Histochemical,ultrastructural and X-ray microprobe analytical studies of localization of calcium in the mucous lining of the rat duodenum

Summary Localization of calcium in a rapid frozen and freeze substituted duodenum of normal, starved or calciumrepleted rat was examined using either of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, a sensitive histochemical calcium stain or electron microscopy. In normallyfed rats, a majority of absorptive cells of the duodenum showed numerous discrete red granular GBHA reactions, approximately 1 m or less in diameter, located primarily along their lateral plasma membranes and within intercellular spaces. Electron microscopy also revealed electron-dense granules, 30–100 nm in diameter, showing a similar distribution as the GBHA granules in the respective absorptive cells, and confirmed their absence in mitochondria and other intracellular compartments. Some of the absorptive cells located exclusively at the tip of each villus contained highly GBHA-reactive tubulo-vesicular structures extending throughout the cytoplasm. However, they displayed virtually no granular GBHA reaction. In these cells, electron microscopy revealed numerous electron-dense granules in the nucleus, mitochondria and in other unidentified organelles. X-ray microprobe analyses of ultrathin sections confirmed the presence of calcium within electron-dense granules associated with both types of absorptive cells. The number and intensity of all GBHA reactions fluctuated according to luminal calcium concentration. In calcium-repleted rats, strong GBHA reactions appeared in a narrow zone of lamina propria at the tip of the villus, overlaid, predominantly, with absorptive cells showing tubulo-vesicular GBHA reactions. these results suggest the existence of distinct types of absorptive epithelial cells in the rat duodenum, with respect to patterns of calcium localization which they display.     

Histochemical localization of calcium in the enamel organ of rat incisors in early-stage amelogenesis

The localization of calcium in the enamel organ of rapidly-frozen, freeze-substituted rat incisors in early-stage amelogenesis was examined by a histochemical calcium-staining method. In secretory ameloblasts, glyoxal bis(2-hydroxyanil) (GBHA) staining revealed intense red reactions in mitochondria and tubulovesicular structures located throughout the cytoplasm, while no reaction was seen in the nucleus and cytosol, nor along the plasma membranes of the respective cells. No significant GBHA reaction was observed in the intercellular compartment and other cells of the enamel organ. Some granular reactions were localized in the cells of the adjacent connective tissue. Control tests confirmed the specificity of GBHA reactions for calcium. Thus, the present observations provide histochemical evidence indicating an exclusive localization of calcium in mitochondria and tubulovesicular structures of the secretory ameloblast, and support their contributions to the translocation of calcium from the proximal to the distal pole of the cytoplasm.     

Calcium containing lysosomes in the normal chick duodenum: a histochemical and analytical electron microscopic study.

Absorptive cells of the normal chick duodenum contain numerous supranuclear vesicular/vacuolar structures. By routine transmission electron microscopy, such structures are membrane bound and demonstrate a granular content. These vesicles appear to move laterally and eventually coalesce with the lateral plasma membrane (exocytosis). The granular contents are resistant to high temperature microincineration, thus revealing their mineral-containing nature. The granular vesicular matrix also stains intensely with osmium pyroantimonate EGTA chelation of pyroantimonate-stained vesicles selectively extracts the granules indicating a high concentration of calcium. X-ray microanalysis also demonstrates a significant intravesicular calcium localization. When tissues were incubated for the presence of acid phosphatase, the supranuclear vesicles were markedly positive for this lysosomal enzyme. A possible role for these calcium-containing lysosomes in the transcellular flux of calcium ions across the intestinal absorptive cell is discussed.     

Calbindin D28k in mammalian intestinal absorptive cells: immunohistochemical evidence

Calbindin D28k and D9k are two cytosolic calcium-binding proteins abundant in intestinal absorptive cells which appear to play a role in calcium translocation. Until today, calbindin D28k was found in avian and reptilian absorptive cells but not in mammalian ones. We have described the presence of calbindin D28k-immunoreactivity in intestinal absorptive cells of pig and jerboa (Jaculus jaculus). Pig calbindin D28k-immunoreactive absorptive cells were prominent in duodenum, they were scattered along the villi and nearly absent in the crypts. Jerboa labelled absorptive cells were located along the colonic mucosal surface. No calbindin D28k could be detected in mouse, rat and goat absorptive cells. Topography of calbindin D28k absorptive cells was compared with calbindin D9k distribution. Our results confirmed the data of the literature showing a gradient of labelling increasing from the crypt to the top of the villus and no positive endocrine cell. Young (48 h old) pigs did not expressed calbindin D28k in absorptive cells although calbindin D9k was detected. Calbindin D28K was also observed in endocrine cells which were numerous in pig and goat duodenum and very rare in mouse and jerboa. Western blot experiments confirmed the presence of calbindin D28k in the adult pig intestine, in the jerboa colon and the absence of cross-reactivity between calbindin D28k antibody and calbindin D9k.     

