Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study.

The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 microns in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine structure of nerve cells in a planarian

The fine structure of the nerve cell types in the white planarian Procotyla fluviatilis were described. Ganglion cells comprise the major portion of the brain. These cells are irregular in shape with several cytoplasmic processes and contain ribosomes, a sparse endoplasmic reticulum, microtubules, lysosomes, and a Golgi apparatus with numerous small vesicles. Granule-containing cells are situated in the peripheral regions of the brain and along the nerve cords. These cells contain ribosomes, rough-surfaced endoplasmic reticulum and a Golgi apparatus with associated dense granules. The granules occupy most of the cytoplasm and are ～ 750A in diameter with moderately dense contents, ～ 750A with opaque contents, and ～ 1000A with contents of medium density. These granules are similar to those in the nervous systems of higher animals that contain epinephrine, norepinephrine, and neurosecretory substance, respectively. Each cell contains predominantly one type of granule although there is some intermixing of granules and intermediate types between the three most abundant granules. Small clear vesicles, resembling cholinergic synaptic vesicles, and all types of dense granules occur in the neuropil and within nerve endings.     

Cytochemical reaction for cationic proteins as a marker of primary granules during development in chick heterophilis.

The ammoniacal silver reaction (ASR) for cationic proteins was used as a cytochemical marker for the primary or A granules in the cytoplasm of developing heterophils of chick bone marrow. The presence of the electron-dense particulate reaction product of silver, which is localized in the fully formed rod-shaped A granules, provides a marker by which the A granules could be distinguished from the B granules of similar size and by which the formation and maturation of both granule types could be followed through the developmental stages. Progressive developmental stages were ascertained on the basis of decreasing cell size, increasing condensation and margination of the chromatin, and the number and morphology of the granules; the stages were divided into promyelocyte, myelocyte, metamyelocyte and heterophil. During the promyelocyte stage, the first appearance of the electron-dense, membrane-bound, spherical granules (0.3--1.0 micrometer in diameter) is observed in the vicinity of an extensive Golgi complex. They occur in a cytoplasm containing rough-surfaced endoplasmic reticulum, ribosomal clusters, centrioles, mitochondria, microtubules, as well as the membranes, saccules, vesicles and vacuoles of the Golgi complex. These granules are considered as primary but their presence as the only granule type appears very brief. The ASR reaction product is first detected on the surface of these primary granules in late promyelocytes or myelocytes. The secondary or B granule, devoid of reaction for cationic protein at all stages, appears as a condensing vacuole in promyelocytes, but after some A granules are already present. The vacuole contents condense to form the B granules which are 0.1--0.6 micrometer in diameter, often oval-shaped, and contain a loose filamentous material surrounded by a membrane. Tertiary C granules or lysosomes appear during the myelocyte stage as dense core vesicles (0.1--0.2 micrometer in diameter) negative for cationic protein.     

Ultrastructural studies on the formation of yolk granules in the statoblast of a fresh-water bryozoan,Pectinatella gelatinosa

The formation of protein-carbohydrate yolk in the statoblast of a fresh-water bryozoan, Pectinatella gelatinosa, was studied by electron microscopy. Two types (I and II) of yolk cells were distinguished. The type I yolk cells are mononucleate and comprise a large majority of the yolk cells. The type II yolk cells are small in number; they become multinucleate by fusion of cells at an early stage of vitellogenesis. In both types of yolk cells, electron-dense granules (dense bodies) are formed in Golgi or condensing vacuoles, which are then called yolk granules. For the formation of yolk granules, the following processes are considered: 1. Yolk protein is synthesized in the rough-surfaced endoplasmic reticulum (RER) of the yolk cells. 2. The synthesized protein condenses in the cisternal space of the RER and is packaged into small oval swellings, which are then released from the RER as small vesicles (Golgi vesicles, 300-600 A in diameter). 3. The small vesicles fuse with one another to form condensing vacuoles, or with pre-existing growing yolk granules. 4. In the matrix of the condensing vacuoles or growing yolk granules, electron-dense fibers are fabricated and then arranged in a paracrystalline pattern to form the dense body. 5. After the dense body reaches its full size, excess membrane is removed and eventually the yolk granules come to mature. Toward the end of vitellogenesis of the yolk cells, the cytoplasmic organelles are ingested by autophagosomes derived from multivesicular bodies and disappear.     

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.     

