Serotonin-containing epithelial cells in rat duodenum. I. Quantitative morphometric study of the distribution density

Summary Serotonin (5HT)-containing epithelial cells in rat duodenum were studied quantitatively by three-dimensional morphometric analysis. Longitudinal sections covering the whole length of rat duodenum were stained by either 5HT immunohistochemistry or by glyoxylic acid fluorescent histochemistry. Three-dimensional values for positive cell density, namely the number of 5HT cells per unit volume of the epithelium, were obtained by stereological morphometry with the aid of a computer-assisted image analyzer. This analytical method provides an absolute value for the distribution density of 5HT-containing cells regardless of thickness of sections, or which of the two histochemical procedures is used. The mean number of such cells per unit volume was higher in the crypts than in the villi but varied little along the duodenum. The density of 5HT cells in a given duodenal region, however, varied greatly among individual animals. The villi of the 10 to 16-mm segment from the pylorus were identified as having the smallest individual variation and therefore as being the most suitable for statistical evaluation in future pharmacohistochemical investigations.     

Serotonin-containing epithelial cells in rat duodenum. I. Quantitative morphometric study of the distribution density

Serotonin (5HT)-containing epithelial cells in rat duodenum were studied quantitatively by three-dimensional morphometric analysis. Longitudinal sections covering the whole length of rat duodenum were stained by either 5HT immunohistochemistry or by glyoxylic acid fluorescent histochemistry. Three-dimensional values for positive cell density, namely the number of 5HT cells per unit volume of the epithelium, were obtained by stereological morphometry with the aid of a computer-assisted image analyzer. This analytical method provides an absolute value for the distribution density of 5HT-containing cells regardless of thickness of sections, or which of the two histochemical procedures is used. The mean number of such cells per unit volume was higher in the crypts than in the villi but varied little along the duodenum. The density of 5HT cells in a given duodenal region, however, varied greatly among individual animals. The villi of the 10 to 16-mm segment from the pylorus were identified as having the smallest individual variation and therefore as being the most suitable for statistical evaluation in future pharmacohistochemical investigations.     

Cultured proliferating rat mammary epithelial cells

Summary Normal epithelial cells from the rat mammary gland proliferated in culture when plated with lethally irradiated cells of the LA7 rat mammary tumor line. Proliferation of the normal rat cells occured as the LA7 cells slowly died from the radiation. By labeling the cultures with³H-thymidine it was determined that most of the proliferating rat cells were those adjacent to the LA7 feeder cells. The epithelial cells from the primary culture proliferated after subsequent passages if the cells were plated at each subculture with newly irradiated LA7 cells. If the cells were plated at a ratio of ∼1:8 rat:LA7 a confluent layer of normal rat cells covered the plastic substrate after 6 to 7 wk. The cells have so far been carried up through Passage 7, which amounted to ∼19 doublings in cell number, and still proliferate vigorously. The growth medium for this culture system was Dulbecco’s modified Eagle’s medium:Ham’s F12 1:1 supplemented with fetal bovine serum, insulin, and antibiotics. The presence in the cells of keratin, desmosomes, and cell junctions attested to their epithelial origin. The cultures were composed of cells with diploid or near diploid chromosome numbers. Samples of the cultured cells were implanted into the cleared fat pads of nude mice. Most of the implants from Passage 2 formed normal mammary ductal structures, but the incidence of outgrowths decreased significantly with later passages until no out-growths resulted from the implantation of cells from Passage 5. The one unusual, feeder-independent cell line that arose from a primary culture seemed to be immortal in culture, contained a hyperdiploid chromosome complement, and formed abnormal structures when implanted into cleared fat pads. This work was supported by the Veterans Administration, Washington, DC, and by CA grant 05388 from the U.S. Public Health Service, Washington, DC.     

Glucose metabolism in proliferating epithelial cells from the rat colon.

1. The effects of fasting and fasting followed by refeeding on the activities of the oxidative pentose pathway (OPP) and the tricarboxylic acid cycle (TCA) in isolated rat colonocytes were estimated by the rate of production of 14CO2 from [1-14C]glucose and [6-14C]glucose, respectively. 2. Refeeding after a fast induced a 2-3-fold increase in glucose flux through the OPP and TCA cycle and the degree of change was similar in colonocytes from the proximal and distal colon. 3. Butyrate at a concentration of 40 mM inhibited the OPP by 20-30% (P less than 0.05) but had no effect on the activity of the TCA cycle. Glutamine at a concentration of 2 mM decreased the glucose flux through both the OPP and the TCA cycle by 30-50% (P less than 0.05). 4. Production of 14CO2 from the oxidation of butyrate or glucose indicated that the former was 5-7 times more active in colonocytes from fasted rats. After refeeding, however, butyrate utilization was similar to fasting values in the proximal colon but significantly lower (P less than 0.05) in the distal colon.     