Immunocytochemical localization of pleurocidin to the cytoplasmic granules of eosinophilic granular cells from the winter flounder gill

The teleost gill is considered to be of significant immunological importance, as it is one of the first tissues exposed to environmental or pathogenic challenge and thus should be well equipped to mount an effective immune response. This study characterizes ultrastructurally and immunocytochemically a tissue granulocyte (eosinophilic granular cell) from the winter flounder gill that was previously determined to be involved in the gene expression and synthesis of a known antimicrobial peptide (pleurocidin). The cell is irregular in shape with a cytoplasm characterized by numerous large, electron-dense, membrane-bounded granules. The nucleus is euchromatic and closely associated with a prominent rough endoplasmic reticulum. The cytoplasm typically contains two to three mitochondria and a centralized Golgi apparatus surrounded by numerous electron-lucent vesicles. Immunogold staining of the cells with an anti-pleurocidin antibody shows large number of gold particles in direct association with the electron-dense granules. These data provide the first evidence definitively showing storage of an antimicrobial peptide in the cytoplasmic granules of an eosinophilic granule cell resident in gill tissue.     

The influence of various cell kinetic conditions on functional differentiation in the small intestine of the rat. A study of enzymes bound to subcellular organelles

The process of cell maturation and cell ageing of absorptive epithelial cells was investigated in normal rat duodenum. The development of a number of enzymes bound to subcellular organelles was studied by using microchemical analyses on various cell compartments dissected from crypts and villi from freeze-dried cryostat sections. The development of the ultrastructural features of the absorptive epithelium was investigated by electron microscopy of various cell positions along the whole length of the crypt and the base of the villus. The data obtained were related to cell position along the crypt and villus and to cell age during migration from the bottom of the crypt to the tip of the villus.The influence of changes in the life-span of the cells and of increasing proliferative activity was studied by comparing normal rat duodenum with that from germfree rats and rats recovering from low radiation doses (72 hr after 400 R).Our data show that the specific activity of nonspecific esterases mainly localized in the endoplasmic reticulum increases when the cells migrate along the upper half of the crypt and the basal part of the villus. Activity of alkaline phosphatase, measured as a marker for the microvilli, is absent in the crypt, but increases linearly from the base of the villus to the tip. The longer life-span of villus cells in germfree animals does not result in a higher activity of these enzymes than in normal animals. An increased proliferative activity in the crypt, as present 72 hr after X-irradiation, is accompanied by a decreased activity of both enzymes but the pattern of activity during cell migration remains the same. The specific activity of enzymes bound to mitochondria or lysosomes (monoamineoxidase and β-N-acetylglucosaminidase) are not affected by changing crypt cell kinetics.Electrophoretic analyses of isolated cell compartments showed that the increase during normal differentiation or the decrease after X-irradiation of esterase activity is due to changes in overall activity, not to the appearance or disappearance of specific isoenzymes. Electron microscopy showed that in the normal intestine there is a gradual development of ultrastructural features during migration of the cell along the crypt while the most drastic changes in cell structure occur at the moment the cell enters the villus. Contrary to our expectation, the ultrastructural development was not influenced by increased proliferative activity in the crypt 72 hr after irradiation, and hence the decrease in enzyme activity found cannot be related to changes in ultrastructure.     