Electron microscopic studies on the pars intermedia of the ferret

Summary The structure of the pars intermedia of the ferret has been studied with the electron microscope, with particular reference to the morphology of the glandular cells and their innervation. Two types of cell were found. The predominant cell is ovoid in shape and contains membrane-bound vesicles of varying size (1,000–5,000 Å) and density, the most electron-dense of which are associated with the Golgi region. The nucleus is indented and the cytoplasm contains rough endoplasmic reticulum. The second cell type is often associated with the colloid material and is elongated or stellate-shaped with long processes which extend between the predominant cells. It is devoid of cytoplasmic vesicles and has a poorly defined Golgi apparatus. Certain other structural features of this cell such as microvilli, cilia or cytoplasmic microfilaments are reminiscent of ependymal cells.Numerous nerve endings are observed throughout the pars intermedia, making synaptic contact with the predominant cell type. The majority contain vesicles with an electron-dense core measuring 750 Å; less frequently terminals contain dense granules measuring 1,000 A or more. Both also contain small electron-lucent vesicles (200–400 Å); occasionally terminals containing only the latter type are found. The pattern of innervation in the ferret is thus comparable to that previously observed in the cat, rather than that seen in rodents or monkeys, and the implications of this finding are discussed.We are indebted to Prof. Sir Solly Zuckerman, O. M., K. C. B., F. R. S., for his help and guidance and to Mr. J. Wallington for his unfailing technical assistance.     

Immunocytochemical localization of surfactant protein D (SP-D) in type II cells, Clara cells, and alveolar macrophages of rat lung.

We investigated the cellular and subcellular distribution of surfactant protein D (SP-D) by immunogold labeling in lungs of adult rats that had been given bovine serum albumin coupled to 5-nm gold (BSAG) for 2 hr to visualize the endocytotic pathway. Specific gold labeling for SP-D was found in alveolar Type II cells, Clara cells, and alveolar macrophages. In Type II cells abundant labeling was observed in the endoplasmic reticulum, whereas the Golgi complex and multivesicular bodies were labeled to a limited extent only. Lamellar bodies did not seem to contain SP-D. Gold labeling in alveolar macrophages was restricted to structures containing endocytosed BSAG. In Clara cells labeling was found in the endoplasmic reticulum, the Golgi complex, and was most prominent in granules present in the apical domain of the cell. Double labeling experiments with anti-surfactant protein A (SP-A) showed that both SP-A and SP-D were present in the same granules. However, SP-A was distributed throughout the granule contents, whereas SP-D was confined to the periphery of the granule. The Clara cell granules are considered secretory granules and not lysosomes, because they were not labeled for the lysosomal markers cathepsin D and LGP120, and they did not contain endocytosed BSAG.     

Exocytotic exposure and recycling of membrane antigens of chromaffin granules: ultrastructural evaluation after immunolabeling

The exocytotic exposure and retrieval of an antigen of chromaffin granule membranes were studied with chromaffin cells isolated from bovine adrenal medulla. Cells were incubated with an antiserum against glycoprotein III followed by fluorescein- or gold-labeled anti-IgG. Immunofluorescence on the cell surface was present in a patchy distribution irrespective of whether bivalent antibodies or Fab fragments were used. During subsequent incubation these fluorescent membrane patches were internalized within 45 min. At the ultrastructural level immunogold-labeled patches were present on the surface of stimulated cells. During incubation (5 min to 6 h) these immunolabeled membrane patches became coated, giving rise to coated vesicles and finally to smooth vesicles. These latter vesicles were found spread throughout the cytoplasm including the Golgi region, but Golgi stacks did not become labeled. Part of the immunolabel was transferred to multivesicular bodies, which probably represent a lysosomal pathway. 30 min after incubation immunolabel was also found in electron-dense vesicles apparently representing newly formed chromaffin granules. After 6 h of incubation immunolabel was found in vesicles indistinguishable from mature chromaffin granules. These results provide direct evidence that after exocytosis membranes of chromaffin granules are selectively retrieved from the plasma membrane and are partly recycled to newly formed chromaffin granules, providing a shuttle service from the Golgi region to the plasma membrane.     