Tight junctions of human uterine epithelial cells change during the menstrual cycle: a morphometric study.

Tight junctions between luminal epithelial cells of the human uterus were studied by freeze-fracture electron microscopy. It was found that junctional complexity decreased during the menstrual cycle, and we explore how this finding may contribute to the role of the uterus in facilitating implantation.     

Nuclear morphometric features of epithelial cells lining keratocysts

OBJECTIVE: To investigate the nuclear morphometric features of epithelial cells lining keratocysts and some other odontogenic cysts. STUDY DESIGN: All cases were selected from the archives of the Department of Pathology, Gülhane Military Medical Academy, as follows: 20 keratocysts and 10 dentigerous and 10 radicular cysts. Nuclear morphometric variables were measured on hematoxylin and eosin-stained histologic slides. Basal and intermediate cells of the epithelium were evaluated separately. Nuclei of the cells were outlined interactively and measured using a specially written macro program. Area, feret ratio (ratio of the longest nuclear axis to the shortest one) and circularity (F circle) of the nuclei were calculated. Additionally, nuclear densitometric analysis was performed on the keratocyst cases. RESULTS: The number of cells in the basal layer (cell density) was higher in keratocysts than in other cysts. The mean nuclear area of basal cells was smaller than of intermediate cells in both keratocysts and other cysts (P < .001). The feret ratio values revealed that basal cell nuclei of keratocysts were more ovoid as compared to those of other cysts (P < .001). Nuclear densitometric findings showed that the DNA indices of all keratocyst cases were close to 1.0, and the cells were considered diploid. CONCLUSION: Increased cell density, a more ovoid nuclear shape and more variation in the size of basal layer cell nuclei in keratocysts were helpful in differentiating these lesions from other odontogenic cysts.     

Human stools as a source of viable colonic epithelial cells.

Human stools consist of a mixture of undigested food residues, colonic microflora, and cellular components shed from the walls of the gastrointestinal tract. The cellular components are made up mostly of terminally differentiated colonic epithelial cells. Using a combination of Percoll density gradient centrifugation and countercurrent centrifugal elutriation, it is now possible to recover these cells as an enriched fraction from fresh human stools. Cells can be visualized on heat-fixed smears of the enriched fractions stained with modified Wright's stain. The enrichment process is optimized by following the segregation of eukaryotic cells as determined by an ELISA technique using monoclonal antibodies against human double-stranded DNA. This work, demonstrating the feasibility of isolating intact colonic cells from stools, has important applications as a noninvasive approach to the biology of exfoliated cells from the gastrointestinal tract.     

Collaborative validation study of the in vivo micronucleus test using mouse colonic epithelial cells

The in vivo micronucleus test using mouse colonic epithelial cells was evaluated as the 11th collaborative study organized by the Collaborative Study Group on the micronucleus test (CSGMT) with three model chemicals that were known to induce chromosome damage in mouse colonic cells. Five laboratories participated in this validation study. All three model chemicals, i.e. 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), N-methyl-N-nitrosourea (MNU), and mitomycin C (MMC), induced micronucleated colonic epithelial cells in a 4-day exposure protocol in all participating laboratories. We confirmed that the present single cell suspension method could be used to detect the model chemicals as micronucleus inducers in mouse colonic epithelial cells. Advantages of this method are that experiments are easy to perform and that intact cells can be analyzed. The present study suggested that the colon micronucleus assay proposed here is useful for mechanistic studies of colon carcinogenesis.     

Long-term culture of normal human colonic epithelial cells in vitro.

