FUNCTIONS OF COATED VESICLES DURING PROTEIN ABSORPTION IN THE RAT VAS DEFERENS

The role of coated vesicles during the absorption of horseradish peroxidase was investigated in the epithelium of the rat vas deferens by electron microscopy and cytochemistry. Peroxidase was introduced into the vas lumen in vivo. Tissue was excised at selected intervals, fixed in formaldehyde-glutaraldehyde, sectioned without freezing, incubated in Karnovsky's medium, postfixed in OsO₄, and processed for electron microscopy. Some controls and peroxidase-perfused specimens were incubated with TPP,¹ GP, and CMP. Attention was focused on the Golgi complex, apical multivesicular bodies, and two populations of coated vesicles; large (> 1000 A) ones concentrated in the apical cytoplasm and small (<750 A) ones found primarily in the Golgi region. 10 min after peroxidase injection, the tracer is found adhering to the surface plasmalemma, concentrated in bristle-coated invaginations, and within large coated vesicles. After 20–45 min, it is present in large smooth vesicles, apical multivesicular bodies, and dense bodies. Peroxidase is not seen in small coated vesicles at any interval. Counts of small coated vesicles reveal that during peroxidase absorption they first increase in number in the Golgi region and later, in the apical cytoplasm. In both control and peroxidase-perfused specimens incubated with TPP, reaction product is seen in several Golgi cisternae and in small coated vesicles in the Golgi region. With GP, reaction product is seen in one to two Golgi cisternae, multivesicular bodies, dense bodies, and small coated vesicles present in the Golgi region or near multivesicular bodies. The results demonstrate that (a) this epithelium functions in the absorption of protein from the duct lumen, (b) large coated vesicles serve as heterophagosomes to transport absorbed protein to lysosomes, and (c) some small coated vesicles serve as primary lysosomes to transport hydrolytic enzymes from the Golgi complex to multivesicular bodies.     

TEM immunocytochemistry of a 48 kDa MW organic matrix phosphoprotein produced in the mantle epithelial cells of the Eastern oyster (Crassostrea virginica)

Immunohistochemical TEM of Eastern oyster (Crassostrea virginica) mantle epithelial cells using a polyclonal antibody to a gel purified 48 kDa MW oyster shell phosphoprotein revealed that it is phosphorylated in the Golgi, packaged into secretory vesicles and subsequently exocytosed across the apical membrane of specialized cells. These phosphoprotein producing cells are concentrated along the mantle side facing the shell, in the region of the outer mantle lobe. A layer of calcium enriched immuno-reactive mucous is associated with the apical microvilli of these cells. The 48 kDa phosphoprotein forms a component of the fibrous organic matrix and appears to be involved in calcium supply thus enabling crystal growth at the mineralization front.     

Morphology of the bovine epididymis

The epididymis of the bull was divided into six regions, and morphological differences between regions were studied. The epithelium of all regions contained four cell types: principal and basal epithelial cells, and intraepithelial lymphocytes and macrophages. The epithelium of regions II-V also contained a few apical cells. Principal cells of all regions possessed an endocytotic apparatus including stereocilia underlain by canaliculi, coated vesicles, and subapical vacuoles (up to 1 micron in diameter); however, large vacuoles with a flocculent content and multivesicular bodies (up to 5 microns in diameter) were most numerous in regions II, III, and IV. The unique features of principal cells of region I were the presence of well-developed Golgi bodies, few lipid droplets, and whorls of smooth endoplasmic reticulum in the supranuclear cytoplasm. Numerous mitochondria, distended cisternae of rough endoplasmic reticulum, and dense granules characterized the infranuclear cytoplasm of the principal cells of regions II-VI; however, these features were more developed in region V. Apical cells were characterized by the apical location of the nucleus, many mitochondria in the apical cytoplasm, and few microvilli at the luminal border. Basal cells with few cytoplasmic lipid droplets were present throughout the length of the epididymis but appeared more numerous in region V. Intraepithelial lymphocytes were present at all levels of the epithelium but were never seen in the lumen. Intraepithelial macrophages containing heterogeneous granules, eccentric nuclei, and pseudopods were invariably seen near the basal area of the epithelium in all regions. These observations are discussed in an effort to define the role of each cell type in the epididymal epithelium.     