Membrane modification during secretory granule formation in rat somatotrophs

Somatotrophs from male rat anterior pituitary were used to investigate the formation of secretory granules. When enzymatically dispersed cells were incubated with cationized ferritin (CF) for 15 min, CF labeled immature secretory granules, but not mature granules of somatotrophs. Most immature granules labeled by CF transformed to the mature types within 120 min. This indicates that the fusion of endocytic vesicles with the immature granules occurs during the maturation process of secretory granules. The internalized CF was distributed not only in the immature secretory granules, but also in the peripheral region of trans Golgi cisternae or GERL. Enzyme cytochemistry revealed that acid phosphatase-positive cisternae (GERL) were the main site for secretory granule formation, and was devoid of thiamine pyrophosphatase (TPPase) activity. A small number of secretory granules were also present in the peripheral regions of TPPase-positive Golgi cisternae. The granule-forming sites, however, lacked TPPase activity, while the remaining region of the same cisterna showed the positive enzyme activity. This indicates that the granule-forming region at the periphery of Golgi cisterna is different from the remaining part of the same cisterna in terms of cytochemical properties. This probably results from the insertion of endocytic vesicle membrane, since the same granule-forming sites preferentially fused with CF-labeled small vesicles which lacked cytochemical TPPase activity. Taken together. Our results suggest that the membrane of secretory granules is modified during the granule formation, at least partly by the fusion of endocytic small vesicles with Golgi cisternae (or GERL), and with immature secretory granules.     

Ultrastructural characteristics of rat peritoneal mast cells undergoing differential release of serotonin without histamine and without degranulation

Summary Rat mast cells pretreated with the tricyclic antidepressant drug amitriptyline and stimulated with compound 48/80 secreted 60% of the total serotonin present in the cells, but only 15% of histamine, another amine stored in the same granules. Ultrastructural studies demonstrated that mast cells undergoing such differential release do not exhibit classical degranulation by compound sequential exocytosis. However, there were changes in granule shape and size, as well as alterations in many morphometric parameters consistent with secretion. Storage granules lost their homogeneity, exhibited greatly reorganized matrix and were surrounded by clear spaces which were often associated with small (0.1–0.01 m) cytoplasmic vesicles, some of which contained electron-dense material. Secretory granules often had bud-like protrusions or were fused together in series. Quantitative autoradiography localized ³H-serotonin outside the storage granules, close to small vesicles, while staining with ruthenium red demonstrated that vesicular structures associated with differential release were not endocytotic. These results suggest that amitriptyline may inhibit regular exocytosis and permit at least serotonin to be moved selectively from storage granules to the cytosol or small vesicles from which it is eventually released.     

The effect of bromocriptine on the intermediate lobe of the rat pituitary: an electron-microscopic,morphometric study

Summary The morphological effect of chronic synthetic and secretory inhibition of the intermediate lobe of the rat pituitary induced by bromocriptine treatment was studied using morphometric techniques in combination with electron microscopy. On the basis of granule diameters, a heterogeneous cell population was shown in the normal intermediate lobe. Bromocriptine treatment did not induce any change in the volume fraction, number or location of electron-dense secretory granules. Instead, there was a shift toward a more homogeneous cell population containing smaller granules, the mean granule volume being reduced by 30%. The volume fraction of electron-lucent granules or vacuoles was markedly reduced, indicating a functional significance of these organelles. The volume of the Golgi apparatus was not significantly altered, but the number of condensing granules within the Golgi area was reduced. The volume of the intermediate lobe was decreased, apparently due to a decrease in the mean cell volume.     

Formation of crystalloid inclusions in the small intestine of neonatal pigs: an immunocytochemical study using colloidal gold

Summary Enterocytes of the small intestine in 1-day-old suckling piglets contain numerous vesicles in the apical cytoplasm and a large granule located beneath the nucleus. Within the next 3 days, these granules transform into electron-dense crystalloid inclusions. These membrane-bound inclusions are up to 10 m in length and 1–2 m in diameter, and they are composed of electron-dense lamellae 3.9 nm apart. Postembedding immunocytochemistry, using rabbit anti-porcine IgG and goat anti-rabbit IgG conjugated to 10 nm colloidal gold, revealed that both the granules and the crystalloid inclusions contained a high concentration of maternal IgG. Although the IgG content of the crystalloid inclusions was detected on epoxy-embedded sections, the use of LR White resin resulted in a much higher density of labelling. Quantification of the labelling density showed that the concentration of IgG in the crystalloid inclusions was approximately ten times higher than that in the lumenal colostrum and approximately three times higher than that in the granules. These observations showed that there are at least three compartments involved in the accretion of IgG in the small intestine of neonatal piglets: smaller apical endocytotic vesicles, large subnuclear granules and crystalloid inclusions. The role of these compartments in maternal immunoglobulin absorption and in the acquisition of passive immunity has yet to be explored.     

