Neonatal estrogen stimulates proliferation of periductal fibroblasts and alters the extracellular matrix composition in the rat prostate

The purpose of this study was to examine whether changes in extracellular matrix (ECM) molecules are associated with the growth inhibition and differentiation defects of the prostate gland following neonatal exposure to estradiol. Using immunocytochemistry (ICC), laminin and collagen IV were localized to the basement membrane (BM) as well to the basal lamina of the periductal smooth muscle of the control developing prostates. In contrast, fibronectin and collagen III were localized throughout the stromal ECM. Exposure to neonatal estrogen altered the staining profile for specific ECM molecules. In the estrogenized rats, a thick layer of cells negative for laminin and collagen IV was observed adjacent to the BM. Electron microscopy and ICC for alpha-actin, fibronectin, and vimentin identified this multicellular layer of periductal cells as differentiated fibroblasts. Peripheral to these fibroblasts, actin-positive smooth muscle formed a second layer of periductal stromal cells. PCNA labeling showed that estrogen exposure increased the fibroblast proliferation. Because many periductal fibroblasts were positive for estrogen receptor alpha (ER alpha) in estrogenized rats, a direct effect of estradiol on their proliferation is suggested. Gelatinolytic gels revealed that estrogen exposure did not alter the activity of matrix metalloproteinases associated with tissue remodeling during prostate morphogenesis. However, the periductal fibroblast layer in estrogenized prostates was devoid of urokinase- and tissue-plasminogen activator, which may potentially alter the localized proteolysis involved in matrix remodeling. It is proposed that proliferation of a multicellular layer of periductal fibroblasts in estrogenized prostates results in a physical barrier that constrains branching morphogenesis and blocks paracrine communications between smooth muscle and epithelial cells which normally regulate differentiation.     

Cytoskeleton and tissue origin in the anterior cynomolgus monkey eye

We studied cytoskeletal proteins and other markers for embryologic origin in the outflow pathways of the aqueous humor, cornea, sclera, and ciliary muscle of the cynomolgus monkey. The corneal endothelium and trabecular cells stained with markers for vimentin, smooth muscle cell alpha-actin, F-actin, spectrin, vinculin, and talin. The endothelium of Schlemm's canal stained with markers for vimentin, spectrin, and F-actin. These results suggest that trabecular cells are a kind of myofibroblast and support the belief that the endothelial cells of Schlemm's canal are vascular in origin. Fibrillary staining with antibodies to vimentin, spectrin, neurofilament protein, and glial acid fibrillary protein was observed along and between the ciliary muscle cells. Cells in the deep sclera adjacent to the supraciliary space stained with antibodies to smooth muscle alpha-actin, alpha-vinculin, talin, and desmin. These cells may anchor ciliary muscle cells into the sclera or may be developmental remnants of ciliary muscle cells. Leu 19 immunoreactivity was found in the corneal endothelium, in all trabecular cells, in ciliary muscle cells, and in keratocytes and fibroblasts in the superficial part of the cornea and sclera. All of these cells are therefore likely to express neural cell adhesion molecules indicating neuroectodermal origin.     

The dynamics of the imprinted H19 gene expression in the mouse model of bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine

The imprinted H19 gene product is an oncofetal RNA molecule in humans. It is expressed in fetal bladder, down-regulated postnatally and is re-expressed in human bladder carcinoma. This study was designed to investigate the dynamics of the expression of H19 in the mouse bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and its relation to stages of neoplastic transformation. BBN was administered to mice in the drinking water for 26-28 weeks. The bladders were removed at 5-10 week intervals for histopathological examination and for in situ hybridization for H19 RNA, using a 35S-labeled probe. Following BBN administration expression of H19 first appeared after 5 weeks in the lamina propria adjacent to the basement membrane, concomitant with mucosal hyperplasia. At 11 weeks focal expression was noted in epithelial cells. Invasive carcinomas, of the transitional and squamous sub-types, were seen after 20 weeks and more of BBN administration. At this stage H19 expression was observed in scattered tumor cells, in the connective tissue stroma of the tumor and in the lamina propria underlying the remaining hyperplastic/dysplastic mucosa. Abundant expression of H19 was evident in fetal bladder but was absent in normal adult bladder. We conclude that, similar to humans, the H19 gene product is an oncofetal RNA molecule in the experimental mouse model of bladder carcinoma. In this model H19 is expressed in the connective tissue of the lamina propria prior to its expression in epithelial cells, concurrent with preneoplastic changes in the transitional epithelium of the bladder.     

