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Binucleate trophoblast giant cells (TGCs) are one characteristic feature of the ruminant placenta. In cows, the frequency of TGCs remains constant for most of the duration of pregnancy. As TGCs are depleted by their fusion with uterine epithelial cells, they need to be constantly formed. It is still unclear whether they develop from stem cells within the trophectoderm or whether they can arise from any uninucleate trophoblast cell (UTC). Within the latter, generally accepted theory, a basally located uninucleate cell (BUC) without contact to the feto-maternal interface would represent a transient cell between a UTC and a TGC. So far, no evidence for the existence of such transient cells or for the presence of stem cells has been shown. The aim of the present study is to morphologically characterize the early stages of TGC development. Placentomal tissue of 6 pregnant cows from different gestational stages (gestational days 51-214) was examined for BUCs, UTCs, and TGCs either in serial sections (light and transmission electron microscopy, TEM, n = 3), in single sections (TEM, n = 2), or by serial block face-scanning electron microscopy (n = 1). These investigations revealed the occurrence of BUCs, as well as young TGCs showing contact with the basement membrane (BM), but without apical contact to the feto-maternal interface. The study morphologically defines these 2 cell types as early stages of TGC development and shows that binucleation of TGCs can precede detachment from the BM., (© 2018 S. Karger AG, Basel.)

Na+/H+ exchangers (NHEs) represent a highly conserved family of ion transporters that regulate pH homeostasis. NHEs as well as other proton transporters were previously linked to the regulation of the Wnt signaling pathway, cell polarity signaling, and mucociliary function. Furthermore, mutations in the gene SLC9A3 (encoding NHE3) were detected as additional risk factors for airway infections in cystic fibrosis patients. Here, we used the Xenopus embryonic mucociliary epidermis as well as human airway epithelial cells (HAECs) as models to investigate the functional roles of NHEs in mucociliary development and regeneration. In Xenopus embryos, NHEs 1-3 were expressed during epidermal development, and loss of NHE function impaired mucociliary clearance in tadpoles. Clearance defects were caused by reduced cilia formation, disrupted alignment of basal bodies in multiciliated cells (MCCs), and dysregulated mucociliary gene expression. These data also suggested that NHEs may contribute to the activation of Wnt signaling in mucociliary epithelia. In HAECs, pharmacological inhibition of NHE function also caused defective ciliation and regeneration in airway MCCs. Collectively, our data revealed a requirement for NHEs in vertebrate mucociliary epithelia and linked NHE activity to cilia formation and function in differentiating MCCs. Our results provide an entry point for the understanding of the contribution of NHEs to signaling, development, and pathogenesis in the human respiratory tract., (© 2018 S. Karger AG, Basel.)

Aim: To study the conjunctival development in hypothyroid genetically epilepsy-prone rats (GEPRs) with serum T3 and T4 significantly lower than in normal rats., Methods: A structural, ultrastructural and histochemical study on the conjunctival epithelium of GEPRs and of control Sprague-Dawley (SD) rats before and after eyelid opening, with particular regard to goblet cell differentiation., Results: From birth to day 12, no goblet cells were demonstrated on the conjunctival surface of both strains, so that the epithelium was formed only by a cuboidal basal layer and by a superficial layer of roundish or flattened cells. On day 16, after the eyelid opening, Alcian blue (AB)-positive goblet cells filled with homogeneous granules were demonstrated isolated, in GEPRs, or clustered, in SD rats, in both the fornices and palpebral conjunctiva. The epithelium showed a basal layer and many layers of flattened cells and was taller in SD rats (8-10 layers) than in GEPRs (6-7 layers). At 3 months, the epithelium in SD rats was higher with generally clustered goblet cells, whilst in GEPRs goblet cells were both isolated or clustered. In both strains, the goblet cells showed a marked AB/periodic acid-Schiff positivity all over the conjunctival surface and were filled with granules of different density. In both strains, goblet cells were absent at birth and their appearance, as AB-positive cells, was concomitant with eyelid opening., Conclusions: Hypothyroid rats showed a conjunctival development different than that of normothyroid rats for both epithelial and goblet cells. It appears that thyroid hormone imbalance may influence conjunctival development., (Copyright © 2010 S. Karger AG, Basel.)

Background: There is general consensus that the effects of intrinsic aging on the oral mucosa are relatively small, though potentially important to understanding the pathologies present in the aged animals., Objective: In this paper, the development of dorsal surface of rat tongue was examined using transmission electron microscopy (TEM) and high-resolution scanning electron microscopy (HRSEM) in order to understand the age-related structural and ultrastructural changes experimentally., Methods: In this study, we used female rats 75 and 720 days old (adult and aging). Tissues of rat tongue were prepared and the specimens submitted to HRSEM and TEM techniques., Results: The analysis of HRSEM and TEM demonstrated that the same characteristic keratinous epithelium was found in aging animals, however with some modifications., Conclusion: We agree that there are obvious changes in the oral mucosa with aging and these modifications can be observed starting from the ultrastructural aspects., (Copyright 2009 S. Karger AG, Basel.)

Background/aims: To evaluate by electron microscopy the effects of methotrexate on the tubal morphology of rabbits., Methods: Biopsies were taken from the isthmic portion of both tubes of 4 rabbits in the control group. 1 mg/kg methotrexate was injected in the left tubes and as a control, a 14-gauge fine needle was used to puncture the right tubes of 10 rabbits in the study group. One month later, tubal biopsies were taken from both tubes of every rabbit in the study group. To mimic pregnancy, 100 IU hCG was injected intramuscularly to every rabbit 24 h before every surgery. On examination by electron microscopy, the effects of methotrexate and of the damage with the fine needle on the tubes were compared to the control group., Results: Young epithelial cells of the methotrexate-injected fallopian tubes kept their normal structure, but others contained important ultrastructural changes. These were: nuclear pyknosis, cytoplasmic vacuolization, dilatation of endoplasmic reticulum cisternae, increased cytoplasmic density and compound cilia., Conclusion: Degenerative changes on epithelial cells caused by the temporary blockage of mitotic activity associated with local methotrexate injection are reversible through the formation of new epithelial cells., ((c) 2008 S. Karger AG, Basel)

Objective: To formulate cytologic features for differential diagnosis of basal cell adenoma (BCA)., Study Design: The usefulness of 5 items for a cytologically definitive diagnosis of BCA was examined. The 5 items in 8 BCA and 22 non-BCA cases (adenoid cystic carcinoma [ADCC], basal cell adenocarcinoma, myoepithelioma, pleomorphic adenoma and polymorphous low-grade adenocarcinoma) that displayed mimicking cytology were examined cytologically., Results: The useful items were < 5.1 microm in mean of epithelial nuclear short diameter; mild atypia on definitive diagnosis; stromal cell cluster combining smooth margin surrounding the epithelial cell cluster or containing the epithelial cell cluster; epithelial clusters surrounded by or adhered to a thick, hyalinized smooth margin without stromal cluster; and closely fastened, tight clusters with denser cytoplasm than ADCC, but an indistinct border, with oval nuclei and no hyaline cells., Conclusion: Five items are useful criteria for BCA.