Studies of normal cellular function as well as the understanding of cellular mechanisms of carcinogenesis and other diseases of the large intestine have been limited, particularly due to the lack of long-term culture of normal human large intestinal epithelial cells (NHLIEC). Using the epithelia from surgically resected human colon, we have dissociated a sufficient number of viable NHLIEC and maintained them in in vitro culture for up to 5 months. Normal-appearing human large intestinal mucosal fragments (1 mm2) were treated with 0.01 mg/ml trypsin, 0.2 mg/ml collagenase + 0.1 mM EGTA or 0.1 mg/ml trypsin + 0.1 mM EGTA in a Stomacher laboratory blender to isolate the cells. Compared with other methods, the use of the Stomacher blender combined with low concentrations of proteolytic enzymes yielded greater numbers of cells per gram of tissue, with up to 84% viable cells. Primary and serially passaged NHLIEC were cultured in CMRL-1066, MEM with 5% serum, and serum-free KGM. These media were all supplemented with insulin, hydrocortisone, epithelial growth factor, and bovine pituitary extract. CMRL-1066 was found to be the best medium for NHLIEC. Contaminating fibroblasts were selectively removed by briefly allowing the cells to adhere to the culture vessel and adding 25 U/ml collagenase to the culture media at the first subculture treatment. The epithelial nature and secretory function of the established cells were confirmed by morphological criteria (light microscopy, phase contrast microscopy and electron microscopy), immunoreactivity to cytokeratin, and positive mucin cytochemistry. We propose that using this methodology for the culture and maintenance of NHLIEC for an extended period of time would serve as a valuable model for a variety of investigations.     

Epidermal growth factor-induced alterations in proliferating mouse epithelial cells

The effects of epidermal growth factor (EGF) on the growth and morphology of mouse embryo epithelial cells (MMC-E) were studied in culture. Growing cultures of epithelial cells were incubated in the media containing EGF or certain other mitogenic peptides. It was found that nanogram (ng) quantities of EGF stimulated growth in these cells and caused reversible phenotypic changes in these cells. These changes were not observed in cultures treated with the other mitogens. The compact growing islands of MMC-E cells were surrounded by elongated border cells [12]. EGF induced the elongated border cells to flatten and spread. The change of the elongated border cells into polygonal, flattened cells was dependent on the dose of EGF. After treatment with higher concentrations of EGF all cells appeared more flattened and their cytoplasm was more granular than that of the controls. Scanning electron microscopic studies (SEM) showed that the elongated border cells in the control cultures were distinctly higher than the cells inside the islands, while after exposure to EGF they flattened and had fewer surface microvilli than control cells. When EGF was removed and the cells were further cultivated in media without EGF, the border cells became smaller and elongated, eventually resembling those in the control cultures. These results show that EGF may act as a regulatory factor in the control of the proliferation and differentiation of mouse epithelial cells.     

A morphometric ultrastructural study of the nucleus of cerebellar granule cells

A quantitative approach to the nuclear ultrastructure of cerebellar granule cells is described here. The study was made using conventional electron microscopy from cerebellar cortices of adult rats by means of a semiautomatic image analyzer. The basic observation is that the nuclei of mature granule cells constitute a homogeneous population in terms of morphometric and stereologic data; in fact, the volume density of condensed chromatin within the nuclei remains practically constant in all nuclear sections. These results seem to indicate the existence of a cell-specific nuclear morphometric phenotype which might be considered as an effective criterion for the typification of this cellular lineage.     

A morphometric and comparative study of the malleus.

This study has attempted to research, in detail, the dimensions and form of the malleus, and to indicate possible differences with regard to race, sex and side of origin (left or right). The ossicles were obtained from 75 adult cadavers and the dimensions were determined with the aid of a reflection microscope. Clear statistically significant differences were found between the Negroid and Caucasoid races, as well as between the right and left ossicles. No meaningful differences were determined between male and female ossicles.     

GNRH induces activation of Leydig-like cells in Pleurodeles waltlii. A morphometric study

The ultrastructure of the interstitial cells of the glandular tissue of Pleurodeles waltlii was studied in testis of animals obtained in early breeding season (January) under gonadotropic releasing hormone (GNRH) treatments and controls. These cells (parenchymal or Leydig-like cells) displayed the structural characteristics of steroid-producing cells. GNRH administration for 24 hours induced a significant decrease of both medial volume and volume density of lipid droplets. On the other hand, cell volume, nucleus, mitochondria, mitochondrial cristae and tubules of smooth endoplasmic reticulum were increased. The surface density of mitochondrial cristae was also increased.     

Desmosomes in uterine epithelial cells decrease at the time of implantation: an ultrastructural and morphometric study

Displacement of uterine epithelial cells is an important aspect of implantation in the rat and other species, allowing invasion of the blastocyst into the endometrial stroma. Desmosomes, which are part of the lateral junctional complex, function in cell-to-cell adhesion, and are therefore likely to be involved in displacement of uterine epithelial cells at the time of implantation. This study used transmission electron microscopy to study rat uterine epithelial cells during the peri-implantation period to investigate the change in the number of structural desmosomes along the lateral plasma membrane of uterine epithelial cells. We found a significant decrease in the number of desmosomes along the entire lateral plasma membrane as pregnancy progressed. Furthermore, there were also significant decreases in the number of desmosomes on the apical portion of the lateral plasma membrane between all days of pregnancy examined. In addition, on day 6 of pregnancy, the time of attachment, desmosomes were larger and seen as "giant desmosomes." For the first time, this study has shown that there is a significant reduction in cell height and actual number of ultrastructurally observable desmosomes at the time of implantation in the rat. It is proposed that this reduction in desmosome number leads to a decrease in lateral adhesion between uterine epithelial cells at the time of implantation, and hence is involved in the loss of uterine epithelial cells to facilitate blastocyst invasion.     