Functional morphology of the mantle of Nautilus pompilius (Mollusca, Cephalopoda)

This study presents histological and cytological findings on the structural differentiation of the mantle of Nautilus pompilius in order to characterize the cells that are responsible for shell formation. The lateral and front mantle edges split distally into three folds: an outer, middle, and inner fold. Within the upper part of the mantle the mantle edge is divided into two folds only; the inner fold disappears where the hood is attached to the mantle. At the base of the outer fold of the lateral and front mantle edge an endo-epithelial gland, the mantle edge gland, is localized. The gland cells are distinguished by a distinct rough endoplasmic reticulum and by numerous secretory vesicles. Furthermore, they show a strong accumulation of calcium compounds, indicating that the formation of the shell takes place in this region of the mantle. Numerous synaptic contacts between the gland cells and the axons of the nerve fibers reveal that the secretion in the area of the mantle edge gland is under nervous control. The whole mantle tissue is covered with a columnar epithelium having a microvillar border. The analyses of the outer epithelium show ultrastructural characteristics of a transport active epithelium, indicating that this region of the mantle is involved in the sclerotization of the shell. Ultrastructural findings concerning the epithelium between the outer and middle fold suggest that the periostracum is formed in this area of the mantle, as it is in other conchiferan molluscs.     

The outer mantle epithelium of Haliotis tuberculata (Gastropoda Haliotidae): an ultrastructural and histochemical study using lectins

The mantle of molluscs has been the subject of many studies as it is the organ that forms the shell. Microscopic studies in particular focus on the outer mantle epithelium, but few studies address this epithelium in a histochemical way. In this study, the outer mantle epithelium in adult specimens of Haliotis tuberculata is studied, that is, in specimens involved in maintaining and repairing the shell rather than in generating it. The epithelial cells are studied by scanning (SEM) and transmission electron microscopy (TEM), and by histochemical techniques, including the use of lectins for their biochemical characterization. The epithelium is composed of pigmented epidermal cells with small microvilli and junctional complexes. It furthermore contains a few ciliated cells, as well as two types of secretory cells which differ in the ultrastructural appearance of their secretory granules and their glycoconjugate content. Histochemical study shows secretory cells containing sulphated glycoconjugates such as glycosaminoglycans or mucins rich in N‐acetylgalactosamine and N‐glycoproteins rich in fucose. Furthermore, the apical regions of the epidermal cells are positive for lectins that label fucose, mannose and N‐acetylglucosamine. The role of epithelial cells in the synthesis of structural components of the shell is discussed.     

Amorphous Calcium Carbonate Precipitation by Cellular Biomineralization in Mantle Cell Cultures of Pinctada fucata

The growth of molluscan shell crystals is generally thought to be initiated from the extrapallial fluid by matrix proteins, however, the cellular mechanisms of shell formation pathway remain unknown. Here, we first report amorphous calcium carbonate (ACC) precipitation by cellular biomineralization in primary mantle cell cultures of Pinctada fucata. Through real-time PCR and western blot analyses, we demonstrate that mantle cells retain the ability to synthesize and secrete ACCBP, Pif80 and nacrein in vitro. In addition, the cells also maintained high levels of alkaline phosphatase and carbonic anhydrase activity, enzymes responsible for shell formation. On the basis of polarized light microscopy and scanning electron microscopy, we observed intracellular crystals production by mantle cells in vitro. Fourier transform infrared spectroscopy and X-ray diffraction analyses revealed the crystals to be ACC, and de novo biomineralization was confirmed by following the incorporation of Sr into calcium carbonate. Our results demonstrate the ability of mantle cells to perform fundamental biomineralization processes via amorphous calcium carbonate, and these cells may be directly involved in pearl oyster shell formation.     