Background: Well-differentiated papillary mesothelioma (WDPM) is an uncommon subtype of mesothelioma that typically occurs in the peritoneum of women without a history of asbestos exposure and usually follows an indolent clinical course. Fine needle aspiration (FNA) of this type of tumor has rarely been reported., Case: A 64-year-old woman with 11-year history of colon cancer and an adrenal nodule was found, on abdominal computed tomography, to have a mass in the right lobe of the liver. Aspirates of the mass were composed of abundant, tight, papillary groups, monolayered, pavementlike sheets; and scattered single cells with minimal atypia. The cell block showed a predominantly papillary growth pattern and a single layer of bland, cuboidal to flattened covering cells with stout, fibrovascular cores containing clusters of foamy histiocytes. Tumor cells in the focal tubulopapillary and solid areas were mingled with inflammatory cells and showed slightly more atypia than did the cells covering the papillae. The differential diagnoses were intrahepatic papillary neoplasm, including well-differentiated mesothelioma and metastatic low grade papillary serous carcinoma. At surgery the tumor was found to be a pedunculated peritoneal mass that arose from the posterior surface of the right lobe of the liver. The mesothelial origin of the tumor was confirmed by both immunoperoxidase study and electron microscopic examination, which demonstrated long, slender, branching microvilli., Conclusion: Familiarity with the cytomorphologic features and clinical presentation of WDPM, knowledge of the exact anatomic location and consideration of the appropriate differential diagnosis combined with ancillary studies are the keys to an accurate diagnosis.

The aim of this investigation was to visualize the epithelial structures of the tongue using confocal laser scanning microscopy (LSM). The human tongue epithelium of 28 healthy subjects, aged 21-67 years, mean age 38 years, 14 women and 14 men, was examined in vivo by LSM. Using LSM, a combination of the Heidelberg Retina Tomograph HRT II and the Rostock Cornea Module, up to 800-fold magnifications were obtained. On the tongue surface both filiform and fungiform papillae and their taste pores were easily identified. The epithelium of the tongue with its subcellular structures could be observed up to a depth of 50 microm, cellular structures up to 150 microm and subepithelial vessels up to 300 microm. Additionally the papillary crests and blood flow were visible. Confocal LSM seems suitable for noninvasive in vivo examination of the tongue. The hydraulic z scan, the manual start setting and the measurement of the depth allow a clear classification of the observed structures.

The four stages of the estrous cycle in Monodelphis domestica, namely proestrus, estrus, postestrus and the transitional metestrus, were analyzed with the scanning electron microscope and compared with the results of the previously published transmission electron-microscopic paper [Cells Tissues Organs 2002;172:276-296]. During the estrous cycle the vaginal epithelium undergoes dramatic changes from a nonkeratinized to a highly keratinized epithelium. The predominant feature of proestrus with the beginning of keratinization is the presence of polygonal flat cells with pavement-like appearance, bordered by raised ridges and covered with microvilli. The epithelium is fully keratinized in estrus, and the superficial layers overlap like shingles. Many cells are still densely covered by microvilli, whereas others develop a complex pattern of microridges. In postestrus different epithelial structures are revealed depending on the actual stage of desquamation. In early postestrus surface cells resemble those present during estrus. In late postestrus, when only few keratinized cells are left, the nonkeratinized cells become exposed to the lumen through desquamation. These cells border the lumen during metestrus, a cycle stage during which numerous leukocytes migrate into the vaginal canal. A number of these uppermost cells is probably not yet prepared to function as metestrus cells and are therefore sloughed off as well. During metestrus compact cell masses stick in the vaginal furrows. Epithelial surface cells are highly irregular and bulging with their microvilli covered surfaces in the vaginal lumen. This study represents the first comprehensive description of alterations on the surface ultrastructure of a marsupial vagina during the estrous cycle, demonstrating considerable differences in comparison to many eutherians., (2004 S. Karger AG, Basel.)

Background: Pleural effusion caused by varicella-zoster virus (VZV) is rare. We report a case of a woman with acute lymphocytic leukemia (ALL) who developed a pleural effusion caused by VZV infection., Case: A 55-year-old woman with ALL treated with consolidation therapy developed skin vesicles and a pleural effusion. Pleural fluid smears contained numerous mesothelial cells, which had ground-glass nuclei or eosinophilic nuclear inclusions. Some multinucleated giant cells were also seen. Electron microscopic examination revealed intranuclear virus particles, about 150 nm in diameter, in some mesothelial cells. Tissue samples from the skin, lungs, pleura, liver, pancreas, kidneys and gastrointestinal tract, obtained at autopsy, contained many virus-infected cells. They were positive for VZV glyco-protein 1 by immunohistochemistry., Conclusion: VZV infection should be considered in the differential diagnosis of an unexplained exudative pleural effusion, especially in immunocompromised hosts.

