The influence of fixation on the relative amount of cytoplasmic ribosomes in mouse epidermal basal keratinocytes

The nature and significance of so-called dark keratinocytes in the epidermis during chemical carcinogenesis is still a matter of concern and debate. Based on ultrastructural observations it has been suggested that dark cells most often are shrunken cells. Reports on skin carcinogenesis, however, claim that dark cells are a sign of ongoing tumor promotion and represent those stem cells in the epidermis from which the tumors originate. It is therefore important to find out whether these cells are simply injured and shrunken cells, or vital cells of great importance for carcinogenesis. Dark cells are assumed to be rich in ribosomes. There is evidence, however, that the observed number of dark cells is highly dependent on tissue fixation. In the present ultrastructural study, morphometric methods were used to compare the effects of two different fixation procedures on the amount of cytoplasmic ribosomes in dark cells from both untreated and carcinogen-treated hairless mouse epidermis. The results show that the ultrastructural features of both dark and clear cells vary considerably with different fixation procedures. In acetone-treated controls typical dark cells are only observed when the fixative has a lower osmotic activity than the plasma. With iso-osmolal fixation typical dark cells are not observed. After an abortive two-stage carcinogenesis treatment, in which a single application of 9,10-dimethyl-l,2-benzanthracene (DMBA) in acetone was followed by a single application of 12-O-tetradecanoyl-13-acetate (TPA) in acetone, signs of cell injury could be found after both fixation procedures. With DMBA/TPA and hypo-osmolal fixation the number of dark cells seemed to increase, whereas only signs of cell injury with occurrence of some heavily altered “clear cells” dominated the picture with iso-osmolal fixation. Morphometry showed that both the numerical and the volumetric densities of cytoplasmic ribosomes in basal keratinocytes varied most significantly with the fixation procedure used. The cytoplasmic volumes did not vary in a way that could explain these differences. One might therefore assume that the number of ribosomes depends on the fixative. Large swelling artifacts occurred when a fixative with low osmotic activity was used, leading to compression of neighboring cells. Hence, an increased ribosomal density reported previously in dark cells is probably related to such cell volume artifacts and does not reflect an actually increased quantity of ribosomes. With both fixation procedures, a single application of DMBA followed by one of TPA appeared to produce an increased number of ribosomes in basal keratinocytes. When hypo-osmolal fixation was used, however, treatment with DMBA/TPA did not influence the cytoplasmic volume or the numerical density of ribosomes, in dark cells. This might indicate that so-called dark keratinocytes following DMBA/TPA treatment are functionally inactive cells that appear more vulnerable than active cells to compression during hypo-osmolal fixation.     

The influence of fixation on the morphology of mouse epidermis. A light and electron microscopical study with special reference to "dark cells" and epidermal carcinogenesis

Different opinions exist on the normal ultrastructure of the epidermis including the significance of so-called basal dark cells. Thus, the dark cells are still assumed to be key elements in experimental skin carcinogenesis. We therefore explored the effects of tissue fixation on the ultrastructure of the epidermis. Untreated normal hairless mouse skin was processed for transmission electron microscopy with two different sets of fixatives, applied either by perfusion-immersion or immersion fixation only. The morphology of both the basal and the lower suprabasal layers of the epidermis, including the extracellular space, the shape and volume of the cells, their electron density, and the organisation of some of the organelles, were profoundly affected by the choice of fixatives. The non-keratinocytes showed comparable changes, including the appearance of a dark phenotype. The incidence of small electron-dense keratinocytes (dark cells) and the nature of their ultrastructure changed markedly with the fixation procedure. We were not able to identify undifferentiated dark cells. The pattern of changes and the quality of the morphological picture were almost unaffected by the mode of fixation. The upper suprabasal and the cornified layers appeared to be more or less unaltered by the change in fixatives and the method of application. The vehicle osmolality of the primary fixative was found to be mainly responsible for the ultrastructural appearances. A low vehicle osmolality may be responsible for the occurrence of the dark cell phenomenon, by inducing swelling artefacts of many cells with compression of some neighbouring cells.     