四种淡水养殖鱼类血细胞的细微结构

四种淡水鱼的血细胞形态基本相似。红血球形态与其他低等脊椎动物基本相似。淋巴球绝大部分是小淋巴球:单核球数量较少;四种鱼的嗜中性白血球形态结构差不多,胞核多为蚕豆形,很少见分叶核,分叶一般也只有二叶,这与哺乳类显然不同;嗜酸性白血球的形态结构与其他脊椎动物基本相似;在少数血涂片中看到了嗜碱性白血球。       

Ultrastructural observations on the granular leucocytes of the tuatara Sphenodon punctatus (gray)

The granular leucocytes of an active, mature female tuatara, Sphenodon punctatus (Gray) were examined in the electron microscope. Eosinophils contained a lobulated nucleus, homogeneous, dense, irregularly shaped granules, assorted smaller granular inclusions, mitochondria and beta-glycogen. Endoplasmic reticulum, Golgi complexes and ribosomes were scanty. Immature neutrophils (myelocytes) were regular in outline and contained a compact nucleus. In the adjacent centrosomal region were paired centrioles with connected microtubules, and Golgi complexes. Ovoid electron-dense granules, mitochondria, lipid droplets and numerous microfilaments arranged randomly or in bundles, lay in the cytoplasm. Mature neutrophils were often highly irregular in outline, had a segmented nucleus and contained possibly a second type of granular inclusion. The basophils were regular in outline with a compact nucleus. Numerous ovoid homogeneous, electron-dense granules, mitochondria, beta-glycogen particles and some microfilaments were seen in the cytoplasm. The granules in many basophils appeared 'altered' or degenerate and most of these contained microtubules. The cytology of the granulocytes of the tuatara is compared with that in other vertebrates.     

Ultrastructure of the haemocytes of Tetrodontophora bielanensis Waga (Collembola)

Five types of haemocytes: prohaemocytes, plasmatocytes, granular haemocytes, spherule cells and phagocytes, have been distinguished on the basis of ultrastructural studies. Prohaemocytes are ovoid cells with a simple structural organization. Plasmatocytes are larger; their cytoplasm contains well-developed rough endoplasmic reticulum, numerous mitochondria and free ribosomes. Granular haemocytes are the most numerous of the blood cells, characterized by the presence of electron-dense granules. The cytoplasm of spherule cells contains many spherules made up of filamentous material of medium electron density. Rough endoplasmic reticulum, free ribosomes and mitochondria are also found in the cytoplasm. Phagocytes are the largest haemocytes. Their cytoplasm contains an abundance of lysosomes and myelin structures. In addition to haemocytes, cells intermediate between plasmatocytes and granular haemocytes have been observed, which indicates that the granular haemocytes are derived from plasmatocytes.     

Fine structure of the parotid gland in the crest-tailed marsupial-rat (Dasyuroides byrnei).

The parotid gland of Dasyuroides byrnei was examined by light microscopy, and transmission and scanning electron microscopy. The acini were composed predominantly of seromucous cells with a few mucous cells. The seromucous cells were light or dark cells containing acidophilic spherical granules of moderate to high electron density and had well-developed cytoplasmic organelles-ordinary mitochondria and large mitochondria with tubular cristae, RER with vesicular or tubular elements, and Golgi apparatus with lamellae, vesicles and vacuoles. The mucous cells had basophilic amorphous granules of low electron density, like those of ordinary mucous cells. The intercalated ducts were composed of simple cuboidal light cells having a few electron-dense granules. The striated ducts consisted of tall columnar light cells containing numerous vesicles and mitochondria with tubular cristae, the same as found in acinar seromucous cells.     

The postbranchial digestive tract of the ascidian, Polyandrocarpa misakiensis (Tunicata: Ascidiacea). 2. Stomach

The organization of the stomach in the compound styelid ascidian, Polyandrocarpa misakiensis, is described, and the morphology and cell types of the stomach is discussed from the phylogenetic viewpoint. The stomach is a sac-like organ whose wall is formed into longitudinal folds. The stomach consists of external and internal epithelium. The internal epithelium is simple columnar, except for the bottom of the folds. There are five cell types: absorptive cells, zymogenic cells, endocrine cells, ciliated mucous cells, and undifferentiated cells. The absorptive cells have numerous microvilli. The apical region of these cells is occupied by coated vesicles. The zymogenic cells have a conical outline and a few microvilli on their apical surfaces. There are secretory granules in the apical region of zymogenic cells. The endocrine cells have low cell height and electron-dense granules around the nucleus. Endocrine cells have one or two cilia and a few microvilli on the apical surfaces. The basolateral part of these cells often bulges into the adjoining cells. Immunoelectron microscopy revealed that some endocrine cells have serotonin-like immunoreactivity. The ciliated mucous cells are restricted to a single ventral groove. They have numerous microvilli and a few cilia on their apical surfaces. Moderately electron-dense granules are accumulated in the apical part of the ciliated mucous cells. Undifferentiated cells, filled with free ribosomes, form a pseudostratified epithelium in the base of each fold. The nucleus of undifferentiated cells has a prominent nucleolus. The pseudostratified epithelium of the pyloric caecum consists of electron-dense and electron-light cells.     