The aim of the present study was to investigate the role of the marsupial shell coat in embryonic development because it may be a potential target for immunocontraceptive control of the brushtail possum. Conceptuses from 52 female possums were collected between 1995 and 1997 in New Zealand and Australia. Development was examined in representative stages from cleavage to the early trilaminar blastocyst. The effect of coat removal by microdissection was examined by comparing development in vivo (n = 29), with development in vitro, both with (n = 10) and without (n = 13) shell coat removal. Conceptuses were monitored and photographed in culture, fixed and examined by transmission electron microscopy. The ultrastructure of uni-, bi- and trilaminar blastocyst stages in vivo and in vitro and of the early stages of hypoblast and embryonic endoderm formation are described for the first time in the possum. Shell coat removal had little impact on most cleavage stages, as the intact mucoid coat appeared to provide structural support to the embryo. Common features of unilaminar coat-free specimens after culture were rounding up and detachment of cells from the blastocyst epithelium and the loss of cell surface projections. The most remarkable features of the shell-free trilaminar blastocysts in comparison with the in vivo and in vitro controls were the hydration of many cells, and the large-scale disruption and modification of numerous epithelia, particularly of the younger, or newly forming populations. The shell appears to be functionally important after blastocyst formation, particularly after breakdown of the mucoid coat. After shell removal, conceptus response in vitro suggested that the shell played a role in maintaining structural integrity. The shell was found to be necessary for normal embryonic development., (Copyright 2003 S. Karger AG, Basel)

The vaginal complex of marsupials differs from that of eutherians. Cervices open separately in a sinus vaginalis or cul-de-sac. Two lateral vaginae adjoin the sinus vaginalis and fuse at the level of the urethra opening and form the sinus urogenitalis. During the estrous cycle the vaginal epithelium undergoes a number of specified morphological changes. This paper is the first to describe these changes on an ultrastructural level in a marsupial. Investigations in Monodelphis vagina reveal that a cyclic switch exists between a keratinized and a stratified nonkeratinized epithelium. Keratinization starts during proestrus and reaches its maximum during estrus. In the postestrus, desquamation of the stratum corneum takes place, mostly in two steps. In metestrus one to two additional layers of the now nonkeratinized surface cells are shed into the vaginal lumen. Typical cell structures, such as keratin filaments, keratohyalin and membrane-coating granules, are involved in the keratinization process. Keratohyalin is found in the cytoplasm as well as in the nucleus of stratum granulosum cells, a phenomenon which is known from other parakeratinized epithelia of rapid turnover. Membrane-coating granules, responsible for the permeability barrier between the epithelial cells, are of the nonlamellated type in the nonkeratinized epithelium and produce an amorphous material in the intercellular spaces after extrusion. At periods, however, when the epithelium is keratinized, membrane-coating granules are of the lamellated type and form a lamellated barrier structure after extrusion in the intercellular space. The loss of the protective keratinized layers asks for an additional defense mechanism for the epithelium. The migration of leukocytes through the epithelium predominantly during post- and metestrus and their presence in the vaginal lumen may play a protective role together with the bacterial content., (Copyright 2003 S. Karger AG, Basel)

Ultrastructural changes in the endometrium associated with the oestrous cycle were studied in the South American marsupial Monodelphis domestica. The most conspicuous changes include the height and the differentiation of the uterine luminal and glandular epithelium, which consists of ciliated and non-ciliated cells. The glandular epithelium attains its maximum development during oestrus, the luminal epithelium at postoestrus. A distinct increase in the number of ciliated cells can be observed during pro-oestrus, reaching a maximum number at oestrus; this is followed by a process of deciliation. The presence of solitary cilia on the apices of non-ciliated cells is very conspicuous during all oestrous stages and can best be seen on the luminal epithelium. These findings differ from the observations in eutherian mammals, where solitary cilia are only found in the immature uterus or after ovariectomy. The secretory activity of non-ciliated cells of the luminal epithelium is hardly noticeable along the apical membrane and stains only very faintly with Alcian blue. The glandular epithelium cells are filled apically with exocytotic vesicles at oestrus and early postoestrus. However, in contrast to the cervical gland cells, they hardly stain with Alcian blue, indicating that mucins of a different type must be present. Mechanisms for the remodelling of the luminal and glandular epithelium are especially conspicuous at metoestrus and early pro-oestrus and include the presence of autolysosomes, residual bodies and apoptotic bodies. In the endometrial stroma, around the uterine glands, macrophages accumulate and attain a typical oestrous stage-dependent appearance during their phagocytotic activities., (Copyright 2002 S. Karger AG, Basel)

Objective: To document the presence of Anitschkow nuclear changes (ANC) in pericardial mesothelium at autopsy after the incidental finding of ANC in pericardial scrapings from a fatal case of overwhelming sepsis., Study Design: Fourteen, nonconsecutive autopsy cases were studied. Using the edge of a scalpel, the visceral pericardium from the left ventricle was scraped, and the sample was smeared onto glass slides, fixed in 95% ethanol, Papanicolaou stained and evaluated for the presence of ANC. Histologic correlation was also performed., Results: ANC were observed in pericardial mesothelial cells in 6 of 14 cases. Sepsis was the cause of death in three. Fatal cardiac arrhythmia, T-cell lymphoma and fulminant hepatic necrosis were found in the remaining cases. While readily seen in cytologic preparations, ANC were found focally in only one case examined histologically., Conclusion: Postmortem cytologic evaluation provides information relevant to the autopsy. In this study, ANC were very clearly seen in six pericardial scrapings. Clinical correlation supports the current theory that ANC represent a nonspecific reactive cell change.

The process of degeneration and regeneration of the lip mucosal epithelium after cryo treatment was observed by transmission electron microscopy. The epithelial cells were degenerated by the formation of ice crystals and subsequently detached from the basement membrane, forming a blister cavity. The separation occurred between the epithelial cells and the lamina densa, leaving a small amount of cell debris on the lamina densa. The surviving cells at the periphery of the blister cavity, especially the cells in the basal half of the epithelium, provided the regeneration cells. They migrated over the cell debris, attached to the lamina densa and gradually phagocytozed it. Finally, they formed hemidesmosomes with the old lamina densa. The connections between the epithelial cells by desmosomes were so tight that desmosomes were preserved even between dead cells and between dead and living cells. Regenerating cells were moving in a multilayered form, remaining connected to each other by the dosmosomes. They were seen to divide by mitosis and thereby increase the number of the cell layer, whilst maintaining their connections with the neighbouring cells., (Copyright 2001 S. Karger AG, Basel.)

We studied corresponding structures on the oral and basal surfaces of the oral epithelial layer, focusing on the microanatomy of gustatory papillae. Specimens for scanning electron microscopy were obtained from the rat soft palates and fixed with a mixture of 2% paraformaldehyde and 1% glutaraldehyde. They were first prepared without postfixation and conductive-staining to study the oral surface. After examination, the epithelium was reinforced by additional sputter coating and treated with 6 N NaOH at 60 degrees C to exfoliate the basal epithelial surfaces without any significant artifacts. The papillae, showing circular, elliptical or fusiform protuberances on the oral surface, were classified into two types: types I and II. The type I and type II papillae contained one and two taste pores, respectively. On the basal epithelial surface, the basal portions of the taste buds were associated with concentrically arranged nerve fibers and Schwann cells. Another characteristic finding on the basal epithelial surface was the presence of excretory ducts of minor salivary glands in a close spatial relationship to taste buds. It is suggested that saliva coming out through the duct is mixed with food, thus enabling intimate contact with the taste pores of the papillae., (Copyright 2001 S. Karger AG, Basel.)

