis , Ratas , Ratas Wistar , Células de Sertoli/ultraestructura , Espermatogonias/ultraestructura , Testículo/irrigación sanguínea , Testículo/químicaRESUMEN
The main oligosaccharide residues and the saccharide linkage in infantile and adult human seminal vesicles were studied by means of lectin histochemistry at light and electron microscopy levels. In adult glands, the epithelial cell cytoplasm and luminal content reacted positively to the following residues: (GlcNAc)n (WGA), Galbeta1,3GalNAc (PNA), GalNAcalpha1,3Gal (SBA), GalNAcalpha1,3GalNAc (HPA), Fucalpha1,2Galbeta1,4GlcNAc (UEA-I), and alphaL-Fuc1,6DGlcNAc-O-Melibiosc (AAA). The presence of intense staining in the luminal content suggest that glycoproteins containing these oligosaccharide moieties are secreted by epithelial cells. Adult epithelial cells also reacted to Neu5Acalpha2,6Gal (SNA), Neu5Acalphaa2,3Galbeta1,4GlcNAc (MAA), Galbeta1,4GlcNAc (DSA), branched mannose chains (ConA), Man1,3Man (GNA), and Fucalpha1,2Galbeta1,4GlcNAcFucalpha1,3GlcNAc (LTA) but reaction to these residues was weak (MAA, DSA, ConA, and LTA) or absent (SNA and GNA) in the gland lumen, which suggests that they belong to intracytoplasmic proteins. The chemical and enzymatic treatments used suggest that the residues recognized by SNA, MAA, PNA, DSA, HPA, and SBA belong to O-linked oligosaccharides; those residues localized by ConA and GNA have an N-glycosidic linkage, and those bound by WGA, LTA, UEA-I, and AAA are linked to both N- and O-oligosaccharides. In prepubertal seminal vesicles, reaction in the epithelial cell cytoplasm was similar to that observed in adults, except for GNA and HPA, which showed a weaker reaction. However, the lumen of prepubertal seminal vesicles showed intense reaction to WGA and SBA only. The chemical and enzymatic treatments suggest that the scanty glycoproteins secreted by the prepubertal glands belong to the mucin-type.
Asunto(s)
Oligosacáridos/análisis , Vesículas Seminales/química , Epitelio Seminífero/química , Adulto , Factores de Edad , Humanos , Lactante , Lectinas , Masculino , Microscopía Electrónica , Vesículas Seminales/ultraestructura , Epitelio Seminífero/ultraestructuraRESUMEN
In this prospective clinical study, 892 patients with normal and impaired semen were examined in order to investigate the correlation between the concentration of fibronectin in seminal plasma and the motility of spermatozoa. The fibronectin concentration in seminal plasma, total sperm motility and linear sperm motility were measured. We report here a significant negative correlation between the fibronectin concentration in seminal plasma and total sperm motility (r=-0.3474). There was no link between varicocele and vasectomy, or between varicocele and variation in the concentration of fibronectin. It is concluded that higher concentrations of the acute-phase protein fibronectin may be a cause of severe reduction in sperm motility. The investigation of fibronectin concentrations in seminal fluid could be a new and helpful clinical tool in the assessment of male fertility.
Asunto(s)
Fibronectinas/análisis , Semen/química , Motilidad Espermática , Adolescente , Adulto , Biomarcadores/análisis , Humanos , Masculino , Persona de Mediana Edad , Valores de Referencia , Análisis de Regresión , Varicocele/fisiopatología , VasectomíaRESUMEN
BACKGROUND: The neuroendocrine cells of the human prostate have been related to proliferative disorders such as prostatic cancer. Their origin, distribution, and development have therefore been studied and discussed in terms of current stem cell concepts in the prostate. METHODS: Prostatic tissue specimens (n = 20) from human fetuses (n = 8), prepubertal and pubertal children (n = 8) and mature men (n = 4) were studied immunohistochemically using antibodies directed against neuroendocrine, epithelial as well as secretory markers. Semiquantitative computer-assisted evaluation of different epithelial and stromal components based on stereological principles was performed on azan-stained sections representative of all developmental stages. RESULTS: By the end of gestational Week 9, neuroendocrine (NE) cells appear in the epithelium of the urogenital sinus and are subsequently closely associated with the formation of urethral prostatic buds. The fetal and postnatal distribution pattern of NE cells within the gland is characterized by a relatively constant number of cells per gland similar to prostatic smooth muscle cells. Likewise, a density gradient exists with the highest density in the large collicular ducts and almost no NE cells in subcapsular peripheral acini. In peripheral ducts, the distribution is random. Maturation of the NE cells precedes that of the secretory cells by about 10-16 years. CONCLUSIONS: A second prostatic stem cell lineage, different from the urogenital sinus (UGS)-lineage is hypothesized originating from immature neuroendocrine cells. Being morphologically indistinguishable from the UGS-derived prostatic secretory cell lineage, it gives rise to neuroendocrine cells. Their presence is apparently important for proliferation regulation of the UGS-derived lineage of the prostate.