Fine structure of the rectal pads in Grylloblatta compodeiformis (walker) (Orthoptera : Grylloblattidae) with reference to fluid transport

The rectal pads of the primitive insect Grylloblatta compodeiformis (Orthoptera : Grylloblattidae) were studied using light and electron microscopy. In this species, the rectal epithelium is thickened to form 6 prominent rectal pads, each of which is composed of tall columnar epithelial cells and laterally placed slender junctional cells, but is devoid of secondary cells. The rectal pads are interconnected by simple rectal epithelium, and are lined by a thin cuticular intima. They are surrounded by an extensive connective tissue space, which contains bundles of delicate connective tissue fibers, neurosecretory axons, and tracheae and tracheoles, which do not penetrate into the pads. The epithelial cells exhibit extensive infoldings of the apical plasma membranes that are closely associated with mitochondria. The lateral membranes are also highly folded around large mitochondria that possess longitudinally oriented cristae. These membrane folds form mitochondrial-scalariform junctional complexes and enclose intercellular channels and spaces. The apical cytoplasm of the epithelial cells contains numerous coated vesicles, dense tubular elements, multivesicular bodies and lysosomes, which suggests receptor-mediated endocytosis of macromolecules. The presence of large whorls of rough endoplasmic reticulum and abundant free ribosomes in the cytoplasm and nuclei with multiple, well-developed nucleoli indicate that the epithelial cells are actively engaged in protein synthesis. The ultrastructural features were examined in relation to their role in fluid transport in a cold habitat.     

Ultrastructural and cytochemical studies in the mantle of Anodonta cygnea

The large central region of the mantle of Anodonta cygnea was studied using transmission and scanning electron microscopy (TEM and SEM) for ultrastructural analysis and light microscopy (LM) and TEM for cytochemical analysis. X-ray diffraction studies of the organic matrix pellicle deposited on the inner surface of the shell were also carried out. Two groups of columnar cells presenting desmosomes on the apical region were observed in the outer epithelium. One group secretes a structural and neutral mucopolysaccharide (MPS) identified with chitin, normally excreted to form the organic matrix of the shell. Another group presents numerous cytoplasmic vesicles and constitutes the predominant cells of this epithelium. Two different types of mucous cells were found in the inner epithelium. One type secretes a faintly acid mucopolysaccharide (MPS) with sulphate groups and another type secretes an association of this polysaccharide with a neutral MPS. Staining for sulfhydryl groups (probably cysteine) was also positive, suggesting the presence of proteoglycans in these secretions. A third type of cells was also observed presenting a very different ultrastructural aspect (columnar form) without large secretion masses. They may correspond to the replacing cells in this highly secretory epithelium. Elastic fibers were found on the base of the outer epithelium and amoebocytes were observed in the interepithelial tissue.     

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.     

Fine structural demonstration of acid phosphatase in rabbit germinal epithelium prior to induced ovulation

The germinal or surface epithelium covering rabbit Graafian follicles contains occasional small, dark, lysosome-like bodies. After an ovulatory dose of human chorionic gonadotropin (HCG) such bodies gradually increase in size and number. At 8 hr after HCG there is a maximal accumulation in the apical follicle cells; then the dense bodies decrease and just prior to ovulation, 9.5 hr after HCG, only few of them remain in the attenuated surface epithelium. Most of the growing membrane-surrounded bodies probably represent lysosomes, since electron microscopy combined with cytochemistry revealed that many of them contain the lysosomal "marker" enzyme, acid phosphatase. The role of sex steroids and prostaglandins regarding lysosomal growth and LABILization is discussed. The close temporal relation between disappearance of the apical surface epithelial lysosomes and disintegration of the underlying tunica albuginea gives further support to our working hypothesis that at least part of the "ovulatory enzymes" emanate from the surface epithelium.     