Objective: To determine the ultrastructural features of diffuse malignant pleural mesothelioma cells in cytologic specimens from pleural effusions., Study Design: We retrospectively studied 35 pleural effusions: 12 diffuse malignant pleural mesotheliomas (8 epithelial type, 4 biphasic type), 12 pulmonary adenocarcinomas and 11 cases of reactive mesothelial cells., Results: In the cytoplasm, reactive and malignant mesothelial cells had more-abundant intermediate filaments (P < .05, P < .01) and fewer free ribosomes (P < .001, P < .001) than adenocarcinoma cells. Reactive mesothelial cells had fewer mitochondria than mesothelioma cells (P < .05). Mesothelioma cells had longer, thinner microvilli on the cell surfaces (P < .001); length/diameter ratios of microvilli were 19.1 +/- 7.0 (mesothelioma) vs. 9.1 +/- 2.2 (adenocarcinoma) and 9.2 +/- 2.4 (mesothelial cells). Giant intercellular junctions (desmosomes or desmosomelike structures > 1 micron in length) were found in eight cases of mesothelioma. Core filaments or rootlets in microvilli were present in two cases of adenocarcinoma., Conclusion: Because cytologic specimens from pleural effusions were easy to obtain, we think ultrastructural cytology is useful in distinguishing mesothelioma from adenocarcinoma and benign effusions.

The ultrastructure of the lens epithelium in Alport's syndrome was studied. In a transmission electron-microscopic examination, a large number of tonofilaments, swollen mitochondria, a dilated Golgi complex, a dilated rough endoplasmic reticulum with electron-dense (osmiophilic) material in some of these irregular structures and myelin figures were observed. Most of the cells had regularly shaped and only a few of them irregularly shaped nuclei. Additionally, polymorphism and marked degeneration in the transitional zone were observed in the scanning electron-microscopic examination of the lens epithelium. After the transitional zone, there were no epithelial cells present under the lens capsule, an observation that has never been reported in the literature.

Objective: To define the composition of cytoplasmic inclusions forming stacks and concentric whorls in histiocytes and mesothelial cells of serous fluids, imparting to them a resemblance to Gaucher cells, and to draw conclusions on the mechanism of their formation., Study Design: Three serous fluids (one pleural and two pericardial) containing a fair number of the cells referred to were progressively subjected to the following studies: (1) cytochemistry for mucopolysaccharides, proteins, phospholipids and hemoglobin; (2) immunocytochemistry for immunoglobulins IgA, IgG, IgM and lysozyme; (3) transmission electron microscopy (TEM), and (4) scanning electron microscopy-based energy dispersive X-ray microanalysis (SEM-EDAX)., Results: All three specimens were blood stained and contained large numbers of histiocytes and mesothelial cells, arranged singly and in groups, with abundant cytoplasmic inclusions. The inclusions stained strongly positive for phospholipids, weakly positive for hemoglobin and negative for all other substances examined by cytochemistry and immunocytochemistry. By TEM the inclusions had a concentric lamellar membranous structure, reminiscent of myelinosomes or lamellar bodies of lipid-forming or -storing cells. There was also phagocytosis by histiocytes and mesothelial cells of red blood cells, which were mostly in a degenerated state. SEM-EDAX of inclusion-bearing cells showed a modest peak for phosphorus and a variable but small peak for iron, which corroborated the cytochemical and TEM findings., Conclusion: Since there was not metabolic or other systemic disease in the patients to account for these cells, we posit that phospholipids derived from cell membranes of phagocytized cells, especially red blood cells, provide the building blocks for the formation of such inclusions as they enter the metabolic pathway of phagocytic cells (mesothelial cells and histiocytes) and appear in their lysosomal structures. It is advantageous for cytologists to be familiar with significance of such changes and not to mistake them for metabolic or other systemic disease.

The conjunctival epithelium of the Mongolian gerbil (Meriones unguiculatus) was studied using the transmission and scanning electron microscopy. The superficial cells of the conjunctival epithelium were isoprismatic in shape and were covered, on their luminal surface, with microplicae. They were connected with the adjacent cells by junctional complexes and desmosomes. Some of the superficial cells were partially or completely detached from the underlying intermediate cells: this morphological pattern was interpreted as an expression of cellular renewal of the conjunctival surface. Goblet cells were either isolated or gathered in clusters: in any case, they were firmly connected with the adjacent epithelial cells. The apical part of the goblet cells was covered with a great number of long microvilli: they showed a variety of morphological aspects, which were interpreted as occurring in a sequential pattern during the secretory process. The Mongolian gerbil can be considered among the laboratory animals used for ophthalmic research.

Aims: To provide a 3-D description and basic morphometric data along the course of subcapsular proximal tubules., Methods: Proximal convoluted (PCT) and straight (PST) tubules were analyzed in series of up to one thousand 4-micron sections of perfusion-fixed rat renal cortex. Nine proximal tubules were traced through the sections by computer-assisted 3-D reconstruction. Selected sections were reembedded and resectioned for ultrastructural morphometry., Results: Subcapsular PCTs (n = 3) had a total length of 12.0 +/- 0.2 mm (SEM), formed tight clusters and contacted the renal surface from 5 to 10 times. In contrast, mid-cortical tubules (total PT length 11.6 +/- 0.1 mm) and in particular deep cortical tubules (total PT length 12.1 +/- 0.9 mm) extended laterally and intermingled considerably with neighboring tubules. Epithelial height gradually decreased from around 11 microns in the PCT to 7.5 microns in the end of the PST. Brush-border height was around 4 microns in the first PT segment (S1), 2.3 microns in the second segment (S2) and 4.5 microns in the end of the proximal tubule. Tubular wall volume, excluding microvilli, decreased from around 1,450 micron3/microns tubule length in early PCT to 825 micron3/microns in terminal PST., Conclusions: The results provide, at precisely defined nephron levels, quantitative ultrastructural data relevant to transport physiology.