Quantitative regional variation in the expression of major histocompatibility class II antigens in enterocytes of the mouse small intestine.

The qualitative and quantitative expression of major histocompatibility class II antigens was investigated in the absorptive epithelium of the duodenum, jejunum, and ileum from mice of C3H/He (H-2k haplotype) and C57BL/6 (H-2b haplotype) strains by peroxidase-antiperoxidase labelling and image analysis. Immunohistochemical labelling revealed that the expression of class II antigens was greatest in the ileum and decreased proximally towards the duodenum. The villus epithelium of the duodenum showed a granular staining pattern in the apices of some cells. In the jejunum, an increased expression was demonstrated in the apical and basal cytoplasm of all cells covering the villus. Cells at the tip of the villus, in addition, showed staining of the lateral surfaces. Ileal enterocytes demonstrated the most intense immunostaining appearing in the cytoplasm and along baso-lateral surface membranes. Quantitative analyses confirmed that a highly significant (p less than 0.0001) difference in expression of class II antigens occurred in the three regions of the small intestine, which corroborated the qualitative findings. This regional variation of class II molecules by the absorptive epithelium may influence regional differences in antigen presenting functions and immune responsiveness to ingested antigens.     

An ultrastructural study of the dorsal lingual epithelium of the crab-eating frog,Rana cancrivora

The amphibian tongue contains two types of papilla which are believed to function in gustation and in the secretion of salivary fluid. Scanning electron microscopy reveals that columnar, filiform papillae are compactly distributed over nearly the entire dorsal surface of the tongue of the frog, Rana cancrivora, and fungiform papillae are scattered among the filiform papillae. Microridges and microvilli are distributed on the epithelial cell surface of the extensive area of the filiform papillae. Light microscopy shows that the apex of each filiform papilla is composed of stratified columnar and/or cuboidal epithelium and its base is composed of simple columnar epithelium. Transmission electron microscopy reveals that most of the epithelium of the filiform papillae is composed of cells that contain numerous round electron-dense granules 1–3 μm in diameter. Cellular interdigitation is well developed between adjacent cells. On the free-surface of epithelial cells, microridges or microvilli are frequently seen. Between these granular cells, a small number of ciliated cells, mitochondria-rich cells and electron-lucent cells are inserted. In some cases, electron-dense granules are present in the ciliated cells. At higher magnification, the electron-dense granules appear to be covered with patterns of spots and tubules. Overall, the morphology and ultrastructure of the lingual epithelium of the three species of Rana that have been studied are quite similar, but they can be easily distinguished from those of Bufo japonicus. Therefore, it appears that lingual morphology is phylogenetically constrained among members of the predominantly freshwater genus Rana to produce uniformity of papillary structure and this morphology persists in Rana cancrivora despite the distinct saline environment in which it lives. © 1993 Wiley-Liss, Inc.     

Release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells

Summary The release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells was studied by electron microscopy. The treatment of the cells with SEB at the concentration of 20 μg/ml caused marked increase of the chromaffin granules that either bound to the plasma membrane by the characteristic rods, measuring 15 to 20 nm in length and showing a tubular structure, or budded off at the free cell surface, surrounded by a layer of rod-containing cytoplasm and enclosed by the plasma membrane. The binding between the granular and plasma membranes by the rods did not lead to membrane fusion and exocytosis of the granular content. Many of the bound granules showed vesiculation with loss of the electron-dense core material; at the same time, some of the binding rods contained intraluminal electron-dense material similar to the granular core material. These findings suggested that the electron-dense material (i.e., norepinephrine) of the bound granules was released extracellularly through channels within the rods. Although the granules were bound to the plasma membrane with equal frequency at the free and contiguous cell surfaces, the granular budding occurred only at the free cell surface, indicating that it occurred incidentally to some granules bound at the free cell surfaces. On the basis of the morphological observations, it is postulated that the electron-dense material of the bound granule is selectively released extracellularly through the rods, leaving the vesiculated granules behind in the cytoplasm. The same mode of release of the granular content was observed, though less frequently, in the untreated control cells. No morphological evidence that indicated that the granular content was released extracellularly by exocytosis was found in the treated and control cells. The present observations indicated that the SEB treatment of PC12 cells stimulated the binding of chromaffin granules to the plasma membrane by the rods and the budding of the bound granules at the free cell surface.     