Asunto(s)
Sistemas Neurosecretores/citología , Próstata/citología , Adolescente , Adulto , Niño , Preescolar , Células Epiteliales/citología , Epitelio/embriología , Epitelio/crecimiento & desarrollo , Humanos , Recién Nacido , Masculino , Próstata/embriología , Próstata/crecimiento & desarrollo , Células Madre/citologíaRESUMEN
BACKGROUND: An antibody directed against a 100 kDa protein was immunoselected from a polyvalent antiserum against human prostasomes. The antibody as well as biochemical characteristics of the respective antigen were used to study the structural relationship of the latter with prostate membrane specific antigen (PMSA), another 100 kDa membrane protein of the prostate. METHODS: The isolated purified 100 kDa protein was characterized by tryptic degradation, aminoacid-sequencing and mass spectroscopy peptide-fingerprinting as well as mono-saccharide analysis and lectin binding and identified as a prostasomal neutral endopeptidase (NEP, EC 3.4.24.11). Immunohistochemistry, immunoelectron microscopy, in situ hybridization, and RT-PCR were performed to analyze the expression and distribution of the protein in normal and malignant human prostatic tissues and cell lines. RESULTS: Prostatic NEP, which has no relationship with PMSA, is a glycosylated, integral membrane protein type II. The prevalent glycosyl residues are NeuNAc, GlcNAc, GalNAc, Gal, Man, Fuc. NEP-mRNA is expressed in human prostatic epithelial and some stromal cells. NEP-immunoreactivity is strong in normal prostatic epithelium and confined to the apical plasma membrane. During apocrine secretion, the enzyme is released from the secretory cells, contributing to the formation of prostasomes. In prostate cancer specimens, immunoreactivity of apical plasma membranes is lost, while generalized cytoplasmic immunoreactivity develops. CONCLUSIONS: Prostatic secretory cells contain a membrane-bound, highly glycosylated neutral endopeptidase which is restricted to the apical plasma membrane. The enzyme is released from the cells in an apocrine fashion and contributes to the formation of prostasomes. In prostate cancer cells a preferential cytoplasmic localization is observed, pointing to alterations in intracellular targeting.
Asunto(s)
Neprilisina/metabolismo , Próstata/enzimología , Vesículas Secretoras/enzimología , Secuencia de Aminoácidos , Secuencia de Carbohidratos , Línea Celular , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Neprilisina/genética , Neprilisina/aislamiento & purificación , Próstata/metabolismo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Semen/enzimología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Fracciones Subcelulares/enzimologíaRESUMEN
Transforming growth factor-beta2 (TGFbeta2) is an important mediator of growth and differentiation. We here describe for the first time the complete sequence of the TGFbeta2 complementary DNA derived from peritubular myoid cells of the rat testis. The size of the rat TGFbeta2 complementary DNA was 1245 bp, and the deduced protein sequence contained 414 amino acids. Sequence comparison with the human and mouse amino acid sequences demonstrated 96.4% and 97.9% sequence identities, respectively. To elucidate the functional role of TGFbeta2 in testicular somatic cells, we studied its secretion in vitro in monocultures and cocultures of mesenchymal peritubular and epithelial Sertoli cells. The highest amounts of TGFbeta2 protein were secreted in the cocultures and by peritubular cells, whereas Sertoli cells secreted only minor amounts. Stimulation experiments with FSH revealed a reduced secretion of TGFbeta2 in cocultures, probably mediated by a paracrine interaction of the FSH-responsive Sertoli cells. In contrast, TGFbeta2 secretion by peritubular cells was increased after stimulation with glucocorticoids and after addition of recombinant TGFbeta2, indicating an autoregulation of TGFbeta2. Furthermore, application of recombinant TGFbeta2 to cocultures resulted in an enhanced aggregation and cell clustering of Sertoli cells, pointing to an important role of TGFbeta2 in the paracrine interaction of peritubular and Sertoli cells of the developing rat testis.