A comparative study of cytoplasmic basophilia and the population density of ribosomes in the secretory cells of mouse seminal vesicle

Summary A comparative cytochemical and electron microscopic study on the ergastoplasm in the secretory cells of the seminal vesicles of castrated, normal, and testosterone-treated mice is reported. Castration induced a progressive decline in cytoplasmic basophilia (identified with ribonucleic acid) and testosterone treatment caused an enhancement over the normal level. There were corresponding changes in total area of ergastoplasmic membranes, but the expected changes in population density of ribosomes (ribonucleoprotein particles) in the intercisternal cytoplasm did not occur. These observations conflict with the currently-accepted view that almost all of the ribonucleic acid responsible for cytoplasmic basophilia in adult mammalian cells is contained in the ribosomes.Other changes in the fine structure of these cells in the experimental animals are briefly described.This research was aided by grants from the American Cancer Society and the United States Public Health Service (B-2145). Preliminary reports were made at the Tenth Annual Meeting of the Histochemical Society (Deane and Porter 1959) and the Tenth International Congress for Cell Biology (Deane and Porter 1960).     

The influence of different fixatives and a tumor promoter, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on the induction of so-called dark cells in mouse epidermis. A light microscopical study

Epidermal "dark cells" (DC) are believed to play a specific role in the so-called promotion phase of experimental skin carcinogenesis. They are recognized by their morphological features both at the light and the electron microscopical level. The possible effects of fixation on the morphology of epidermal cells and hence on the number of DC have not yet been thoroughly studied. In the present light microscopical study we used a semiquantitative method together with simple cell counting to evaluate the influence of fixation on the specific cellular morphology which is traditionally used to determine the number of DC. The use of cacodylate vehicled prefixatives, either formaldehyde or glutaraldehyde, led to a higher incidence of DC, and furthermore both to an increased width of the intercellular spaces (ICS) and a more heavy staining of the keratinocytes than when s-collidine vehicled glutaraldehyde was used. Differences in yield of DC solely due to the prefixative itself (formaldehyde or glutaraldehyde) were not detected. Exposure to TPA or the use of a hyperosmolal prefixative vehicle both yielded higher DC numbers than did controls or conventional prefixative vehicles, respectively. After prefixation with hyperosmolal vehicles, however, TPA treatment did not induce higher DC yield than in a control series. Phenomena usually accompanying exposure to TPA, such as intercellular oedema (widening of the ICS) and cytoplasmic vacuolization, varied in parallel to the number of DC. Hence, there is reason to believe that the induction of epidermal DC is mainly associated with volume reduction of keratinocytes. Such shrinkage may be due to the cytotoxic properties of TPA and degenerative phenomena appearing during tissue processing.     

Polarization microscopic evidence for an oriented cytoplasmic structure in the "dark" variants of adrenalin-storing cells.

A diffuse cytoplasmic birefringence confined to "dark" adrenalinstoring cells has been described. The main optical characteristics of the birefringence factor include: regular orientation of birefringence relative to the base-apex axis of cells; additive anisotropic staining with methods based on the principle of topo-optical staining reactions; dependence of birefringence on labile morphologic properties. On the basis of the capacity of the macromolecular matrix of chromaffin granules to form lamellar liposomal structures in vitro it has been proposed that a reorientation of molecular organization in the matrix of chromaffin cells is responsible for the observed optical phenomenon. The direction of birefringence was explained by a preferential direction of contractile forces acting during "dark" cell formation.     

The role of fibronectin in adhesion of metastatic melanoma cells to endothelial cells and their basal lamina

Vascular endothelial cells synthesize an extracellular matrix or basal lamina composed of collagens, proteoglycans and glycoproteins such as fibronectin (FN). Using affinity-purified anti-FN, we have examined the role of FN in adherence of metastatic B16 melanoma cells to endothelial cell monolayers which lack FN on apical cell surfaces and to their basal lamina which contains FN. B16 melanoma cells, which do not contain significant amounts of FN, attached at much higher rates to endothelial basal lamina and polyvinyl-immobilized FN compared with intact endothelial cell monolayers. Anti-FN failed to inhibit attachment of melanoma sublines of low (B16-F1) or high (B16-F10) metastatic potential to intact endothelial cell monolayers, inhibited slightly B16 cell attachment to basal lamina and completely abolished attachment of B16 cells to polyvinyl-immobilized FN. The antibiotic tunicamycin which inhibits glycosylation of B16 cell surface glycoproteins and blocks experimental metastasis [18] inhibited B16 attachment to endothelial cells, basal lamina and immobilized FN. The results suggest that FN mediates, only in part, the adhesion of B16 melanoma cells to basal lamina through glycoprotein receptors on B16 cells.     

