Histochemical studies of Ca-ATPase, succinate and NAD+-dependent isocitrate dehydrogenases in the shell gland of laying Japanese quails: with special reference to calcium-transporting cells

In order to elucidate the problem of which cells are involved in calcium transport and to estimate the role of mitochondria in calcium transport in the avian shell gland, the fine structure and the Ca-ATPase, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide (NAD+)-dependent isocitrate dehydrogenase (NAD+-ICDH) activity of the shell gland of egg-laying Japanese quails were examined. The surface epithelial cells, consisting of ciliated cells with cilia and microvilli and non-ciliated cells with microvilli, had many large and electron-dense granules. The tubular-gland cells occupied the proprial layer and lacked secretory granules. When an egg was in the shell gland, the well-developed mitochondria of tubular-gland cells characteristically tended to accumulate in the apical cytoplasm, while they were scattered throughout the cytoplasm when an egg was not in the shell gland. Intense Ca-ATPase activity was found on the microvilli of tubular-gland cells, and moderate activity was found on the lateral-cell surface. In the surface epithelial cells, the basolateral cell surface showed moderate enzymatic activity. Both SDH and NAD+-ICDH activity were found in tubular-gland cells when an egg was in the shell gland. These results strongly suggest that calcium for eggshell calcification is actively transported by the tubular-gland (depending on Ca-ATPase activity) and that the mitochondria of gland cells may play an important role in this process as an energy source.     

Histochemical studies of Ca-ATPase, succinate and NAD+-dependent isocitrate dehydrogenases in the shell gland of laying Japanese quails: with special reference to calcium-transporting cells

Summary In order to elucidate the problem of which cells are involved in calcium transport and to estimate the role of mitochondria in calcium transport in the avian shell gland, the fine structure and the Ca-ATPase, succinate dehydrogenase (SDH) and nicotinamide adenine dinucleotide (NAD⁺)-dependent isocitrate dehydrogenase (NAD⁺-ICDH) activity of the shell gland of egg-laying Japanese quails were examined. The surface epithelial cells, consisting of ciliated cells with cilia and microvilli and non-ciliated cells with microvilli, had many large and electron-dense granules. The tubular-gland cells occupied the proprial layer and lacked secretory granules. When an egg was in the shell gland, the well-developed mitochondria of tubular-gland cells characteristically tended to accumulate in the apical cytoplasm, while they were scattered throughout the cytoplasm when an egg was not in the shell gland. Intense Ca-ATPase activity was found on the microvilli of tubular-gland cells, and moderate activity was found on the lateral-cell surface. In the surface epithelial cells, the basolateral cell surface showed moderate enzymatic activity. Both SDH and NAD⁺-ICDH activity were found in tubular-gland cells when an egg was in the shell gland. These results strongly suggest that calcium for eggshell calcification is actively transported by the tubular-gland (depending on Ca-ATPase activity) and that the mitochondria of gland cells may play an important role in this process as an energy source.     

The connective tissue index of <Emphasis Type="Italic">Helix aspersa</Emphasis> as a metal biomarker

The digestive gland of adult land snails, Helix aspersa, sampled from four different sites in São Miguel island (Azores) was submitted to chemical analyses, autometallography and haemalum/eosin staining, in order to quantify the relative abundance of heavy metals, calcium cells and connective tissue cells. Metals were visualized, through light microscopy, as black silver deposits mostly in the connective tissue cells. Metal levels, essentially of Cu and Fe, were related to the relative volumetric density of connective tissue cells but not to the relative volumetric density of calcium cells from the digestive gland epithelium. Thus, the connective tissue index presented herein is suggested as a biomarker of Cu exposure in terrestrial mollusks.     

Immunohistochemical localization of a calcium pump and calbindin-D28k in the oviduct of the laying hen.

The localization of a plasma membrane calcium pump in the oviduct of the laying hen was investigated by immunohistochemical techniques, utilizing a monoclonal antibody (5F10) produced against the human erythrocyte calcium pump. This antibody was shown to react with an epitope of the pump in oviductal tissue, and prominent staining was observed on the microvilli of the tubular gland cells of the hen shell gland (uterus) and the isthmus. The Ca2+ pump was not detectable in the infundibulum or the magnum. Calbindin-D28k, also localized by immunohistochemical means, was observed to be present in the tubular gland cells of the shell gland and the distal isthmus (adjacent to shell gland) but not in either the proximal isthmus (adjacent to the magnum), the magnum or the infundibulum. The localization of the Ca2+ pump in the oviduct corresponds to known sites of mineral deposition during egg shell formation. The distribution of calbindin-D28k differed, co-localizing with the Ca2+ pump in the shell gland and distal isthmus but not in the proximal isthmus. This might reflect a greater rate of active Ca2+ secretion in the distal isthmus and shell gland as compared to the proximal isthmus.     