Characterization of a monoclonal antibody recognizing a DAG-containing epitope conserved in aphid transmissible potyviruses: evidence that the DAG motif is in a defined conformation

Fetal and postnatal bovine bladders were examined for expression of elastic fiber components by immunohistochemistry as well as by measurement of steady state mRNA levels. Expression of fibrillin-1, microfibril-associated glycoprotein (MAGP) and elastin during the fetal period were compared with that of postnatal two year old animals (heifers) and adults. Each bladder was separated into two distinct tissue samples: 1) the outer smooth muscle layer (detrusor) and 2) the inner epithelium (urothelium) lined lamina propria (urotherial-lamina propria). Each of these samples was analyzed separately. Distribution of the elastic fiber components, determined by immunohistochemistry with matrix-specific antibodies, was different depending upon the region of the bladder wall examined and its developmental stage. In particular, MAGP and fibrillin-1 were conspicuously present in the urothelium during the later fetal stages. RNA products of elastic fiber genes were detectable both in the detrusor smooth muscle and urothelial-lamina propria fractions. The highest level of expression occurred in the urothelial-lamina propria fraction during the late second-early third trimester. Elastin expression was different from that of MAGP and fibrillin-1. The highest levels of steady-state elastin mRNA occurred at the earliest developmental stages examined and then progressively decreased through term. A high level of elastin expression occurred within the inner or lamina propria layer of the bladder. Since this layer is the functional capacitance layer within the bladder, its flexibility is likely related to the structural integration of elastin and associated microfibrillar components.     

Instructive induction of prostate growth and differentiation by a defined urogenital sinus mesenchyme

Instructive influences of fetal mesenchyme were examined in heterotypic tissue recombinants consisting of urogenital sinus mesenchyme (UGM) from male and female rats and distal ductal tips from adult rat prostate. Tissues were grown under the renal capsule of male hosts for periods up to 28 days. Resultant growths exhibited typical prostate histology. Expression of lobe-specific proteins for the ventral (prostatic steroid binding protein [PSBP]) lateral (seminal vesicle secretion II [SVS II]), and dorsal prostate (secretory transglutaminase [TGase]) were examined by immunocytochemistry. Male or female UGM combined with terminal segments of the ventral or dorsal prostate and immunolabeled with antibodies to lobe-specific proteins demonstrated expression of all three secretory products. The pattern of staining was consistent with a compound inductive response from the UGM. Unique to this study was our ability to use a defined mesenchymal tissue (female ventral mesenchymal pad [VMP]). This tissue is specifically associated with ductal branching morphogenesis and cytodifferentiation of the ventral prostate. Distal ductal tips from the dorsal lobe of the adult male prostate when recombined with female VMP and grown in vivo exhibited transformation of secretory phenotype, and the epithelium expressed mRNAs for PSBP. Immunocytochemistry of serial sections did not demonstrate labeling for TGase in the new epithelial growth. Ultrastructural analysis of the heterotypic recombinants indicated that the epithelium had similar characteristics to those of normal ventral prostate. Early stages of the mesenchymal-epithelial interactions resulted in dedifferentiation of the adult epithelium to solid cords of stratified cells. These findings illustrate the potent instructive capacity of a defined fetal UGM to influence development and cytodifferentiation of adult prostate epithelium.     