The authors studied the overall microscopical anatomy of the middle ear and of the eustachian tube in 20 tympanic bullae of 10 Wistar rats. Large hexagonal epithelial flat cells covered the roof of the bulla, the two upper thirds of both lateral walls, the upper half of the eustachian tube, and the upper third of the tympanic orifice; ciliated and secretory cells lined the inferior thirds of both walls of the bulla while a ciliated epithelium with strong cilia, all directed towards the tubal orifice, located on the floor of the tympanic cavity together with groups of non-ciliated cells was detectable near the tubal and antral areas and on the floor of the eustachian tube. Non-ciliated cells covered by microvilli were found near the pharyngeal orifice. These observations seem to demonstrate the following: (1) the ciliated elements present a well-defined topographic distribution; (2) a preferential pathway for mucociliary clearance made up of strong ciliated cells located on the floor of the bulla is clearly detectable, and (3) the roof of the bulla seems to be mainly involved in the ventilatory function.

In order to identify a new cell surface antigen as a potential marker of renal epithelial cell injury, we produced a monoclonal antibody (Mab), 1F3, by immunizing mice with cultured human glomerular cells. Immunofluorescence (IF) and immunoelectron microscopy (IEM) studies demonstrated that 1F3-recognizing antigen (1F3 antigen) was strongly expressed on the cell surface of glomerular podocytes and very weakly on parietal epithelial cells. 1F3 antigen was not expressed in any other cells in the normal kidney. Immunoprecipitation analysis using metabolically labeled glomeruli revealed that 1F3 recognized a 125-kD protein under reducing conditions. IF studies of biopsy specimens from patients with a variety of glomerular and tubulointerstitial diseases showed that 1F3 antigen was almost negative in cellular crescents but was strongly expressed in fibrocellular crescents. When glomerular sclerosis appeared, the expression of 1F3 antigen decreased in sclerotic areas of glomeruli. 1F3 antigen became positive in atrophic tubules that were seen in diseased kidneys. Severity of tubular atrophy correlated well with the extent of tubular expression of 1F3 antigen. These results indicate that Mab, 1F3 marks phenotypic changes of renal epithelial cells under disease conditions and may be a useful marker for progressive kidney diseases.

Exposure to tobacco smoke can cause irritation of the conjunctiva. We conducted this study to identify the effect of tobacco on rat conjunctiva. Animals were divided into experimental and control groups and we exposed the experimental group to tobacco smoke. Control group rats inhaled only room air. Spectrophotometric analysis of the smoke-air mixture revealed that many toxic substances were present in this compound. We found very high levels of plasma thiocyanate after exposure to smoke in experimental group animals but no increase in the control group. So, this data indicates that these animals inhaled smoke effectively in our method. After 3 months conjunctivas were examined by light and electron microscopy. In the experimental group, conjunctivas were thinned, atrophied and microvillous projections and desmosomal connections were absent in comparison with the control conjunctivas. This pathologic change is very similar to conjunctival response to chronic irritants of any type.

The participation of apical membranes of uterine epithelial cells in the process of blastocyst adhesion makes them an interesting object in the study of changes occurring during early pregnancy. In the study of these changes alkaline phosphatase (AIP), a typical brush border enzyme, was chosen for demonstration with the scanning electron microscope (SEM) by means of a backscatter detector. Thus the temporal and spatial pattern of enzyme activity on the uterine luminal surface was made visible with lead salt procedures. AIP activity was shown to be located on apical membranes and microvilli of endometrial epithelial cells with high activity on day 2 of pregnancy decreasing to virtually no activity on day 5. This decrease in overall AIP activity was shown to be asymmetrical with respect to the uterine cavity. It begins on the antimesometrial half of the uterine lining on day 2. A distribution pattern demarcating a presumptive implantation site along the uterine horn was not found. However, on day 5 of pregnancy, a characteristic pattern of surface folds was found, dividing the uterine horn into 'implantation segments'. In addition, SEM investigation revealed a marked variation of AIP activity from one individual cell to the next on day 2 of pregnancy resulting in a mosaic-like pattern. This pattern is lost with the decrease of AIP activity on day 5. Thus heterogeneity of uterine epithelial cells in AIP activity is apparently a feature of nonreceptive epithelium in contrast to the homogeneous epithelium on day 5. It is proposed that epithelial cell homogeneity could be a marker for uterine receptivity.

We developed a method to culture mouse corneal epithelium. Cultured cells tested by 1-D-SDS-PAGE exhibited protein mobility patterns similar to freshly isolated epithelia. Western blots with antibodies broadly recognizing cytokeratins showed a similar pattern for both fresh and cultured cells, but only the fresh sample stained with J7, specific for a 55-kD 'corneal' cytokeratin. Cultured cells examined at confluency by transmission electron microscopy exhibited desmosomal contacts typical of epithelia. The ability to culture mouse corneal epithelial tissue will be useful for studies requiring large amounts of material by reducing animal numbers.

The ultrastructure of rabbit tracheal epithelium was studied 20 min after injection of 0.04 and 0.5 mg of atropin. The tracheae were lined with altered ciliated pseudostratified columnar epithelium. The degree of damage to the epithelial cells was dose dependent. The injury to the tracheal epithelium due to treatment with 0.04 mg of atropin was considered mild. The administration of the higher dose of atropin caused moderately severe epithelial damage. Both experimental groups exhibited morphological signs of impaired self-cleaning ability.

Important and precisely regulated transitions in tissue phenotype from epithelium to mesenchyme and from mesenchyme to epithelium occur in the developing embryo. The gene for E-cadherin has been shown to cause fibroblastic cell lines to become epithelioid in culture. We asked whether or not the activities of the E-cadherin gene could cause a definitive embryonic mesenchyme to transdifferentiate into an epithelial phenotype. Primary corneal fibroblasts from 6- to 7-day-old chick embryos were contransfected by impact loading with plasmids containing E-cadherin and Neo genes and selected in G418. The fibroblasts expressing E-cadherin aggregate, localize E-cadherin to lateral surfaces, and form stratified epithelia that develop zonulae occludentes and adherentes, connexin 43, cytokeratin, desmoplakin, and desmosomes. Vimentin intermediate filaments persist and no basement membranes appear, even though the cells synthesize laminin and type IV collagen. Our study is the first to demonstrate the ability of E-cadherin to induce fibroblasts to form desmosomes and stratified epithelia. The primary embryonic fibroblasts apparently have more developmental potential to transdifferentiate into epithelia than do the fibroblastic cell lines previously studied. We conclude that E-cadherin is likely to play an important role in transformation of mesenchyme to epithelium in the embryo.