Glycoprotein synthesis in colonic epithelial cells from rats

Viable colonic epithelial cells from rats were isolated by a non-enzymatic procedure using EDTA. The isolated cells were fractionated by sedimentation through a 15% to 35% discontinuous Ficoll gradient to yield cells differing in proliferative capacity from three different density regions of the gradient. Glycoprotein synthesis of these fractionated cells was examined in terms of their ability to absorb and incorporate labeled glucosamine and fucose into trichloroacetic acid precipitable material. Glycoprotein synthesis was highest among cells with intermediate densities, banding in the middle of the gradient (Fraction II). Cytomorphological examination showed that these cells were predominantly goblet type.     

A cell cycle study of human mammary epithelial cells.

The growth regulation of human mammary epithelial cells (HMEC) cultured in a growth factor/hormone-enriched (e.g. EGF, insulin) medium with bovine pituitary extract as the only undefined supplement was studied. The doubling times of the cultures, in which the cells appear in colonies, was 55-72 h, and a considerable intercolonial heterogenecity in proliferative activity could be demonstrated. However, every colony, irrespective of the size of the growth fraction, comprised a sub-population of rapidly growing cells which had a mean generation time of approximately 22 h. When insulin was removed from the culture medium, HMEC proliferation was inhibited. This growth inhibition was shown to be a result of a cell cycle-specific block.     

The hyaluronate receptor is preferentially expressed on proliferating epithelial cells

In the present study, we have examined the distribution of the hyaluronate receptor as well as hyaluronate itself in a variety of adult tissues. The hyaluronate receptor was localized with a monoclonal antibody, termed K-3, while hyaluronate was localized using proteolytic fragments of cartilage proteoglycan. Staining with the K-3 monoclonal antibody revealed that the hyaluronate receptor was present in a variety of epithelia including the skin, cheek, tongue, esophagus, vagina, intestines, oviduct, and bladder. However, it was notably absent from epithelial cells of the cornea and stomach as well as from endothelial cells of blood vessels. When present, the hyaluronate receptor was preferentially located in regions of active cell growth, such as in the basal layers of stratified epithelium and at the base of the crypts of Lieberkuhn in intestinal epithelium. A similar phenomenon was observed in cultured 3T3 cells. Cultures of 3T3 cells that were actively proliferating were found to have greater amounts of the receptor than their nonproliferating counterparts. When the various tissues were examined for hyaluronate, it was found to have a widespread distribution, being present in most of the basement membranes and between the cells in stratified epithelium. Indeed, in many cases, the distribution of hyaluronate closely paralleled that of the hyaluronate receptor. These results suggest that the interaction between hyaluronate and its receptor is involved in cell-to-substratum adhesion.     

Comparative proteomic analysis of proliferating and functionally differentiated mammary epithelial cells

Proliferation and differentiation of mammary epithelial cells are governed by hormonal stimuli, cell-cell, and cell-matrix interactions. Terminal differentiation of mammary epithelial cells depends upon the action of the lactogenic hormones, insulin, glucocorticoids, and prolactin that enable them to synthesize and secrete milk proteins. These differentiated cells are polarized and carry out vectorial transport of milk constituents across the apical plasma membrane. To gain additional insights into the mechanisms governing differentiation of mammary epithelial cells, we identified proteins whose expression distinguishes proliferating from differentiated mammary epithelial cells. For this purpose we made use of the HC11 mammary epithelial line, which is capable of differentiation in response to lactogenic hormones. Using two-dimensional gel electrophoresis and mass spectrometry, we found about 60 proteins whose expression levels changed in between these two differentiation states. Bioinformatic analysis revealed differential expression of cytoskeletal components, molecular chaperones and regulators of protein folding and stability, calcium-binding proteins, and components of RNA-processing pathways. The actin cytoskeleton is asymmetrically distributed in differentiated epithelial cells, and the identification of proteins involved in mRNA binding and localization suggests that asymmetry might in part be achieved by controlling cellular localization of mRNAs. The proteins identified provide insights into the differentiation of mammary epithelial cells and the regulation of this process.