The frontal ganglion of Periplaneta americana L. (insecta)

Summary The frontal ganglion, part of the stomatogastric nervous system, contains about 60 to 80 neurons, 25 to 30 m in diameter. A well developed Golgi system, producing dense-core vesicles, lysosomes, multivesicular bodies and dense bodies are abundant. Glia elements are sparsely distributed. Many nerve fibres contain granules of different size and electron density. Five groups of fibres can be distinguished: Fibres with granules of about 200 nm (type A), fibres with granules of about 160 to 170 nm (type B), fibres with granules of about 80 to 100 nm (type C) and those with synaptic vesicles of 50 nm (type D) respectively. A fifth very small type contains neither vesicles nor granules. Special attention was paid to synaptic contacts. The divergent dyad seems to be the main type in the frontal ganglion. Frequently, neurosecretory endings are observed in presynaptic position. Immunocytochemical staining of neurosecretory material closely corresponds to the distribution of type A fibres, as observed electron microscopically. Immunoelectrophoresis of extracts from frontal ganglia with polyspecific anti-neurosecretion-serum reveals a single precipitation line, indicating that the immunocytochemical localization of neurosecretory material is due to reaction with a specific as well as a crossreagent antibody.Supported by the Ministerium für Wissenschaft und Technik der DDRThe authors wish to thank Mrs. B. Cosack and Mrs. A. Schmidt for excellent technical assistance     

Submicroscopic characterization of proctolin-like immunoreactivity in the nervous system of the cockroach Periplaneta americana L.

Summary Proctolin-like immunoreactivity (PLI) was found in different parts of the arthropod central nervous system and in nerve fibers of muscles. In order to examine whether this PLI is related to a uniform type of secretory vesicle, hindgut musculature and frontal and hypocerebral ganglia were examined with the immunogold technique. PLI occurs exclusively within membran-bounded secretory granules. Neither granular ER nor Golgi stacks show PLI. In some cases close relationships between PLI-bearing granules and lysosomes were observed. In presynaptic areas, PLI-reactive granules are associated with numerous clear synaptic vesicles and restricted to an area distinctly separate from the presynaptic membrane. Three types of granules were found, differing in diameter and electron density: (1) dense, 80 nm; (2) dense, 150 nm; (3) low density, 150 nm. The results demonstrate that: (1) the PLI of the produced peptide occurs shortly after its separation from the Golgi stack; (2) the occurrence of PLI in three different granule types could be the morphological expression of the common occurrence of proctolin with other neuroactive substances. However, a possible cross-reactivity with other, hitherto unknown substances must be considered as well.     

CYTOPLASMIC FINE STRUCTURE OF SCIARA SALIVARY GLANDS : I. Secretion

Cells from the anterior segment of the salivary glands of Sciara coprophila were found to synthesize and secrete into the gland lumen three morphologically distinct types of granule: 1) A large, electron-lucid granule, up to 1 µ in diameter, staining only faintly with pH 2 fast green and the PAS reaction; 2) an ellipsoid granule of moderate density, strongly fast green and PAS positive; and 3) a small spherical granule of high electron density. The cells contained numerous Golgi areas, up to an estimated 8,000 per cell. Evidence is presented for the transfer of material from the endoplasmic reticulum to the Golgi areas via small vesicles. Three types of Golgi areas were distinguishable, each containing intercisternal material resembling one of the three types of secretion granule. Patterns of secretion granule synthesis varied with the developmental stage of the larva as determined by counts of eye spots in the eye anlage. Lucid granules were most abundant in the youngest larvae, and decreased in abundance as larvae grew older, becoming virtually absent in prepupae. The small, dense granules were present in all gland cells, but became more prevalent in older larvae and prepupae. Ellipsoid granules were only occasionally present, and were independent of larval stage. It is suggested that lucid granules are digestive in function, since their abundance correlates with feeding patterns. Other granules may produce the external slime coating of the larvae, and also the mucoprotein component of the pupal cocoon.     

Depletion of polyamines prevents the neurotrophic activity of the GABA-agonist THIP in cultured rat cerebellar granule cells

Effects of polyamine depletion by -difluoromethylornithine (DFMO) were studied on the GABA-agonist mediated enhancement of the morphological development of cultured rat cerebellar granule cells. An increase in the number of neurite extending cells and in the cytoplasmic density of organelles relevant for protein synthesis was observed upon culturing in the presence of 4,5,6,7-tetrahydro-isoxazolo[5,4-c]pyridin-3-ol (THIP) for 4 days. The intracellular concentrations of putrescine, spermadine, and spermine in these cultures were similar to the concentrations of the polyamines observed in cultures grown in a plain culture medium for 1, 2, 3 or 4 days, respectively. Upon culturing in the simultaneous presence of THIP and DFMO, the concentrations of putrescine and spermadine were reduced to less than 20% of the levels in the controls. This depletion was associated with a severely impaired morphological development of the granule cell cultures. Thus, the number of neurite extending cells was reduced to 50% of the number in the control cultures upon culturing in the presence of DFMO alone or in combination with THIP. Moreover, the THIP mediated increase in the cytoplasmic density of rough endoplasmic reticulum, Golgi apparatus and different types of vesicles was prevented by the exposure to DFMO.     