Ionic homeostasis is essential to lens clarity and the lens epithelium plays a large part in homeostasis, through vectorial transport. In most epithelia maintenance of vectorial function depends on the cytoskeleton. Capsule/epithelium preparations from human donor lenses have been used for immunohistochemical investigations of both normal epithelial cell cytoskeletal structure, and of structural changes induced by increasing cell calcium. A sustained increase in intracellular calcium, induced by incubation with A23187 and W7, led to a loss of cytoskeletal organisation. Changes induced depolymerisation of cytoplasmic actin filaments, disaggregation of microtubules and initial thickening, followed by breakdown, of vimentin intermediate filaments. In addition, spectrin staining, normally confined to the basal-lateral plasma membranes, became diffuse within the cytoplasm. Such disruptions of cytoskeletal structure would decrease ionic control, leading to increasing osmotic stress.

The distribution of calcium in the human eye lens was investigated using the oxalate-pyroantimonate technique. In epithelial cells and superficial cortical fibers calcium is sequestered and tightly bound to proteins in the endoplasmic reticulum, nuclear envelope, Golgi cisterns and mitochondria. In intermediate fibers these cell organelles are broken down and the liberated calcium is ionically bound to the phospholipids at the extracellular side of the fiber-limiting membrane. It is argued that calcium is probably additionally bound to beta-crystallin. This site-specific variation in calcium storage might explain the deeper cortical localization of early focal dot opacities and retrodots.

The atomic force microscope (AFM) was invented in 1986. It created the possibility to study the surfaces of biological material at the molecular level. The AFM is not only a microscope with atomic resolution (at least when applied to suitable samples) but is also an instrument with a broad variety of other applications. We present five examples demonstating the application of the AFM technique in different ways, mainly in renal epithelium. First, it was used as a high-resolution near-field microscope which generated images of canine kidney chromosomes. Second, dynamic changes of the plasma membrane of canine kidney cells were visualized with a resolution at least 10-fold greater than that obtainable by light microscopy. Third, the AFM was used as a tool which interacted directly with the plasma membrane of canine kidney cells. Fourth, due to its excellent resolution in the third dimension (namely height), continuous measurements of changes in 'height' of multimers of cloned plasma membrane potassium channel proteins of rat kidney (ROMK1) in response to ATP were performed with a time resolution of about 100 ms in physiological buffer. Fifth, the AFM continuously followed dissociation processes of multimeric macromolecules (such as transcription factors) under physiological conditions and, on the basis of individual molecular volume measurements, allowed estimation of the number of monomers in a multimeric complex. Taken together, the AFM offers a wide spectrum of novel approaches for the experimental nephrologist.

Using lectin histochemistry we assessed whether chronic bladder infection modifies carbohydrate residues of glycoconjugates on uroepithelial cells in rats. The bladder infection was produced by implanting a knotted silk thread with Escherichia coli into the bladder. One or 4 weeks after the implantation the bladder was excised, incubated with sixteen biotinylated lectins and stained. The bladder epithelia as a whole stained more strongly positive for eight lectins in the infected rats than in the control rats having a sterile silk thread in the bladder. In the infected rats, the superficial epithelial layer that stained negative for Arachis hypogaea (PNA) in the controls became strongly positive for PNA, whereas the middle and deep epithelial layers increased in staining for Canavalia ensiformis and six other lectins. These results indicate that chronic bladder infection increases carbohydrate residues on uroepithelial cells and may facilitate bacterial adherence to uroepithelial cells.

Epithelial cells are the most important cell type in the development of human malignancies. More than 90% of all malignant tumors are carcinomas, and thus of epithelial origin. Aberrant growth and the ability to invade the underlying tissues are intrinsic properties of the fatally altered cells. Multiple genetic alterations that can influence growth and genetic stability of the carcinoma cells have been characterised during tumor progression. Loss of epithelial morphology and the acquisition of mesenchymal characteristics are typical for carcinoma cells late in tumor progression and correlate with metastatic potential. In vitro, epithelial-mesenchymal transitions can be induced by interference with the integrity of the adherens junction, by signalling via tyrosine kinases, and by oncogene expression. In carcinoma cells, loss or downregulation of E-cadherin expression are frequently observed in carcinomas, and correlate with the malignancy of the tumor. In general, this change in expression is regulated at the transcriptional level. However, tumor types or cell lines exist which show mesenchymal characteristics but nevertheless express E-cadherin protein or mRNA. A more-detailed analysis demonstrated that other mechanisms that interfere with E-cadherin-mediated cell adhesion can be operative. Mutations in the E-cadherin gene and loss or mutation of the second, intact copy as well as mutation in the catenin genes, which encode proteins that interact with the cytoplasmic portion of E-cadherin, can be observed. In addition, transient or unregulated phosphorylation by receptor tyrosine kinases or oncogenic tyrosine kinases, respectively, can interfere with the epithelial morphology and induce a mesenchymal conversion. Since tyrosine phosphorylation of beta-catenin correlates with the epithelial-mesenchymal transition that is observed, E-cadherin-mediated cell adhesion might be modulated by such a mechanism. Interestingly, the same molecules implicated in the control of malignant properties turn out to play fundamental roles in the control of normal epithelial growth, differentiation and morphogenesis.

In this article, the development and application of a modified technique of impression cytology for the electron-microscopic examination of corneal epithelium is presented. Corneal epithelium of controls and diabetic patients was obtained using millipore filters, which had been presoaked with EDTA to weaken intercellular adhesions. Light and electron microscopy (EM) of the control samples showed sheets of well-preserved corneal epithelial cells. Therefore, this modified technique is supposed to be useful for the analysis of alterations of the corneal epithelium. EM of corneal cells of diabetic patients showed irregularly formed epithelial cells. The corneal epithelium of diabetic patients revealed thickening of the basement membrane and a decreased number of hemidesmosomes. The alterations observed in specimens of diabetic patients represent the morphological equivalent of an impaired epithelial barrier function, an increased fragility of epithelial lining and the disturbed healing properties occurring in diabetic keratopathy.