A morphometric analysis of Prunus spinosa, P. domestica ssp. insititia, and their putative hybrids in Denmark

The taxonomy of Prunus subgenus Prunus is notoriously problematic and reliable discriminating characters for species and subspecies identification are missing. Principal Component Analysis (PCA) was performed on different morphological data sets and useful diagnostic characters for Prunus spinosa and P. domestica ssp. insititia were found. Many traditionally used morphological characters were found unreliable. The hypothesis of widespread hybridization between Prunus spinosa and P. domestica ssp. insititia in Denmark were examined using PCA and HYWIN. It was found that hybrids between the two taxa are rare in Denmark.     

The influence of fixation on staining of glycosaminoglycans in glial cells.

Cytoplasmic staining of glial cells by Alcian blue in 0.40 M magnesium chloride can be demonstrated in sections of brain tissue fixed in formol-acetic acid and in Bouin's solution. Phosphate-buffered aldehyde fixatives, whether at neutral or low pH, fail to preserve stainable material.     

The influence of fixation procedure, embedding medium and section thickness on morphometric data in thyroid gland.

In this study, the effects of fixation procedures, embedding medium and section thickness on stereological measurements of normal thyroid were analysed. The following conclusions were drawn: A) the use of a single section for the analysis of a lobe is sufficient if this section is located in the central part of the lobe. B) fixation and embedding with glutaraldehyde-Epon leads to a larger shrinkage than Bouin-paraplast, but the difference between the two procedures is not significant. C) osmium post-fixation reduces the shrinkage induced by glutaraldehyde and lowers the axial deformation produced by sectioning. D) Bouin's fixative and paraplast embedding induce considerable shrinkage of the interstitial tissue. The shrinkage obtained with glutaraldehyde-Epon is less. However, it is still not known whether this difference is due to the fixative, or to the embedding procedure or to both. E) only in glutaraldehyde and osmium-fixed material, embedded in Epon, can follicles and colloids be assumed to be spherical in shape without significant errors.     

The morphology of the denuded epidermal basal cell layer of the hairless mouse after different preparation methods. A scanning and transmission electron microscopical study

The denuded basal cell layer of the hairless mouse epidermis is described in the present scanning (SEM) and transmission electron microscopical (TEM) study. The suprabasal layers were removed mechanically after trypsinization or by extracellular calcium depletion. Trypsinization before removal of the suprabasal cells caused the basal cells to shrink. Characteristic surface plication and hemi-desmosomal attachment to the basement membrane were generally preserved. SEM revealed partly maintained intercellular bridging, whereas by TEM such contacts were absent because half desmosomes were internalized. Total calcium depletion induced more serious damage to the basal cell surface, which was smooth with apparent perforations. However, cell bridges, and occasional desmosomes were present. The cell interior demonstrated important cellular injury. If the calcium deprived explants were allowed to recover in calcium-containing medium, the cells acquired an activated "regenerative" morphology, without junctions, similar to that observed in wound healing. Epidermal non-keratinocytes were seen only after trypsinization. Control experiments revealed that they adapted poorly to organ culture conditions. By TEM, we observed several interesting aspects of the differences, between dark and clear basal keratinocytes. This was unexpected because fixation studies had shown, that with the present fixation method, typical dark and clear cells do not occur in untreated epidermis. We believe that membrane injury through mechanical stripping of partly adhering epidermal layers induced "clear cells", whereby the neighboring cells appeared darker. This provides additional evidence as to the origin of the two sub-populations, dark and clear basal cells. The clear cells may be injured cells, caused by cell damage, and not by processes of cellular differentiation. The results of the present investigation supports the view that basal keratinocytes have a polygonal shape with numerous free surface extensions and they are anchored to the basement membrane with "foot pads". Our study also shows that SEM of the epidermal basal layer might be feasible. Various artifacts, however, must be considered, depending on the denudation method used. We prefer trypsinization to calcium depletion because it is less time-consuming and results in a cell morphology which in TEM is comparable to that of basal cells in untreated whole epidermis. Extra-cellular calcium depletion, however, might be useful as a method to prepare single cell suspensions for flow cytometry. Restoration of a normal calcium concentration after stripping, provides an opportunity to mimic wound healing in situ, as an alternative t     

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.     