The intracellular distribution of calcium in the mucosa of the avian shell gland

The intracellular distribution of calcium has been studied in the mucosa of the avian shell gland, a tissue which transports large quantities of calcium during discrete time intervals. Ca⁴⁵ was administered to hens either in a single dose followed by sacrifice 5 min later or in repeated doses over an extended period followed by sacrifice 2 hr or 24 hr after the last injection. Subcellular fractions were isolated by differential centrifugation and analyzed for Ca⁴⁵. The Ca⁴⁵ was located principally in the particulate fractions; the concentration (CPM Ca⁴⁵/mg N) was highest in the mitochondrial fraction. Comparisons of (1) the Ca⁴⁵ distribution in shell gland cells with that of liver cells, (2) the alterations which occur due to the phase of the egg laying cycle, (3) the effects due to the time elapsed since the last injection of Ca⁴⁵, and (4) the Ca⁴⁵ distribution of the short term experiments with that of the long term experiments revealed that the mitochondrial fraction of the shell gland appeared to be active in the movement of calcium. The microsomal fraction showed increased values in CPM Ca⁴⁵/mg N when calcification was occurring, which may indicate that the subcellular components of this fraction have a role in calcium transport. The nuclear and supernatant fractions did not seem to be involved in the transport process. The implications of these results concerning the manner by which calcium may be controlled on a cellular level in this system are discussed.     

Electron microscope studies of Fasciola hepatica. X. Egg formation

Evidence obtained would suggest that the processes involved in egg formation in Fasciola hepatica occur in the following sequence. The egg constituents, namely an ovum and some vitelline cells, pass through the proximal ootype and as they do so they are smeared by the secretions of Mehlis' gland which have accumulated there. A temporary interface is set up between the Mehlis' gland secretion and the fluid which surrounds the egg constituents. Shell globules are released by the vitelline cells and coalesce on the inner aspect of the interface. At the same time some of the Mehlis' secretion diffuses across the interface, thereby bringing about the dissociation of the interface. It is suggested that Mehlis' gland secretion brings about the fusion of the shell layer. During the initial stages of the process the developing egg is supported by the cells of the ootype epithelium. Later the egg passes into a wider, more distal part of the ootype where the process of shell deposition is continued. When complete, or almost complete, the egg passes into the uterus where changes indiciating the initial process of tanning are seen to take place. A thin, very uniform layer of Mehlis' gland secretion can still be identified on the surface of mature eggs.     

Immunohistochemical localization of a calcium pump and calbindin-D28k in the oviduct of the laying hen

Summary The localization of a plasma membrane calcium pump in the oviduct of the laying hen was investigated by immunohistochemical techniques, utilizing a monoclonal antibody (5F10) produced against the human erythrocyte calcium pump. This antibody was shown to react with an epitope of the pump in oviductal tissue, and prominent staining was observed on the microvilli of the tubular gland cells of the hen shell gland (uterus) and the isthmus. The Ca²⁺ pump was not detectable in the infundibulum or the magnum. Calbindin-D_28k, also localized by immunohistochemical means, was observed to be present in the tubular gland cells of the shell gland and the distal isthmus (adjacent to shell gland) but not in either the proximal isthmus (adjacent to the magnum), the magnum or the infundibulum. The localization of the Ca²⁺ pump in the oviduct corresponds to known sites of mineral deposition during egg shell formation. The distribution of calbindin-D_28k differed, co-localizing with the Ca²⁺ pump in the shell gland and distal isthmus but not in the proximal isthmus. This might reflect a greater rate of active Ca²⁺ secretion in the distal isthmus and shell gland as compared to the proximal isthmus.     

Autometallographical localisation of Cu and Zn within target cell compartments of winkles following exposure to Cu&Zn mixtures