Organization of the lamina propria mucosae of rat intestinal mucosa, with special reference to the subepithelial connective tissue

Light microscopy, scanning electron microscopy and transmission electron microscopy have been used to delineate the structure and function of the lamina propria mucosae in the rat jejunum. In silver-impregnated sections, the adepithelial surface of the lamina propria mucosae was framed by a sheet of reticular fibers (reticular sheet). Short-term (3-hour) immersion of jejunal tissues in 2 N NaOH solution enabled us to simultaneously view networks of reticular fibrils and fibroblasts residing in the subepithelial connective tissue under a scanning electron microscope. The reticular fibrils, which measured about 40 nm in diameter and were interwoven in dense networks, formed a sheet 2-3 microns thick. In the villi, this sheet contained numerous foramina ranging from 3 to 7 microns in diameter, through which lymphocytes, macrophages, basal extensions of epithelial cells and fat particles traversed. The reticular sheet in the domes of isolated lymphoid nodules was markedly porous, and many lymphocytes migrated into or out of the epithelium through the foramina. The formaina of the reticular sheet may participate in the communication between the intestinal epithelium and the lamina propria mucosae. It was noted that the foramina of the reticular sheet in the villi were surrounded by end feet of the cytoplasmic processes of fibroblasts. In addition, these fibroblasts were combined with lymphocytes or dendritic cells in the lamina propria mucosae.     

Cytoskeletal and cytocontractile protein composition of smooth muscle cells in developing and obstructed rabbit bladder

The differentiation patterns of smooth muscle cells (SMC) in rabbit bladder during development and in the hypertrophic response to partial outflow obstruction induced in adult animals were evaluated by biochemical and immunochemical techniques and by using a panel of monoclonal antibodies specific for desmin, vimentin, alpha-actin of smooth muscle (SM) type, SM myosin, and nonmuscle (NM) myosin isoforms. Desmin and SM alpha-actin were homogeneously distributed in SMC of developing, adult, and obstructed bladders. Conversely, marked changes in the ratio and antigenicity of SM myosin isoforms were observed by SDS electrophoresis and Western blotting, respectively. In particular, the 205 K (SM1) isoform was down-regulated with development whereas the 200 K (SM2) isoform was up-regulated around 7 days after birth and down-regulated in the obstructed bladder. Vimentin was expressed in SMC of the fetal bladder and declined markedly during postnatal, physiological hypertrophy of SMC, which occurs concomitantly with diminution of DNA synthesis. This polypeptide became detectable, however, in SMC of obstructed bladders. The 196 K (NM) myosin isoform recognized by NM-A9 antibody, present only in endothelium of blood vessels and in mucosa of normal fetal and adult bladders, became expressed in detrusor muscle, when SMC underwent a process of pathological hypertrophy. The reexpression of vimentin and the de novo appearance of NM myosin isoform in hypertrophic bladders can be reversed when the tissue mass is reduced, such as in bladders after 1-month recovery from partial obstruction. Thus, a specific NM myosin isoform can be used as a marker of SMC hypertrophy in obstructed bladder. In addition, the combined use of anti-vimentin and NM-A9 antibodies can distinguish between SMC which are in the physiological or in the pathological condition of adaptive bladder hypertrophy.     

Differential expression and localization of IGF-I and IGF binding proteins in inflamed rat colon

Recent studies indicate increased insulin-like growth factor I (IGF-I) expression and altered expression of IGF binding proteins (IGFBP) in the bowel during experimental colitis. This study analyzes the cellular sites of altered IGF-I and IGFBP-expression in large bowel of rats with experimental colitis. Colitis was induced by colonic instillation of 2, 4, 6-trinitrobenzenesulfonic (TNB) acid in ethanol. Animals were sacrificed at 7 days after induction of colitis. Cryostat sections of colon from TNB-treated and control rats were hybridized with 35S-labeled antisense probes for IGF-I, IGFBP-3, IGFBP-4 and IGFBP-5. IGF-I mRNA was up-regulated in lamina propria cells, submucosa and smooth muscle of inflamed colon. IGFBP-3 mRNA was localized to lamina propria and was down-regulated in inflamed colon. IGFBP-4 and IGFBP-5 mRNAs were both up-regulated in inflamed colon. IGFBP-4 mRNA was increased in lamina propria, submucosa and smooth muscle, whereas IGFBP-5 mRNA was increased in smooth muscle. Increased IGF-I expression in mesenchymal layers of colon during experimental colitis supports the hypothesis that IGF-I contributes to hyperplasia and fibrosis in response to inflammation. Altered expression of IGFBP-3, IGFBP-4 and IGFBP-5 in specific bowel layers during colitis suggests that they play a role in modulating IGF-I action.     