The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO2 fixation in the light and dark.

1. The pH in the stroma and in the thylakoid space has been measured in a number of chloroplast preparations in the dark and in the light at 20 degrees C. Illumination causes a decrease of the pH in the thylakoid space by 1.5 and an increase of the pH in the stroma by almost 1 pH unit. 2. CO2 fixation is shown to be strongly dependent on the pH in the stroma. The pH optimum was 8.1, with almost zero activity below pH 7.3.Phosphoglycerate reduction, which is a partial reaction of CO2 fixation, shows very little pH dependency. 3. Low concentrations of the uncoupler m-chlorocarbonylcyanide phenylhydrazone (CCCP) inhibit CO2 fixation without affecting phosphoglycerate reduction. This inhibition of CO2 fixation appears to be caused by reversal of light induced alkalisation in the stroma by CCCP. 4. Methylamine has a very different effect compared to CCCP. Increasing concentrations of methylamine inhibit CO2 fixation and phosphoglycerate reduction to the same extent. The light induced alkalisation of the stroma appears not to be significantly inhibited by methylamine, but the protons in the thylakoid space are neutralized. The inhibition of CO2 fixation by higher concentrations of methylamine is explained by an inhibition of photophosphorylation. It appears that methylamine does not abolish proton transport. 5. It is shown that intact chloroplasts are able to fix CO2 in the dark, yielding 3-phosphoglycerate. This requires the addition of dihydroxyacetone phosphate as precursor of ribulosemonophosphate and also to supply ATP, and the addition of oxaloacetate for reoxidation of the NADPH in the stroma. 6. Dark CO2 fixation in the presence of dihydroxyacetone phosphate and oxaloacetate has the same pH dependency as CO2 fixation in the light. This demonstrates that CO2 fixation in the dark is not possible, unless the pH in the medium is artificially raised to pH 8.8.     

A human epidermal differentiation-specific keratin gene is regulated by calcium but not negative modulators of differentiation in transgenic mouse keratinocytes.

Keratins K1 and K10 represent the major differentiation products of the maturing epidermal keratinocytes. Primary epidermal cell cultures from newborn K1 transgenic mice containing a 12-kilobase human K1 genomic fragment were established in order to examine the expression of both human and mouse K1 in the presence of known modulators of epidermal differentiation. Elevated levels of Ca2+ in the culture medium induced both mouse K1 and human K1. Supplementing the medium with retinoic acid or 12-O-tetradecanoylphorbol-13-acetate or introducing a Harvey viral ras oncogene (v-rasHa) into the cells completely suppressed mouse K1 but not human K1. Our results suggest that: (a) the human 12-kilobase insert contains all the necessary cis-acting elements to respond to the Ca2+ signal, and (b) other cis-acting elements, not present within this insert, may function independently to regulate the response of K1 to retinoids, 12-O-tetradecanoylphorbol-13-acetate, and v-rasHa transformation. This transgenic model provides an approach to identify elements required for the regulation of an epidermal differentiation-specific gene.     

The coating of mouse myocardial cells. A cytochemical electron microscopical study.

The coating of mouse myocardial cells has been investigated with a variety of cytochemical methods. The coating of the surface membrane gives a positive reaction with ruthenium red, colloidal thorium, phosphotungstic acid (PTA) at low pH, silver methenamine after periodic oxidation (PA-silver technique) and with silver proteinate after periodic oxidation and thiocarbohydrazide treatment (PA-TCH-silver technique). The coating of the T system gives almost similar results. The nexuses do not react with PTA nor with the PA-silver and PA-TCH-silver techniques, but they are strongly stained with ruthenium red which reveals periodic structures in their gaps. The specificities of the colloidal thorium technique and PAT staining have been tested by chemical treatments (methylation, acetylation, saponification), enzymatic digestions (pronase, trypsin, hyaluronidase, neuraminidase) and carbohydrate extractions (with 0.1 N NaOH and 0.05 M H2SO4). These cytochemical data indicate, considering the specificity of the reactions, that the coating of the membrane surface and the T system contains polyanionic groups. A part of them, at least, would belong to a carbohydrate-containing material (glycoproteins), whereas at the level of nexuses the sugar residues would probably be absent.     