Effects of serotonin (5HT) and complement C3 on the synthesis of the surface membrane precursors of adult Schistosoma mansoni

The syncytial surface epithelium of Schistosoma mansoni plays an important role in immune evasion. This syncytium is covered by an unusual double-membrane complex consisting of an apical plasma membrane (APM) and an overlying envelope (En) that have been shown to have different rates of synthesis and turnover. It has been suggested that discoid bodies (DBs) and multilamellar bodies (MLBs), the major syncytial inclusion bodies of schistosomes, may be the precursors of the APM and En, respectively. In this ultrastructural study, we examined the effects of serotonin (5HT) and complement C3, which have been shown to stimulate synthesis and turnover of the APM and En, respectively, on the synthesis of DBs and MLBs in vitro. With short-time incubations (20 or 40 min), 5HT stimulated the synthesis of the DBs by 2-fold, whereas C3 accelerated synthesis of the MLBs by 2-fold. Furthermore, when microtubules within the cytoplasmic connections between the syncytium and the underlying cell bodies (the site of membrane synthesis) were disrupted with colchicine, the DBs and MLBs synthesized in response to 5HT or C3 accumulated in the cell bodies. This suggests that the transport of the organelles to the syncytium is dependent upon the microtubules but not the signaling mechanism in response to 5HT or C3. These observations also support the suggestion that the DBs and MLBs are synthesized in subsyncytial cell bodies and serve as precursors of the APM and En, respectively. The rapid synthetic response to 5HT and C3 is also consistent with rapid synthesis and turnover of the APM/En, as suggested by previous studies.     

Cloning and expression of a pivotal calcium metabolism regulator: calmodulin involved in shell formation from pearl oyster (Pinctada fucata)

The shells of bivalves are mainly composed of calcium carbonate, a product of calcium metabolism. In the process of shell formation, the uptake, transport and recruitment of calcium ion are highly regulated and involved in many factors. Among these regulatory factors, calmodulin (CaM), a pivotal multifunction regulator of calcium metabolism in nearly all organisms, is thought to play an important role in the calcium metabolism involved in shell formation. In this study, a full-length CaM cDNA was isolated from the pearl oyster (Pinctada fucata). The oyster calmodulin encodes a 16.8 kDa protein which shares high similarity with vertebrate calmodulin. The oyster CaM mRNA shows the highest level of expression in the gill, a key organ involved in calcium uptake in oyster calcium metabolism. In situ hybridization results revealed that oyster CaM mRNA is expressed at the folds and the outer epithelial cells of the dorsal region of the mantle, suggesting that CaM is involved in regulation of calcium transport and secretion. Oyster CaM also showed a typical Ca2+ dependent electrophoretic shift characterization and calcium binding activity. Taken together, we have identified and characterized a pivotal calcium metabolism regulator of the oyster that may play an important role in regulation of calcium uptake, transport and secretion in the process of shell formation.     

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.     

Endocytosis in the uterine luminal and glandular epithelial cells of mice during early pregnancy

We have localized horseradish peroxidase (HRP) in the mouse uterus after intravenous administration on days 1 and 5 of pregnancy in an effort to understand how serum proteins reach the uterine lumen. Direct movement of HRP into uterine and glandular lumina was blocked by the epithelial tight junctions on both days. In luminal and glandular epithelial cells at both times, HRP was localized in endocytic vesicles along the basolateral membranes, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. The uptake of HRP was most extensive in the luminal epithelium on day 1: the number of tracer-containing apical vesicles and bdb was largest, and there were also clusters of vesicles containing the tracer above the nucleus. Acid phosphatase was localized on day 1 in mvb and bdb in both cell types, indicating that these structures are lysosomes. It appeared that HRP followed two pathways after basolateral endocytosis by the epithelial cells: it was transported to the apical region of the cells, where it was present in small vesicles that may release their contents into the uterine or glandular lumina, or it was transported to lysosomes. To investigate whether macromolecules may be transported from the uterine lumen to the stroma, we also studied endocytosis at the apical pole of luminal epithelial cells after intraluminal injection of HRP. There was no detectable uptake of HRP from the lumen on day 1, and no tracer was detected in the intercellular spaces or basement membrane region. On day 5, a large amount of HRP was taken up from the lumen into apical endocytic vesicles, mvb, and dense bodies, but tracer was not present in the Golgi apparatus, lateral intercellular spaces, or the basement membrane region at the times studied. These observations indicate that there was no transport of luminal macromolecules to the uterine stroma on day 1, while the possibility of transport on day 5 requires further study.     