Regeneration of bronchial epithelium on chronic inflammatory changes under laser treatment

Investigation of structural and metabolic changes in 188 biopsy specimens from the epithelium of large bronchi of 76 patients with chronic lung disease was carried out. It was shown that endobronchial treatment by helium-neon laser irradiation induced the proliferative and metabolic processes in damaged epithelium, which after getting through the series of transitional forms restores its normal structure and differentiation into ciliated and goblet cells of a normal ultrastructure. The hyperemia, intensive diapedesis of leucocytes, the formation of leucocytic infiltrations and granulations develop in the lamina propria of the bronchial mucosa. Proliferative and metabolic activity of the endotheliocytes and stromal cells increases. Finally, delicate-fibrous connective tissue forms. The simultaneous reorganizations of the epithelium and underlying connective tissue is interpreted from the point of view of the concept of parenchymal-stromal interactions.     

Nitrergic innervation of the rat larynx measured by nitric oxide synthase immunohistochemistry and NADPH-diaphorase histochemistry

We evaluated the involvement of nitric oxide (NO) in the laryngeal innervation of rats using NADPH-diaphorase (NADPH-d) histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. The findings obtained by NADPH-d histochemistry were identical with those obtained by nNOS immunohistochemistry, indicating that NADPH-d is nNOS in the laryngeal innervation system. We found NADPH-d-positive nerve fibers in every region of the larynx. In the epithelia of the mucosa, a small number of NADPH-d-positive nerve fibers were detected. The plexus of NADPH-d-positive nerve fibers was commonly found in the lamina propria, and some of these fibers were clearly associated with blood vessels. We also noted NADPH-d-positive nerve fibers in the region of laryngeal glands. Some of these fibers appeared to terminate in the glandular cells. We found NADPH-d-positive nerve fibers with varicosities in the intrinsic laryngeal muscle and free-ending nerve fibers on the muscle fiber. Motor end plate-like structures were positive for NADPH-d histochemistry. The NADPH-d-positive nerve fibers appeared to terminate at motor end plate-like structures in two of nine rats examined. A cluster of NADPH-d-positive neurons were occasionally present in the lamina propria of the laryngeal mucosa, in the connective tissue between the thyroid cartilage and intrinsic laryngeal muscle, and in the connective tissue near the cricoarytenoid joint. The present findings suggest that NO participates in the autonomic, sensory, and motor innervation of the larynx.     

Myofibroblast-derived smooth muscle cells during remodelling of rabbit urinary bladder wall induced by partial outflow obstruction