Ultrastructural characterization of human large granular lymphocyte subsets defined by the expression of HNK-1 (Leu-7), Leu-11, or both HNK-1 and Leu-11 antigens

A peroxidase-colloidal gold double labeling system in immunoelectron microscopy was used to investigate the ultrastructural features of human large granular lymphocytes (LGL) subpopulations. Three subsets of LGL, Leu-7+-Leu-11-, Leu-7+-Leu-11+, Leu-7- -Leu-11+, were characterized using combinations of the monoclonal antibodies anti-Leu-7 and anti-Leu-11. They showed different ultrastructural patterns. In fact, Leu-7+-Leu-11- cells showed a high nuclear/cytoplasmic ratio (N/C), a round nucleus, a cytoplasm with few organelles, and a rather even surface. Moreover, most of them lacked electron-dense granules. On the other hand, Leu-11+ cells displayed a low N/C, an irregular-shaped nucleus, and a cytoplasm containing a well-developed Golgi apparatus, many mitochondria, vacuoles, vesicles, and numerous electron-dense granules. Moreover, they exhibited an irregular cell surface. Thus, Leu-7+-Leu-11- cells seemed to represent an immature form of LGL, while cells expressing the Leu-11 antigen showed a fine structure specific for functional NK cells. Our findings suggest that the expression of HNK-1 (Leu-7) and Leu-11 antigens respectively represents subsequent stages in NK cell differentiation.     

Oöcytes degeneration in the queen honey bee after infection by Nosema apis

Terminal o?cytes containing yolk in both healthy and nosema infected queen honey bees were studied. In the healthy queens the terminal o?cytes exhibited a layer of follicular cells which were covered by a smooth-surfaced ovariole sheath. In the o?plasm were numerous electron-dense yolk granules and lipid yolk droplets. The elecron-dense yolk granules exhibited a crystalline structure. Stacks of endoplasmic reticulum were observed in the yolk granules throughout the o?plasm. Numerous mitochondria possessing well defined cristae were also observed. O?cytes in the ovary of queen honey bees appeared degenerated after 7 days of infection by Nosema apis. The ovariole sheath was wrinkled. In the o?plasm, yolk granules were broken down into small spheres and granular substances. Numerous ribosomes without stacks of endoplasmic reticulate were observed. Lysosomes were abundant and numerous electron-dense materials surrounded by a membrane were detected. The o?cytes appeared to be extensively autolysed. The significance of these observations is discussed.     

Fine structure of the dorsal lingual epithelium of the lizard, Gekko japonicus (Lacertilia, Gekkonidae)

Three different types of lingual papilla were observed by scanning electron microscopy on the dorsal lingual epithelium of the lizard Gekko japonicus. Dome-shaped lingual papillae were located at the apex. Flat, fan-shaped lingual papillae were seen in the widest area of the lingual body. Long, scale-like lingual papillae were arranged on the latero-posterior dorsal surface. At higher magnification, microvilli and microridges were seen to be widely distributed over the surface of the papillae. By light microscopy, the epithelium of the dome-shaped papillae was composed of single, columnar epithelial cells filled with secretory granules. The tip of the epithelium of the fan-shaped and scale-like papillae was composed of stratified squamous epithelial cells without granules. The major part of the epithelium of these two types of papilla, except the tip area, was also composed of single, columnar epithelial cells with secretory granules. By transmission electron microscopy, a nucleus without a defined shape was seen to be located in the basal part of each of the single, columnar epithelial cells. Rough-surfaced endoplasmic reticulum and Golgi apparatus were well developed around the nucleus. The other, major part of the cytoplasm was filled with the spherical secretory granules, a large number of which had very electron-dense cores and moderately electron-dense peripheral regions. In the stratified squamous epithelium, a nucleus, which tended to be condensed on the free-surface side, was located in the center of each cell. Mitochondria, endoplasmic reticulum, and vesicles were observed in the cytoplasm.     