Membrane-bound ribosomes of myeloma cells. III. The role of the messenger RNA and the nascent polypeptide chain in the binding of ribosomes to membranes

Mild ribonuclease treatment of the membrane fraction of P3K cells released three types of membrane-bound ribosomal particles: (a) all the newly made native 40S subunits detected after 2 h of [3H]uridine pulse. Since after a 3-min pulse with [35S]methionine these membrane native subunits appear to contain at least sevenfold more Met-tRNA per particle than the free native subunits, they may all be initiation complexes with mRNA molecules which have just become associated with the membranes; (b) about 50% of the ribosomes present in polyribosomes. Evidence is presented that the released ribosomes carry nascent chains about two and a half to three times shorter than those present on the ribosomes remaining bound to the membranes. It is proposed that in the membrane-bound polyribosomes of P3K cells, only the ribosomes closer to the 3' end of the mRNA molecules are directly bound, while the latest ribosomes to enter the polyribosomal structures are indirectly bound through the mRNA molecules; (c) a small number of 40S subunits of polyribosomal origin, presumably initiation complexes attached at the 5' end of mRNA molecules of polyribosomes. When the P3K cells were incubated with inhibitors acting at different steps of protein synthesis, it was found that puromycin and pactamycin decreased by about 40% the proportion of ribosomes in the membrane fraction, while cycloheximide and anisomycin had no such effect. The ribosomes remaining on the membrane fraction of puromycin-treated cells consisted of a few polyribosomes, and of an accumulation of 80S and 60S particles, which were almost entirely released by high salt treatment of the membranes. The membrane-bound ribosomes found after pactamycin treatment consisted of a few polyribosomes, with a striking accumulation of native 60S subunits and an increased number of native 40S subunits. On the basis of the observations made in this and the preceding papers, a model for the binding of ribosomes to membranes and for the ribosomal cycle on the membranes is proposed. It is suggested that ribosomal subunits exchange between free and membrane-bound polyribosomes through the cytoplasmic pool of free native subunits, and that their entry into membrane-bound ribosomes is mediated by mRNA molecules associated with membranes.     

The influence of dietary protein deficiency on haemopoietic cells in the mouse.

These experiments examined the effect of a diet limited only in protein (4% by weight) on haemopoietic stem cells in mice. This diet places severe restrictions on growth and cell proliferation and this was reflected in lower numbers of colony forming units (CFUs) and in vitro colony forming cells (CFCs). Differences were apparent in the response of different organs to this stress; for instance, the incidence of spleen CFUs fell sharply from around 40/mg spleen tissue to 1-4/mg spleen tissue after 3 weeks on a low protein diet. This selective loss did not occur in bone marrow where total CFUs remained proportional to cellular content. Yet a third pattern was shown by thymus CFUs--although the numbers were low these increased from 16/thymus in normal mice to 132/thymus in deprived mice. This was the only organ examined which showed an increase. The effects of a return to a high protein (18%) diet showed that the spleen was the most responsive organ. By day 5 after the return to 18% protein the spleen contained as many CFUs per million cells as the bone marrow. During this time the content of CFU in the spleen had increased some 50-fold whereas bone marrow CFUs only doubled. The spleen assumes the major reconstructive role during the refeeding process.     

Dark hair cells in the inner ear of the rainbow trout. A study of the influence of different fixation methods

The occurrence of dark staining cells in different tissues has been suggested to be artefactual and caused during the fixation process. In inner ear sensory epithelia, dark hair cells (DHC) have been suggested to be apoptotic cells. We have examined whether dark cells represent dying cells or whether they are the results of fixation artefacts. The effects of buffer osmolarity and different fixation methods on the incidence of dark hair cells in the inner ear macula sacculi of the rainbow trout (Oncorhynchus mykiss) were investigated by light and electron microscopy. Glutaraldehyde in phosphate buffer with osmolarities of 0, 135, 225, 425, and 560 mosmol were used for fixation by immersion. For comparison, fixation by vascular perfusion as well as the effects of mechanical injury and delayed fixation were studied. DHC were found in all examined saccular maculae except for the delayed fixation protocol where almost all the sensory cells were lost. The number of DHC accounted for 2.5–12.9‰ of the sensory cells. Neither the buffer osmolarity nor the fixation method had significant effects on the frequencies of DHC. Mitotic cell division events were seen exclusively in the apical cell strata of the sensory epithelium. The DHC are suggested to be associated with apoptosis rather than fixation artefacts.