Calcium localization in the shell-forming tissue of the freshwater snail,Biomphalaria glabrata: a comparative study of various methods for localizing calcium

Summary The routes calcium might take across the mantle to the shell have been investigated with various electron-microscopical techniques in the freshwater snailBiomphalaria glabrata (Planorbidae, Basommatophora).In chemically-fixed tissue, calcium was precipitated with a tannic acid-antimonate technique in predominantly the intercellular spaces of the outer mantle epithelium and the interstitium below it. Some vacuoles of the outer mantle epithelium and one type of mucus cell in the inner mantle epithelium also contained precipitate. The presence of calcium in the precipitates was proved by electron energy loss spectroscopy combined with electron spectroscopic imaging. Incubation with lead acetate and uranyl acetate revealed binding-sites for calcium in the intercellular spaces of the epithelia interstitium and the mucus cells of the inner mantle epithelium. Precipitates were also seen after all incubations in the calcium spherites of the connective tissue.The concentrations of calcium and other elements were analysed in freeze-dried ultrathin sections of cryofixed mantle tissue by means of energy-dispersive X-ray microanalysis. Only in mitochondria of the musculature could high amounts of calcium and phosphorous be detected.     

Zona Localization of Shell Matrix Proteins in Mantle of <Emphasis Type="Italic">Haliotis tuberculata</Emphasis> (Mollusca,Gastropoda)

Organic matrix from molluscan shells has the potential to regulate calcium carbonate deposition and crystallization. Control of crystal growth thus seems to depend on control of matrix protein secretion or activation processes in the mantle cells, about which little is known. Biomineralization is a highly orchestrated biological process. The aim of this work was to provide information about the source of shell matrix macromolecule production, within the external epithelium of the mantle. An in vivo approach was chosen to describe the histologic changes in the outer epithelium and in blood sinus distribution, associated with mantle cells implicated in shell matrix production. Our results characterized a topographic and time-dependent zonation of matrix proteins involved in shell biomineralization in the mantle of Haliotis.     

Rapid in vivo transport of proteins from digested allergen across pre-sensitized gut

Although the route of sensitization to food allergens is still the subject of debate, it is generally accepted the gut immune system plays a pivotal role. However, hitherto the transport of allergens across the normal, pre-sensitized gut epithelium remained largely unknown. Our aim was to identify the route through which protein bodies and soluble proteins from digested peanuts penetrated the pre-sensitized gut epithelium in vivo and the specific cell types involved in the transport. Digestion of peanuts released a large number of protein bodies that are exclusively transported across the epithelium by specialized antigen-sampling M cells and delivered to the lymphoid tissue of Peyer's patch. Intracellular transport of soluble protein also occurred almost exclusively via M cells and it was negligible across absorptive enterocytes. We hypothesize that these conditions which are known to favour strongly the induction of immune responses rather than oral tolerance may play a significant role in the genesis of allergic reactions.     

Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants.

Vertebrate mechanosensory hair cells contain a narrow "pericuticular" zone which is densely populated with small vesicles between the cuticular plate and cellular junctions near the apical surface. The presence of many cytoplasmic vesicles suggests that the apical surface of hair cells has a high turnover rate. The significance of intense membrane trafficking at the apical surface is not known. Using a marker of endocytosis, the styryl dye FM1-43, this report shows that rapid apical endocytosis in zebrafish lateral line sensory hair cells is calcium and calmodulin dependent and is partially blocked by the presence of amiloride and dihydrostreptomycin, known inhibitors of mechanotransduction channels. As seen in lateral line hair cells, sensory hair cells within the larval otic capsule also exhibit rapid apical endocytosis. Defects in internalization of the dye in both lateral line and inner ear hair cells were found in five zebrafish auditory/vestibular mutants: sputnik, mariner, orbiter, mercury, and skylab. In addition, lateral line hair cells in these mutants were not sensitive to prolonged exposure to streptomycin, which is toxic to hair cells. The presence of endocytic defects in the majority of zebrafish mechanosensory mutants points to a important role of apical endocytosis in hair cell function.     