BACKGROUND: Fibrosis of serosa, along with smooth muscle (SM) cell hypertrophy, has been shown to occur in the rabbit bladder after partial outflow obstruction. Identification of cells involved in the serosal thickening can be of primary interest to elucidate the functional changes that this organ undergoes. EXPERIMENTAL DESIGN: Cytoskeletal protein composition of cells present in the thickened serosa at different times from the onset of obstruction (7, 15, 30 and 60 days) was evaluated. This was accomplished by means of a panel of monoclonal antibodies specific for a number of differentiation markers of mesenchymal cells (vimentin, desmin, alpha-actin of SM type, nonmuscle (NM) and SM myosins), and by immunocytochemical and immunochemical techniques. RESULTS: The immunocytochemical study revealed that cells in serosal thickening follow a two-step maturation process from pre-existing vimentin-positive cells. In the first time period (7 to 15 days of obstruction), these cells predominantly achieved an immunophenotype corresponding to that of a specific myofibroblast subtype (i.e., containing vimentin, NM myosin, and SM alpha-actin). After 30 days from the onset of obstruction, the cytoskeletal protein content of serosal cells, as also revealed by Western blotting experiments, shifted towards that of fetal-type SM cells (i.e., presence of vimentin, NM myosin, SM alpha-actin, and SM myosin isoforms). Distribution of vimentin, desmin, SM alpha-actin, and SM myosin in tissue culture as well as the ultrastructure in vivo very closely resembled that of SM cells. Bromodeoxyuridine incorporation studies indicated that cells accumulated in the serosa of obstructed bladders did not derive, at least initially, from SM cells of the detrusor muscle. CONCLUSIONS: These findings are consistent with the existence of a differentiation process in which resident mesenchymal cells of bladder serosa may transform to myofibroblasts and, subsequently, in fetal-type SM cells during experimental outflow obstruction.     

Ultrastructure of sea urchin tube feet. Evidence for connective tissue involvement in motor control

An analysis of the ultrastructure of the tube feet of three species of sea urchins (Strongylocentrotus franciscanus, Arbacia lixula and Echinus esculentus) revealed that the smooth muscle, although known to be cholinoceptive, receives no motor innervation. The muscle fibers are attached to a double layer of circular and longitudinal connective tissue which surrounds the muscle layer and contains numerous bundles of collagen fibers. On its outside, the connective tissue cylinder is invested by a basal lamina of the outer epithelium to which numerous nerve terminals are attached. These are part of a nerve plexus which surrounds the connective tissue cylinder. The plexus itself is an extension of a longitudinal nerve that extends the whole length of the tube foot. It is composed of axons, but nerve cell bodies and synapses are conspicuously lacking, suggesting that the axons and terminals derive from cells of the radial nerve. Processes of the epithelial cells penetrate the nerve plexus and attach to the basal lamina. There is no evidence that the epithelial cells function as sensory cells. On the basis of supporting evidence it is suggested that the transmitter released by the nerve terminals diffuses to the muscle cells over a distance of several microns and in doing so affects the mechanical properties of the connective tissue.     

Complementation tagging of cooperating oncogenes in knockout mice

In the testis, 'hyalinization' of the lamina propria of seminiferous tubules is often accompanied by similar changes within the walls of testicular blood vessels. The aim of our study was to investigate the structure of small blood vessels in hyalinized human testes by means of immunohistochemistry, electron microscopy and image analysis methods. Results of immunohistochemical analysis indicated that, despite hyalinization, testicular small blood vessels retained positive immunostaining for desmin and actin. Their basement membranes remained immunopositive for collagen IV and laminin. No proliferative (Ki-67) activity was observed in the blood vessel walls in testes from both control and infertile men. P-170 glycoprotein was found to be expressed only in primary spermatocytes. No difference in expression and localization of this antigen was observed between control and affected testes. Electron microscopy revealed a number of testicular arterioles with a notably narrow lumen due to enlarged endothelial cells in infertile men. Such arterioles also had a thickened subendothelial layer and an abundant tunica adventitia rich in connective tissue fibres and ground substance. Some venules in hyalinized testes displayed increased connective fibres and ground substance in the subendothelial layer, between the smooth muscle cells of the tunica media and the tunica adventitia. However, no changes were found in the capillary network, when compared to controls. Image analysis data showed a statistically significant increase in the surface of tunica intima and adventitia of arterioles and tunica media of venules. It is concluded that hyalinization mostly affects testicular arterioles and venules, but not capillaries. Our immunohistochemical data indicate that the 'nature' and/or extent of hyalinization in testicular small blood vessels differs from that described previously for the lamina propria of seminiferous tubules.     