Asunto(s)
Comunicación Celular/fisiología , Mesodermo/fisiología , Testículo/citología , Testículo/fisiología , Factor de Crecimiento Transformador beta/fisiología , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Células Cultivadas , Técnicas de Cocultivo , ADN Complementario/genética , Dexametasona/farmacología , Células Epiteliales/fisiología , Epitelio/fisiología , Glucocorticoides/farmacología , Masculino , Mesodermo/citología , Datos de Secuencia Molecular , Ratas , Ratas Wistar , Células de Sertoli/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta2RESUMEN
BACKGROUND: Calcitonin-related peptides have been found in the human prostate, and calcitonin (CT) and calcitonin gene-related peptide (CGRP) have been demonstrated in subpopulations of neuroendocrine (NE) cells. The purpose of this study was to determine the concentrations of CT and CGRP as well as the densities of NE cells in normal prostates, benign prostatic hyperplasia (BPH), and carcinoma of the prostate (CAP). METHODS: In 42 specimens of radical prostatectomy, the number of CT- and CGRP-immunoreactive NE cells in areas of normal and BPH tissue was determined, and compared with CAP tissue using immunocytochemistry. In addition, by radioimmunoassay (RIA), tissue levels of CT and CGRP were analyzed in extracts from areas of normal, BPH, and CAP tissue, as verified by adjacent histologic sections. RESULTS: A significant decrease in CT-immunoreactive NE cells was observed in hyperplastic nodules of BPH in comparison to normal tissue. These findings were in parallel with a significant reduction in tissue CT level in BPH compared to normal tissue. There was also a marked, but statistically nonsignificant, reduction in CT levels in CAP tissue. In contrast, levels of CGRP in BPH and CAP tissue did not show any significant differences compared to normal tissue. CONCLUSIONS: CT and CGRP are present in NE cells of the human prostate. Calcitonin levels are significantly reduced in BPH, in parallel with a decreased number of CT-immunoreactive NE cells, whereas no significant changes in tissue levels of CGRP were observed. The functional significance of these findings is discussed.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/análisis , Calcitonina/análisis , Carcinoma/patología , Próstata/química , Hiperplasia Prostática/patología , Neoplasias de la Próstata/patología , Anciano , Calcitonina/genética , Péptido Relacionado con Gen de Calcitonina/genética , Carcinoma/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Próstata/patología , Próstata/fisiología , Prostatectomía , Hiperplasia Prostática/genética , Neoplasias de la Próstata/genética , RadioinmunoensayoRESUMEN
Macrophage migration inhibitory factor (MIF), originally described as a T-cell product, has recently been identified in several endocrine organs. In the rat testis, MIF is secreted by the Leydig cells into testicular interstitial fluid that directly contacts Sertoli and peritubular cells. To investigate whether MIF is involved in calcium-dependent signal transduction, we have isolated rat Sertoli and peritubular cells. Despite progress in understanding functional properties of MIF, the molecular mechanism of MIF action in target cells is almost completely unknown. Here we find that recombinant MIF evokes a transient increase in calcium levels in peritubular cells but not in Sertoli cells from dissociated rat testis. Concentrations in the range between 12.5 ng/ml and 120 ng/ml of recombinant MIF were found to be effective, with 50 ng/ml yielding the largest increase in intracellular calcium. Preincubation of MIF with a neutralizing monoclonal antibody specifically blocked the response. Incubation of the peritubular cells in calcium-free buffer clearly decreased the evoked response in intracellular calcium concentration. However, the calcium response was greatly decreased by thapsigargin, an inhibitor of the Ca(2+) ATPase of the endoplasmic reticulum. The results strongly indicate that calcium is mobilized from reticulum stores during MIF-mediated signal transduction in the testis. In conclusion, our results provide the first characterization of MIF signal transduction in the testis and suggest that signaling from Leydig cells to peritubular cells through MIF is mediated by receptors coupled to release of intracellular calcium.