Subepithelial fiber components of the round window membrane (RWM) of the guinea pig were studied by the following methods: transmission electron microscopy (TEM), scanning electron microscopy (SEM) with NaOH treatment, high-voltage electron microscopy (HVEM) and immunohistochemical staining. SEM observation revealed intersecting collagen fibers and a vascular network in the middle layer. TEM observation showed that at the attachment region to the bone fibroblasts, collagen fibers and elastic fibers were intermingled with each other, and mesenchymal cells (bone-lining cells) were scattered on the surface of the bone. The cytoplasmic processes of fibroblasts and bone-lining cells were connected by interdigitations. HVEM demonstrated that collagen fibers of the RWM were directly linked to the fibers of the bone matrix. Immunohistochemically, the middle layer was positive for antibody to type I collagen. Immunoreaction for fibronectin was also positive at the middle layer and most intense in the region attached to the bone. Thus, the RWM is securely attached to the bone by type I collagen fibers together with the bone-lining cells and fibroblasts.

The occurrence of phagocytosis of spermatozoa in the epididymis is a controversial issue in male reproductive biology. In this paper we documented, by scanning and electron microscopy, the presence of spermatophages in the lumen of the proximal region of stallion epididymis. Sperm phagocytosis involving the principal cells that line the cauda epididymidis region was also observed. The possible meaning of this process in the removal of damaged, aged or surplus spermatozoa is discussed. These observations were made in samples obtained from random cases, during the course of research work carried out on the stallion male seminal duct system.

The interactive effects of clomiphene citrate (CC) and exogenous oestrogen (E2) or progesterone (P4), on the vaginal epithelium of ovariectomised rats were examined using scanning electron microscopy and light microscopy. Results were compared with P4E2 treatment. Statistical analysis showed no significant difference between the two combination CC treatments. All treatment groups had the ability to stimulate a dormant epithelium and cause surface modifications in the form of individually elongated microvilli and rosette formations. The results of this study indicate that while P4 enhances the oestrogenicity of CC, there is no cumulative oestrogenic effect when E2 and CC are combined, but there is modification of cell surface organisation and evidence of anti-oestrogenic activity.

Podocytes are highly differentiated cells that appear to be postmitotic. However, it is unclear whether cell division or the proliferation of podocytes takes place in the setting of glomerular injury. In this study, we analyzed the presence of mitotic figures and of binucleate or multinucleate podocytes in diseased human kidneys by light and electron microscopy. Among the 164 cases analyzed, mitotic figures in podocytes were found in only one cell, in a case of focal and segmental glomerulosclerosis, and 6 cases provided evidence of two or more nuclei in a single podocyte. Serial sections by light microscopy confirmed that the nuclei in multinucleate cells were separate and of approximately the same size. Electron-microscopic analysis of the binucleate podocytes revealed hypertrophy and effacement of foot processes. These findings indicate that, in human podocytes, mitosis without cytokinesis (cell division) results in binucleate cells in cases of glomerular injury.

The method of in vivo cryofixation with freeze substitution (VC-FS) was used to examine the natural ultrastructure of the corneal superficial epithelium under the transmission electron microscope, and the results have been compared with the conventional method. With the VC-FS method, the ultrastructure down to 8 microns in depth was well preserved in the specimen. There was a layer of homogeneous material thought to be tear film on the corneal surface. Except for the intercellular spaces presenting under the first and sometimes also the second layers of the epithelial cells, other adjacent cells were in close contact without any spaces between them or between the desmosomes. In contrast, when using the conventional method, intercellular spaces were found between adjacent cells in all the layers of epithelium, and no tear film remained on the corneal surface. VC-FS is considered to be a better way to preserve the structure of tissue in its natural state than the conventional method and to be a useful method for the morphological study of the corneal epithelium and the tear film. Intercellular spaces, a kind of artifact, can be reduced by this method.

Evidence from retroviral marking techniques and immortalized cell lines indicates that multipotential stem cells exist in many areas of the developing central nervous system. However, the factors that influence the commitment of these stem cells into distinct neuronal or glial lineages are not known. We have created an immortalized hypothalamic cell line derived from embryonic day 14 hypothalamic cells with a replication-defective retroviral construct containing a temperature-sensitive allele (tsA58) of the large T antigen of the simian virus 40. The clonality of this cell line, which we have named V1, was established by single cell cloning and by Southern blot analysis. V1 cells exhibit two different morphologies: the vast majority of cells are flat and stellate, and a smaller number are phase-bright round cells with processes. V1 cells express nestin and neural-cell adhesion molecule, typical of proliferating neuroepithelial cells. They also express glial fibrillary acidic protein and S100 as well as the low molecular weight neurofilament protein. In addition, the phase-bright, process-bearing V1 cells stain intensely for many typical neuronal proteins, such as low, medium and high molecular weight neurofilament proteins, tau protein, microtubule-associated protein-2, and neuron-specific enolase. The phase-bright cells also have condensed chromatin and display mitotic spindles, indicating that they are in mitosis. When V1 cells are transferred from the permissive temperature (33 degrees C) to the restrictive temperature (39 degrees C), there is a decrease in expression of NF-L and an increase in expression of NF-H and glial fibrillary acidic protein in the flat V1 cells. The enhanced expression of neuronal antigens in mitotically active V1 cells is novel and may represent a more general property of the differentiation process. We suggest that V1 cells arise from a mixed neural/glial neuroepithelial progenitor cell that expresses both neuronal- and glial-specific proteins in the developing hypothalamus.

Secretory particles, designated as 'vesiculosomes', were isolated and purified from bovine seminal vesicle fluid. The protein pattern was characterized using one- and two-dimensional gel electrophoresis. Native vesiculosome fractions were used for immunization of female New Zealand rabbits. The resulting vesiculosome antiserum was used for immunohistochemical studies, designed to obtain data on the intracellular formation, the localization and the potential release from secretory cells of the respective proteins present in these particles. The specificity and sensitivity of the antiserum was tested by Western blot analysis and by an ELISA system. Western blot analysis of isolated vesiculosomes separated on SDS-PAGE showed several heterogeneous immunoreactive bands in the range of 16 kD and above 66 kD, indicating a polyvalent mixture of immunoglobulins. Immunohistochemical staining of different bovine tissues, nevertheless, revealed a strong reaction exclusively at the apical portion of secretory cells of the seminal vesicle and the ampulla of the vas deferens and of intraluminal secretion. The antiserum recognizes both secretory granules and plasma membrane proteins of seminal vesicle epithelium, supporting the concept of vesiculosomes to represent cytoplasmic particles formed from plasma membranes surrounding apical 'blebs' of the cells that are released physiologically during secretion.