The tissue and cell defense mechanisms of the oral mucosa

The modern concepts of tissue and cell defense mechanisms in oral mucosa are discussed. These mechanisms include (a) physical barriers (of epithelium and lamina propria), (b) non-specific antimicrobial humoral factors, (c) non-specific cellular defense mechanisms, (d) specific immune humoral and cell-mediated defense mechanisms, (c) saliva, containing both non-specific and specific antimicrobial factors. Special reference is given to antigen-presenting dendritic cells and various lymphocyte subpopulations both in epithelium and in lamina propria. The interaction of different cell and tissue defense mechanisms and their regional variations are briefly characterized.     

In utero and lactational exposure of the male rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs prostate development. 2. Effects on growth and cytodifferentiation

In the male Holtzman rat, in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure decreases prostate weight without inhibiting testicular androgen production or decreasing circulating androgen concentrations. Therefore, the present study sought to characterize effects of TCDD exposure on prostate development, from very early outgrowth from the urogenital sinus (Gestation Day [GD] 20) until rapid growth and differentiation are essentially complete (Postnatal Day [PND] 32). Pregnant Holtzman rats were administered a single dose of TCDD (1.0 microgram/kg po) or vehicle on GD 15 and offspring were exposed via placental transfer (GD 20 euthanasia) or placental and subsequent lactational transfer until euthanasia (if before PND 21) or weaning. Results show that the prostatic epithelial budding process was impaired by in utero TCDD exposure, as evidence by significant decreases in the number of buds emerging from dorsal, lateral, and ventral aspects of the GD 20 urogenital sinus. Ventral prostate cell proliferation index was significantly decreased on PND 1 but was similar to or higher than control at later times, whereas apoptosis was an extremely rare event in ventral prostates from both control and TCDD-exposed animals. Delays were noted in the differentiation of pericordal smooth muscle cells and luminal epithelial cells. In addition, ventral prostates from approximately 40% of TCDD-exposed animals examined on PNDs 21 and 32 exhibited alterations in the histological arrangement of cell types that could not be explained by a developmental delay. Compared to controls, these ventral prostates exhibited a disorganized, hyperplastic epithelium containing fewer luminal epithelial cells and an increased density or continuous layer of basal epithelial cells, as well as thicker periductal smooth muscle sheaths. In addition, in ventral prostates from TCDD-exposed animals, the intensity of androgen receptor staining was relatively low in the central and distal epithelium, and the number of androgen receptor-positive cells was relatively high in the periductal stroma. These data suggest that in utero and lactational TCDD exposure interferes with prostate development by decreasing very early epithelial growth, delaying cytodifferentiation, and, in the most severely affected animals, producing alterations in epithelial and stromal cell histological arrangement and the spatial distribution of androgen receptor expression that may be of permanent consequence.     

Structural changes in the intramuscular connective tissue during development of bovine semitendinosus muscle

Structural changes in the intramuscular connective tissue during development of bovine semitendinosus muscle were investigated using the cell-maceration method for scanning electron microscopy, by which cellular elements were eliminated and collagen fibrils and fibres were exposed. The endomysium was discontinuous and showed various shapes and sizes in the muscle of 7-month fetuses. The perimysium consisted of collagen fibres in loose contact with each other. In the muscle of neonatal calves, the endomysium consisted of cylindrical sheaths and displayed a honeycomb structure, and the perimysium was composed of several layers of collagen fibres. Collagen fibrils in the endomysium bound ever more closely with each other, and collagen fibres in the perimysium increased in thickness, and the wavy pattern of collagen fibres became more regular with growth of cattle. We have examined the mechanical strength of the intramuscular connective tissue by our new method, 'intramuscular connective tissue (IMCT) model'. The IMCT model is composed of collagen fibrils and fibres which maintains the organization in the endomysium and perimysium in situ. The shear-force value of the model increased rapidly from the 7th fetal month to the neonatal stage, and increased linearly with postnatal ageing thereafter. Changes in the arrangement of collagen fibrils and fibres seem to closely related to an increase in the mechanical strength of the intramuscular connective tissue during development of bovine skeletal muscle.