Asunto(s)
Calcio/metabolismo , Factores Inhibidores de la Migración de Macrófagos/farmacología , Testículo/metabolismo , Animales , Anticuerpos Monoclonales/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Técnicas de Inmunoadsorción , Células Intersticiales del Testículo/metabolismo , Masculino , Ratas , Ratas Wistar , Proteínas Recombinantes/farmacología , Células de Sertoli/metabolismo , Transducción de Señal , Testículo/citología , Testículo/efectos de los fármacos , Tapsigargina/farmacologíaRESUMEN
BACKGROUND: An in vitro model of prostatic stromal cells suitable for experimental studies of the pathogenesis of BPH is still lacking. We therefore standardized the isolation, cultivation, and characterization of human prostatic stromal cell lineages. METHODS: Stromal cells were isolated from a surgical specimen of BPH. Using antibodies specific for either epithelial or stromal cells of the human prostate, the isolated cells were morphologically and immunohistochemically characterized. Viability and functional activity were assessed by proliferation assays and stimulation experiments. Gene expression was monitored by RT-PCR. RESULTS: In early passages (P8), cells showed a high purity (>/=98%) for stromal markers; about 60% displayed the characteristics of fibroblasts, and the remaining 40% were classified as smooth muscle cells. In late passages (P20), the proportion of muscle cells declined to 10%. Stimulation experiments including basic fibroblast growth factor (bFGF) resulted in enhanced proliferation, whereas dihydrotestosterone (DHT), estrogen, and flutamide did not influence proliferation. Gene expression studies demonstrated a positive signal for androgen receptor and keratinocyte growth factor (KGF). CONCLUSIONS: Prostatic stromal cells can be propagated several times and show karyotypic stability for up to 18 subculture experiments. The ratio of myoid and fibroblastic cells can be used for standardization of cell cultures with stable characteristics.
Asunto(s)
Próstata/patología , Células del Estroma/patología , División Celular/efectos de los fármacos , Línea Celular , Células Cultivadas , Factor 2 de Crecimiento de Fibroblastos/farmacología , Fibroblastos/patología , Hormonas/farmacología , Humanos , Inmunohistoquímica , Cariotipificación , Masculino , Persona de Mediana Edad , Músculo Liso/patología , Próstata/ultraestructura , Receptores Androgénicos/genética , Células del Estroma/efectos de los fármacos , Células del Estroma/fisiología , Células del Estroma/ultraestructuraRESUMEN
Epithelial-like Sertoli cells isolated from immature rat testis aggregate to form tubule-like structures when cultured on a monolayer of mesenchyme-derived peritubular cells. At the end of this morphogenetic process both cell types are separated by a basement membrane. In this study the gene expression of monocultures and direct cocultures of peritubular cells and Sertoli cells was examined using DD-RT-PCR. One of the isolated cDNA clones showed high homology to the cDNA encoding the basement membrane component entactin-1 (nidogen-1). Even though the entactin-1 (nidogen-1) gene is transcribed in peritubular cells, Sertoli cells, and in direct cocultures, the mRNA is translated only by the peritubular cells. No entactin-1 (nidogen-1) was detected in the Sertoli cells by Western blotting. Moreover, peritubular cell monocultures and cocultures showed the presence of one single band at 152 kDa in the supernatant, whereas in cell lysates two bands were detectable at 152 kDa and 150 kDa. Perturbation experiments using monoclonal antibodies directed against entactin-1 (nidogen-1) were performed with peritubular cells and Sertoli cells, respectively, and demonstrated loss of cell adhesion of the peritubular cells, while the Sertoli cells remained adherent. From these data we conclude that entactin-1 is exclusively produced and secreted by mesenchymal peritubular cells, and affects adhesion of peritubular cells in an autocrine manner.