This investigation attempts to determine the usefulness of autometallography to localise particular metals in certain key tissues of molluscs exposed to metal mixtures. For this purpose, winkles (Littorina littorea) removed from shell were exposed to very high concentrations of either copper (Cu), zinc (Zn) or a mixture of both metals (Cu&Zn) dissolved in sea-water for short periods of time. Protein-bound metals were detected by autometallography as black silver deposits (BSD) on histological sections of gills, foot, mantle, digestive gland/gonad complex, stomach and kidney. Copper was localised within cytoplasmic granules of gill ciliated cells, nephrocytes and stomach epithelial cells as well as within digestive cell lysosomes. Zinc was essentially found in the basal lamina (histological sense) of gill, stomach, kidney and digestive gland epithelia. BSD were also evidenced in cytoplasmic granules of pore cells present in parenchymal connective tissue of mantle, foot, gill, digestive gland and stomach. Copper and zinc concentrations were additionally calculated for the whole soft body as well as for certain organs by atomic absorption spectrophotometry (AAS). According to AAS, a synergistic phenomenon would contribute to increase the rate of Cu and Zn accumulation in presence of each other. However, after exposure to Cu&Zn autometallography did not evidence any synergistic phenomenon, and Cu and Zn were localised in their respective accumulation sites. In conclusion, autometallography might indicate the presence of certain metals in the environment irrespective of factors, such as "metal-metal interaction-like" phenomena, affecting metal concentrations in soft tissues.     

Histopathology of the digestive gland of the bivalve mollusk Crenomytilus grayanus (Dunker, 1853) from southwestern Peter the Great Bay, Sea of Japan

The histomorphology of the digestive gland of the bivalve mollusk Crenomytilus grayanus from Sivuchya Bay, which is located in the southwest of Peter the Great Bay and subjected to the effect of polluted waters of Tumannaya River, was studied. Pathological changes of the digestive tubules, channels, and connective tissue of the gland were recorded in all the mussels studied. The epithelium of the tubules and channels was characteristic with erosive disturbances and by heavy vacuolization of digestive cells; connective tissue of the gland was specified by cells with lipofuscin (granulocytomes) and by foci of cells necrosis and lysis. Nervous fibers running in the gland were swollen in some mollusks. Strongly basophilic spherical formations, presumably one of the development stages of a parasitic plasmodium, were found in the granulocytomes and among vesicular cells of connective tissue of all the mussels. It was concluded that pathological changes in digestive gland of Gray’s mussel might be caused by chronic pollution of the bay and by parasitic invasion.     

Seasonal variation of xanthine oxidoreductase activity in the digestive gland cells of the mussel Mytilus galloprovincialis: a biochemical, histochemical and immunochemical study

The activity and the tissue distribution of the oxygen radical producing enzyme xanthine oxidoreductase (XOR) were measured in the digestive gland of the common marine mussel Mytilus galloprovincialis Lmk along an annual cycle. No xanthine oxidase (XOX) activity could be measured, the enzyme only displaying xanthine dehydrogenase (XDH) activity in all the cases. This is interpreted as a mechanism to avoid the harmful effects of the oxygen radicals that would be produced by XOX during periods following anoxic conditions at low tide. The highest XDH activities coincided with the late spring/early summer months, the activity maxima being recorded from May to July. Histochemically XOR activity was very pronounced in duct and stomach epithelial cells as well as in the surrounding connective tissue and hemolymph vessels, the activity increasing towards the summer months. These seasonal variations in XDH or XOR activities are possibly linked to hormonal changes governing the reproductive cycle and to changes in food availability. The localization of the protein in the connective tissue lining the hemolymph vessels was confirmed immunohistochemically using a polyclonal antibody against rat liver protein that cross-reacted specifically with a polypeptide of 150 kDa of molecular mass in homogenates of the digestive gland. This polypeptide was linked to cytosolic fractions isolated by differential centrifugation from mussel digestive glands. In paraffin sections the antibody labeled the digestive cells of digestive tubules, as well as the connective tissue surrounding the hemolymph vessels, gonadal follicles, digestive epithelia and certain protozoan parasites. Taken together our results suggest that in the digestive gland of bivalve molluscs XOR is involved in the metabolism of purines and in the scavenging of oxygen free radicals.     

Zinc Secretion in the Oviduct of the Coturnix Quail

The oviduct from laying quail were used to investigate mechanisms of trace mineral secretion and the possible role of metallothionein in this process. Secretion of zinc occurred maximally at pH 5.4, which is close to the normal pH of the oviduct. Secretion occurred to a much greater extent in the isthmus and shell gland than in the magnum, the major protein-secretory section of the oviduct. Intraperitoneal administration of cadmium resulted in a marked reduction in Zn secretion from the oviduct of laying quail. This effect could not be correlated with metallothionein since metallothionein could not be detected in any section of the oviduct in control or Cd-induced quail. Small-molecular-weight metal-binding ligands were present in the isthmus and shell gland, which may play a role in trace mineral mobilization. Histological evaluation by light and elelctron microscopy show that Zn is transported from the smooth muscle cells through the connective tissue matrix in the extracellular space to the epithelial goblet cells. Presumably, Zn and other trace minerals are secreted from the secretory goblet cells into the egg.     