ACTIVE TRANSPORT BY THE CECROPIA MIDGUT : II. Fine Structure of the Midgut Epithelium

A morphological basis for transcellular potassium transport in the midgut of the mature fifth instar larvae of Hyalophora cecropia has been established through studies with the light and electron microscopes. The single-layered epithelium consists of two distinct cell types, the columnar cell and the goblet cell. No regenerative cells are present. Both columnar and goblet cells rest on a well developed basement lamina. The basal portion of the columnar cell is incompletely divided into compartments by deep infoldings of the plasma membrane, whereas the apical end consists of numerous cytoplasmic projections, each of which is covered with a fine fuzzy or filamentous material. The cytoplasm of this cell contains large amounts of rough endoplasmic reticulum, microtubules, and mitochondria. In the basal region of the cell the mitochondria are oriented parallel to the long axes of the folded plasma-lemma, but in the intermediate and apical portions they are randomly scattered within the cytoplasmic matrix. Compared to the columnar cell, the goblet cell has relatively little endoplasmic reticulum. On the other hand, the plications of the plasma membrane of the goblet cell greatly exceed those of the columnar cell. One can distinguish at least four characteristic types of folding: (a) basal podocytelike extensions, (b) lateral evaginations, (c) apical microvilli, and (d) specialized cytoplasmic projections which line the goblet chamber. Apically, the projections are large and branch to form villus-like units, whereas in the major portion of the cavity each projection appears to contain an elongate mitochondrion. Junctional complexes of similar kind and position appear between neighboring columnar cells and between adjacent columnar and goblet cells as follows: a zonula adherens is found near the luminal surface and is followed by one or more zonulae occludentes. The morphological data obtained in this study and the physiological information on ion transport through the midgut epithelium have encouraged us to suggest that the goblet cell may be the principal unit of active potassium transport from the hemolymph to the lumen of the midgut. We have postulated that ion accumulation by mitochondria in close association with plicated plasma membranes may play a role in the active movement of potassium across the midgut.     

Morphological studies on the calcification process in the fresh-water mussel Amblema

Light microscopy, transmission electron microscopy, scanning electron microscopy, various histochemical procedures for the localization of mineral ions, and analytical electron microscopy have been used to investigate the mechanisms inherent at the mantle edge for shell formation and growth in Amblema plicata perplicata, Conrad. The multilayered periostracum, its component laminae formed from the epithelia lining either the periostracal groove or the outer mantle epithelium (of the periostracal cul de sac), appears to play the major regulatory and organizational role in the formation of the component mineralized layers of the shell. Thus, the inner layer of the periostracum traps and binds calcium and subsequently gives rise to matricial proteinaceous fibrils or lamellar extensions which serve as nucleation templates for the formation and orientation of the crystalline subunits (rhombs) in the forming nacreous layer. Simultaneously, the middle periostracal layer furnishes or provides the total ionic calcium pool and the matricial organization necessary for the production of the spherical subunits which pack the matricial ‘bags’ of the developing prismatic layer. The outer periostracal layer appears to be a supportive structure, possibly responsible for the mechanical deformations which occur in the other laminae of the periostracum. The functional differences in the various layers of the periostracum are related to peculiar morphological variables (foliations, vacuolizations, columns) inherent in the structure and course of this heterogeneous (morphologically and biochemically) unit. From this study, using the dynamic mantle edge as a morphological model system, we have been able to identify at least six interrelated events which culminate in the production of the mature mineralized shell layers (nacre, prisms) at the growing edge of this fresh-water mussel.