Asunto(s)
Glicoproteínas de Membrana/fisiología , Testículo/citología , Testículo/fisiología , Animales , Secuencia de Bases , Membrana Basal/fisiología , Adhesión Celular/fisiología , Células Cultivadas , Proteínas de la Matriz Extracelular/fisiología , Masculino , Mesodermo/citología , Datos de Secuencia Molecular , Ratas , Ratas Wistar , Alineación de Secuencia , Células de Sertoli/citología , Células de Sertoli/fisiologíaRESUMEN
In this study, the occurrence of the glucocorticoid receptor in the rat testis during early stages of postnatal development and its potential functional significance were investigated. Quantitative analyses of immunohistochemically labelled paraffin sections revealed that the receptor was present during all stages of postnatal development in the nuclei of interstitial cells such as Leydig cells, macrophages and fibroblasts, and endothelial cells of blood vessels. The labelling index increased initially, with maximum levels reached within the second week of postnatal development, and decreased thereafter. Within the seminiferous tubules, the glucocorticoid receptor could be detected in the nuclei of germ cells as well as Sertoli cells, reaching the highest levels in 3-week-old rats, mainly due to immature germ cell staining. In contrast, approximately 50% of the peritubular cell nuclei were stained throughout postnatal development. In vitro experiments on immature and immortalized peritubular cells demonstrated a dose-dependent and significant decrease in proliferation and fibronectin secretion after administration of dexamethasone. The data of this study suggest that glucocorticoids have a consistently repressive effect on peritubular cells throughout postnatal development. In summary, labelling of germ cells, especially in immature rats, might indicate an inhibition of spermatogenesis by corticosteroids.
Asunto(s)
Receptores de Glucocorticoides/metabolismo , Testículo/crecimiento & desarrollo , Testículo/metabolismo , Animales , División Celular/efectos de los fármacos , Línea Celular , Dexametasona/farmacología , Femenino , Fibronectinas/metabolismo , Inmunohistoquímica , Técnicas In Vitro , Masculino , Ratas , Ratas Wistar , Espermatogénesis/efectos de los fármacos , Testículo/efectos de los fármacosRESUMEN
In the present study the activity of a 20% methanolic extract of stinging nettle roots (Urtica dioica L., Urticaceae) on the proliferative activity of human prostatic epithelial (LNCaP) and stromal (hPCPs) cells was evaluated using a colorimetric assay. A concentration-dependent and significant (p < 0.05) antiproliferative effect of the extract was observed only on LNCaP cells during 7 days, whereas stromal cell growth remained unaltered. The inhibition was time-dependent with the maximum of growth reduction (30%) at a concentration of 1.0E-6 mg/ml on day 5 compared to the untreated control. On day 4 and 6, the reduction in proliferation of LNCaP cells showed the minimal effective dose at 1.0E-9 mg/ml. No cytotoxic effect of ME-20 on cell proliferation was observed. The antiproliferative effect of ME-20 of stinging nettle roots observed both in an in vivo model and in an in vitro system clearly indicates a biologically relevant effect of compounds present in the extract.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , División Celular/efectos de los fármacos , Extractos Vegetales/farmacología , Plantas Medicinales/química , Neoplasias de la Próstata/patología , Línea Celular , Ensayos de Selección de Medicamentos Antitumorales , Humanos , MasculinoRESUMEN
OBJECTIVES: To compare the serum levels of insulin-like growth factor-1 (IGF-1) in patients with prostate cancer and in control patients with no malignancy, and to evaluate any possible influence of testicular androgen withdrawal on the level of IGF-1 in patients with prostate cancer. PATIENTS AND METHODS: IGF-1 was measured in serum samples from 238 patients using both a chemiluminescence method and a radio-immunoassay. From a subgroup of 19 patients presenting with newly diagnosed carcinoma of the prostate, IGF-1 and testosterone values were measured before and during the course of testicular androgen withdrawal, achieved by the administration of luteinizing hormone-releasing hormone (LHRH) analogues combined with anti-androgens. RESULTS: There were no significant differences in the mean serum levels of IGF-1 patients with and without prostate cancer (158.6 and 159.1 ng/mL, respectively). There were no significant differences in mean IGF-1 levels before and after antiandrogen therapy; the mean (median, SD, range) levels of testosterone (microg/L) and IGF-1 (ng/mL) before androgen withdrawal were 4.81 (4.84, 1.26, 3.11-6.93) and 157.1 (152.5, 26.7, 122.8-195. 1). After androgen withdrawal the corresponding values were 0.303 (0. 218, 0.24, 0.13-0.81) and 169.7 (31.7, 168.6, 124.9-227.6). A linear regression analysis (P = 0.76) and Spearman rank order correlation test (correlation coefficient -0.0613, P = 0.64) showed no association between levels of testosterone and IGF-1. Freeze and thaw cycles applied to the samples had no effect on the IGF-1 values measured. CONCLUSIONS: There was no significant association between IGF-1 serum levels and prostate cancer. Short-term androgen withdrawal using LHRH analogues combined with anti-androgens had no effect on the levels of IGF-1.