The digestive gland of Viviparus ater (Mollusca, Gastropoda, Prosobranchia): an ultrastructural and histochemical study

The digestive gland of Viviparus ater was studied using histochemical and ultrastructural methods. Only one cell type was observed in the tubule epithelium of the gland. The cells are involved in an endocytotic process mediated by clathrin-coated vesicles and in the intracellular digestion of food materials (thus they can be regarded as digestive cells). The different stages of digestion and exocytotic extrusion of residual bodies into the tubule lumen were shown by electron microscopy. Very few, small mucocytes are scattered among the digestive cells. Calcium concretions, glycogen-containing cells and endocrine cells are scattered in the area of connective tissue present among the digestive tubules.     

The fine structure of the secretory cells of the uterus (shell gland) of the chicken

The secretory processes in the shell gland of laying chickens were the subject of this study. Three cell types contribute secretory material to the forming egg: ciliated and non-ciliated columnar cells of the uterine surface epithelium, and cells of tubular glands in the mucosa. The ciliated cells as well as the non-ciliated cells have microvilli, which undergo changes in form and extent during the secretory cycle. At the final stages of shell formation they resemble stereocilia. It is postulated that the microvilli of both cells are active in the production of the cuticle of the shell. The ciliated cell which has both cilia and microvilli manufactures secretory granules which arise from the Golgi complex in varying amounts throughout the egg laying cycle. Granule production reaches its greatest intensity during the early stages of shell deposition. The ciliated cell probably supplies proteinaceous material to the matrix of the forming egg shell. The non-ciliated cell has only microvilli. Secretory granules, containing an acid mucopolysaccharide, arise from the Golgi complex. Some granules are extruded into the uterine lumen where they supply the egg shell with organic matrix. Others migrate towards the supranuclear zone. Here a number of them disintegrate. This is accompanied by the formation of a large membraneless space, which is termed “vacuoloid.” Subsequently the vacuoloid regresses and during regression an extensive rough endoplasmic reticulum with numerous polyribosomes of spiral configuration appears. It is suggested that material in the vacuoloid originating from the disintegrating granules is resynthesized and utilized for the formation of secretory product. The uterine tubular gland cells have irregular, frondlike microvilli. During egg shell deposition, these microvilli form large blebs and are probably related to the elaboration of a watery, calcium-containing fluid.     

Localization of osteopontin in oviduct tissue and eggshell during different stages of the avian egg laying cycle

The avian eggshell is an acellular bioceramic containing organic and inorganic phases that are sequentially assembled during the time the egg moves along the oviduct. As it has been demonstrated in other mineralized tissues, mineralization of the eggshell is regulated by extracellular matrix proteins especially the anionic side chains of proteoglycans. Among them, osteopontin has been found in the avian eggshell and oviduct. However, its precise localization in the eggshell or in different oviduct regions during eggshell formation, nor its function have been established. By using anti-osteopontin antibody (OPN 1), we studied its immunolocalization in the isthmus, red isthmus and shell gland of the oviduct, and in the eggshell during formation. In the eggshell, osteopontin was localized in the core of the non-mineralized shell membrane fibers, in the base of the mammillae and in the outermost part of the palisade. In the oviduct, OPN 1 was localized in the ciliated epithelial but not in the tubular gland cells of the isthmus, in the ciliated epithelial cells of the red isthmus, and in the non-ciliated epithelial cells of the shell gland. The occurrence of osteopontin in each of the oviduct regions, coincided with the concomitant presence of the egg in such region. Considering the reported inhibitory function of osteopontin in other mineralized systems, together with its main occurrence in the non-mineralized parts of the eggshell and at the outermost part of the shell, suggests that this molecule could be part of the mechanism regulating the eggshell calcification.     

Effects of methyl mercury at different dose regimes on eggshell formation and some biochemical characteristics of the eggshell gland mucosa of the domestic fowl