A method is described for the sequential detergent and high ionic strength extraction of human amnion with the progressive enrichment of the intermediate filament (IF) cytoskeleton and its associated structures including hemidesmosomes (HD). TEM of the extracted epithelium in situ reveals IF bundles beneath the apical cell surface, around the nucleus and at the lateral edges of the cells where association with desmosomes occurs. IF bundles are also very prominent within basal cell processes where they loop through the cytoplasm adjacent to the HDs. A novel connecting filament network is observed running between the IFs and the hemidesmosomal dense plaque. The adjacent IF network contains both cytokeratin and vimentin, the latter revealed much more fully as a result of the extraction protocol. The hemidesmosomal plasma membrane contains integrin subunits alpha 6 and beta 4 and these are quantitatively retained as the basal cell surface during extraction, while nonjunctional plasma membrane is solubilised. Integrin beta 1 is found at the basolateral cell surface but, like actin, is extracted quantitatively and is not present in HDs. The extracted epithelial cells may be recovered by scraping and the IF network depolymerised to produce a particulate fraction containing short residual IFs, associated thin filaments and plaque material. This fraction contains immunoreactive cytokeratin and vimentin. Integrin alpha 6 beta 4 has been used as a biochemical criterion of the presence of HD material in the fraction. Both subunits are highly enriched. The fraction also contains the hemidesmosomal components HD1, BP230 and BP180. This method is likely to be useful in further characterisation of the HD.

Retinal pigment epithelial (RPE) cells are largely postmitotic. They continuously phagocytose the outer tips of the photoreceptor outer segments (POS). Over the life span of an individual, this activity results, although surprisingly slowly, in the intralysomal accumulation of lipofuscin, or age-pigment. Native lipofuscin shows orange-red autofluorescence when exposed to blue light. The loss of energy resulting from the conversion of excitatory blue light into emitted orange-red light may induce photo-oxidative reactions. We exposed neonatal rabbit RPE cells in culture to purified POS from cow eyes. The material were either native or peroxidized by irradiation with UV-light before being added to the RPE cultures. Lipofuscin accumulation was studied by transmission electron microscopy and measured by microfluorometric registration of its autofluorescence. Cells exposed to peroxidized POS accumulated much more lipofuscin than those exposed to native POS, indicating that peroxidized outer segments are not digestable by lysosomal enzymes. Furthermore, lipofuscin-loaded RPE cells were considerable more sensitive to visible blue light than unloaded control cells. The former ones showed lysosomal membrane destabilization with ensuing leakage of lytic enzymes and eventually cell death. We suggest that photo-oxidation of lysosomal membranes surrounding accumulated lipofuscin may be of importance for the development of age-related macular degeneration.

Morphology and distribution of epithelial foot processes (FP) were studied in hamster neonatal lung bronchioles. To correlate them with growth, bronchiolar mitotic index was determined, rat small intestine used as a reference tissue and the distribution of foot processes compared to the distribution of immunoreactivity against proliferating cell nuclear antigen (PCNA). In bronchioles, where growth occurs through dispersed mitoses, FP also occur dispersed throughout the whole perimeter of the epithelium; whereas in small intestine, where growth is localised in a specific region (crypts), FP are likewise confined to a distinct, but different, zone (villi). Staining the bronchioles for PCNA labels dispersed grouplets of nuclei; most of which being only weakly stained. In the intestines, strong immunostaining mainly occurs in the crypts, while the villi mainly display a weak nuclear stain. Additionally, in both types of tissue the distribution of FP and weak PCNA staining are parallel. Thus, an apparent association between FP, lack of proliferation and nucleoplasmic PCNA staining arises; the basis of which being presently unclear. However, a possible interrelating factor might be a growth inhibitory influence of epithelio-stromal interactions on the epithelial cells.

Rat kidneys undergoing tubular regeneration after ischaemic injury have been examined with regard to EGF, EGF receptor and vimentin, using immunohistochemical techniques. Renal ischaemia was induced in male Sprague-Dawley rats by 35-min clamping of renal arteries. Groups (n = 4-6) of experimental animals were killed at different time intervals (12, 24, 48, 72 h, 7 and 14 days) after reperfusion. One hour before sacrifice, each rat received i.p. 200 mg/kg 5-bromo-2'-deoxyuridine (BrdU) for the immunocytological demonstration of DNA synthesis. Renal necropsies were processed to reveal by immunohistochemistry EGF, EGF receptor, vimentin, and BrdU incorporated into DNA of S-phase cells. Tubular necrosis particularly involved proximal straight tubules in the outer stripe of the outer medulla and was followed by tubular regeneration, with a peak of cell proliferation at 48-72 h and an apparent dedifferentiation of tubular epithelium. As soon as 12 h after ischaemia, there was a substantial reduction of EGF immunostaining and the incidence of distal tubules showing EGF immunoreactivity reached a nadir at 48 h. In control kidneys, EGF receptor was mostly immunolocalized in proximal tubules although juxtaglomerular cells also exhibited immunolabelling. EGF receptor immunostaining in tubular epithelium showed no major change during the episode of tubular necrosis (12-24 h) but disappeared in tubular profiles undergoing regenerative hyperplasia (48-72 h). No vimentin immunostaining was found in tubules of control kidneys. Tubular epithelium remained mostly vimentin negative during the early phase of tubular necrosis/regeneration (12-72 h). By contrast, 7 days after ischaemia numerous dedifferentiated tubules expressed vimentin. In conclusion, tubular regeneration after ischaemia is associated with a typical sequence of transient events: (1) reduction of EGF immunostaining; (2) disappearance of EGF receptors during the mitogenic response; (3) expression of vimentin in tubular epithelium, and (4) return to a normal appearance.

The renal epithelium appears to be an important target tissue for retinoic acid and epidermal growth factor (EGF). We report here that retinoic acid enhances the proliferative effect of EGF on glomerular epithelial cells (GEC) in vitro and also increases EGF binding to GEC. When GEC were exposed to EGF (> or = 1 ng/ml), cellular DNA synthesis was markedly increased. Moreover, the stimulating effect of EGF was synergistically increased by retinoic acid at 5 micrograms/ml. 125I-EGF binding to cultured GEC was increased approximately 3-fold after addition of retinoic acid to cultures for 48 h. Analysis of 125I-EGF binding revealed 8.1 x 10(4) receptors per control (untreated) cell, while retinoic acid-treated cells demonstrated an increase to 14.3 x 10(4) receptors per cell with no detectable change in receptor affinity. These findings suggest that interactions between retinoic acid and EGF may play an important role in the regulation of GEC growth.