Distribution of Ca2+-ATPase, ATP-dependent Ca2+-transport, calmodulin and vitamin D-dependent Ca2+-binding protein along the villus-crypt axis in rat duodenum

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Ca2+-ATPase activity and ATP-dependent Ca2+-transport rates in basolateral membranes from rat duodenum were measured during migration along the crypt-villus axis. In addition, vitamin D-dependent calcium-binding protein and calmodulin content were measured in homogenates of six cell populations which were sequentially derived from villus tip to crypt base. Alkaline phosphatase activity was highest at the tip of the villus (fraction I) and decreased more than 20-fold towards the crypt base (fraction VI). (Na+ + K+)-ATPase activity also decreased along the villus-crypt axis but in a less pronounced manner than alkaline phosphatase. ATP-dependent Ca2+-transport in basolateral membranes was highest in fraction II (8.2 +/- 0.3 nmol Ca2+/min per mg protein) and decreased slightly towards the villus tip and base (fraction V). The youngest cells in the crypt had the lowest Ca2+-transport activity (0.9 +/- 0.1 nmol Ca2+/min per mg protein). The distribution of high-affinity Ca2+-ATPase activity in basolateral membranes correlated with the distribution of ATP-dependent Ca2+-transport. The activity of Na+/Ca2+ exchange was equal in villus and crypt basolateral membranes. Compared to the ATP-dependent Ca2+-transport system, the Na+/Ca2+ exchanger is of minor importance in villus cells but may play a more significant role in crypt cells. Calcium-binding protein decreased from mid-villus towards the villus base and was undetectable in crypt cells. Calmodulin levels were equal along the villus-crypt axis. It is concluded that vitamin D-dependent calcium absorption takes primarily place in villus cells of rat duodenum.     

Immunohistochemical localization of Na+-dependent glucose transporter in the rat digestive tract

Summary Glucose is actively absorbed in the intestine by the action of the Na⁺-dependent glucose transporter. Using an antibody against the rabbit intestinal Na⁺-dependent glucose transporter (SGLT1), we examined the localization of SGLT1 immunohistochemically along the rat digestive tract (oesophagus, stomach, duodenum, jejunum, ileum, colon and rectum). SGLT1 was detected in the small intestine (duodenum, jejunum and ileum), but not in the oesophagus, stomach, colon or rectum. SGLT1 was localized at the brush border of the absorptive epithelium cells in the small intestine. Electron microscopical examination showed that SGLT1 was localized at the apical plasma membrane of the absorptive epithelial cells. SGLT1 was not detected at the basolateral plasma membrane. Along the crypt-villus axis, all the absorptive epithelial cells in the villus were positive for SGLT1, whose amount increased from the bottom of the villus to its tip. On the other hand, cells in the crypts exhibited little or no staining for SGLT1. Goblet cells scattered throughout the intestinal epithelium were negative for SGLT1. These observations show that SGLT1 is specific to the apical plasma membrane of differentiated absorptive epithelial cells in the small intestine, and suggest that active uptake of glucose occurs mainly in the absorptive epithelial cells in the small intestine.     

Ultrastructure of the liver cells in Salamandra salamandra (L.) in the end of Winter

Following hibernation, most salamander liver cells undergo alterations. The liver cells containing the structure of their active period present a normal nucleus, glycogen granules disseminated throughout the entire cytoplasm and granular mitochondria with several peripheral cristae. Most cells, however, possess altered mitochondria, transformed into electron-dense masses, or clear vesicles with the possibility of digesting the ambient organelles. Other cells have clear vacuoles with similar digestive possibilities. The endoplasmic reticulum is poorly represented in the liver cells that have suffered advanced alteration. The nucleus of these cells is homogeneously granular. The sytoplasm is populated by electron-dense bodies and lipid vacuoles. The nucleolemma and plasmalemma are thickened. The pigmented cells, altered concomitantly with the liver cells, contain granular aggregates. Some of the pigmented cells exhibit electron-clear formations similar to the reflecting platelets of the iridocytes of the integument of lower vertebrates.