Eggshell formation and egg production in domestic fowl were studied following the administration of methyl mercury (two dose regimes: 5 mg daily for 6 consecutive days and 1 mg daily for 50 consecutive days). A daily oral dose of 5 mg of methyl mercury for 6 consecutive days induced significant eggshell thinning and deformation and inhibited egg production. Uptake of ⁴⁵Ca and synthesis of prostaglandins by a homogenate of eggshell gland mucosa from methyl-mercury-treated birds were significantly reduced, as was the calcium content of blood plasma. A daily oral dose of 1 mg of methyl mercury administered for 50 consecutive days also induced eggshell deformation and thinning and reduced egg production. This dose did not, however, have significant effects on the following: ⁴⁵Ca uptake and prostaglandin synthesis by a homogenate of the eggshell gland mucosa; ⁴⁵Ca uptake by a homogenate of duodenal mucosa; the Ca content of the blood plasma, shell gland mucosa or shell gland lumen; the HCO₃⁻ content of the shell gland lumen or the specific gravity of tibia. Methyl mercury added in vitro to a homogenate of eggshell gland mucosa significantly stimulated the synthesis of prostaglandins PGF_2α and PGE₂. Addition of mercury chloride to the same type of preparation stimulated the synthesis of PGF_2α at the expense of thromboxane (T × B₂) synthesis. Administration of 5 mg methyl mercury for 6 consecutive days seemed to reduce the availability of calcium for eggshell formation. This effect could have been due to a direct inhibitory effect of methyl mercury on calcium uptake from the gastrointestinal tract and/or to mobilization of medullary bone. The administration of 1 mg methyl mercury for 50 consecutive days probably induced the reproductive effects by another mechanism. The effects of methyl mercury on avian eggshell formation are quite different from the effects p,p′-DDE exerts on that process.     

Calcium homeostasis and vitamin D metabolism and expression in strongly calcifying laying birds

Egg laying and shell calcification impose severe extra demands on ionic calcium (Ca2+) homeostasis; especially in birds characterized by their long clutches (series of eggs laid sequentially before a "pause day"). These demands induce vitamin D metabolism and expression. The metabolism of vitamin D is also altered indirectly, by other processes associated with increased demands for calcium, such as growth, bone formation and egg production. A series of intestinal, renal or bone proteins are consequently expressed in the target organs via mechanisms involving a vitamin D receptor. Some of these proteins (carbonic anhydrase, calbindin and calcium-ATPase) are also found in the uterus (eggshell gland) or are believed to be involved in calcium transport in the intestine or kidney (calcium channels). The present review deals with vitamin D metabolism and the expression of the above-mentioned proteins in birds, with special attention to the strongly calcifying laying bird.     

The distribution of some hydrolytic enzymes in the cells of the digestive gland of certain lamellibranchs and gastropods

Five hydrolytic enzymes (acid phosphatase, aryl sulphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, and non-specific esterase) have been studied histochemically in the cells of the digestive gland of Mytilus edulis, Helix aspersa , and certain other lamellibranchs and gastropods. All the enzymes studied have basically similar distributions.
In the digestive cells, the enzymes occur in cytoplasmic granules which are believed to be primary lysosomes; in vacuoles which contain phagocytosed food material; and in vacuoles containing lipofuscin granules, which are the residues of digestive activity.
In the basiphil cells of M. edulis , most of the enzymes are localized in a few cytoplasmic granules; non-specific esterase, however, is found throughout the cytoplasm. In the calcium cells of H. aspersa and the other pulmonate gastropods studied, the enzymes are either in cytoplasmic granules, or distributed diffusely throughout the cytoplasm. Acid phosphatase is also found in the calcium spherules, especially in H. aspersa.
In the excretory cells of H. aspersa and the other pulmonates studied, the enzymes are found in granules in the cytoplasm, and in the lipofuscin granules which lie in the vacuoles of these cells.     

Studies on the effects of dietary beryllium at two different calcium concentrations in Achatina fulica (Pulmonata)

The mortality was highest in snails fed beryllium in the diet containing the sub-optimal concentration of calcium. There was no increase in weight over an 8 week period. Snails fed the diet containing the optimal concentration of calcium without added beryllium showed maximum weight increase but calcium alone may not be responsible for elevated growth rate. Dietary calcium enhancement appears to be responsible for the reduced concentration of zinc, magnesium, phosphate and beryllium in the tissues. Beryllium treatment did not affect the calcium concentration in the digestive gland tissue but increased zinc and magnesium in the shell. The results are discussed in relation to uptake studies and possible enzyme systems involved.     

Histochemical studies on eggshell formation in Paramphistomum cervi (Digenea: Paramphistomatidae)

The formation of the egg mass in the ootype, containing the oocyte, vitelline cells, numerous spermatozoa and Mehlis' gland secretion marks the beginning of eggshell formation. Mehlis' gland cells pour their lipoprotein secretion into the ootype; this secretion forms a thin template around the egg mass and upon this template the shell globules are deposited. Eggshell formation begins in the proximal uterus and is completed in the middle uterus. The eggshells are devoid of phenols and phenolase indicating that hardening is not by quinone tanning. The eggshells contain keratin and are stabilized by disulphide linkages.