GROWTH AND DIFFERENTIATION OF CYTOPLASMIC MEMBRANES IN THE COURSE OF LIPOPROTEIN GRANULE SYNTHESIS IN THE HEPATIC CELL : I. Elaboration of Elements of the Golgi Complex

The synthesis of "very low" density lipoprotein in liver cells is characterized by the fact that the synthesized products, mostly triglycerides, are processed in the form of discrete, size-limited granules or globules, about 400 A in diameter. The present investigation has been made possible in part by the use of a fixative (OsO₄ in bidistilled H₂O at pH 6.0, in the absence of electrolytes) particularly effective in preserving cytoplasmic membranes and lipids, and giving them high stainability and differential contrast. Under these technical conditions, the lipoprotein granules retain their morphology and high density to electrons practically unaltered, and may serve as tracers in determining their route of transport from the sites of synthesis, starting at the rough-smooth ER junctions, to the lumen of Golgi concentrating vesicles. From the observations, it may be deduced that, along with lipoprotein granule synthesis and transport, there are also production and transfer of new membranes in the form of tubular extensions of smooth ER network which, by progressive fusion and coalescence, participate in the elaboration of fenestrated plates and solid Golgi sacs. In contradistinction to the entire process of liver lipoprotein granule synthesis, transport, and segregation, as reported in the present paper, appears to constitute a developmental sequence which includes the following communicating compartments, in consecutive order: cisternae of rough ER where proteins and possibly phospholipids are synthesized, smooth ER network where triglycerides are synthesized and transported in the form of dense granules, fusion of smooth ER tubular extensions into Golgi fenestrated plates, and further coalescence into solid Golgi sacs, ending in the segregation of the granules in appended concentrating vesicles, or detached "secretory vesicles." It seems that it is this progressive evolution in growth and configuration of membranes which is reflected in the so called polarity, from forming to mature faces, of the Golgi apparatus.     

Schistosoma mansoni: cytochemistry and morphology of the gastrodermal Golgi Apparatus

The gastrodermal Golgi apparatus of adult Schistosoma mansoni displays two distinct morphologies. In one type, there is an identifiable cis (forming) face where vesicles from the endoplasmic reticulum fuse to form the cisternae. A morphological change occurs in the cisternae as the trans (emitting) face is approached with the cisternae becoming progressively flattened. The cisternae at the emitting face produce a membrane-bound secretory granule with moderately electron-dense contents and a vacuolar structure that may be analogous to a condensing vacuole as reported in several vertebrate secretory cells. In a second type, vesicles possessing a thicker membrane than those of the transfer vesicles are observed at the emitting face. They are not observed when the secretory granules are present. Several cytochemical markers were used to aid in studying the polarity of the Golgi apparatus. Enzymes studied were thiamine pyrophosphatase (TPPase) (EC 3.6.1.1), nucleoside diphosphatase (NDPase) (EC 3.6.1.6) using uridine diphosphate as a substrate, and nicotinamide adenine dinucleotide phosphatase (NADPase) (EC 3.1.3.2). Reaction products from all enzyme markers were observed in the cisternae and, to some extent, in the transfer vesicles. At times, NADPase and TPPase reaction products were observed in all cisternae and in the transfer vesicles of the Golgi. When this distribution was evident, the latter vesicles were observed in clusters occasionally fusing with lipid-like globules dispersed throughout the gastrodermis. Heterogeneity in cisternae was observed when NDPase, TPPase, and osmium reduction techniques were used. NDPase activity was limited to the middle cisternae while reduced osmium was observed in the outer two cisternae and in some transfer vesicles. TPPase reaction product was also observed in the secretory granules and in the condensing vacuoles. It is hypothesized that a functional bipolarity may be demonstrated by the Golgi. Under certain stress conditions, the forming face of the Golgi may package lysosomal enzymes while the emitting region of the Golgi appears to be responsible for the packaging of the secretory granules. The fusion of transfer vesicles and, at times, secretory granules with lipid-like globules is postulated to represent a mechanism by which enzymes may be transported to the lumen of the cecum.     

Ultrastructure of esophageal glands and their secretory granules in the root-knot nematodeMeloidogyne incognita

Summary The dorsal and subventral esophageal glands and their secretory granules in the root-knot nematodeMeloidogyne incognita changed during parasitism of plants. The subventral esophageal glands shrank and the dorsal gland enlarged with the onset of parasitism. While secretory granules formed by both types of glands were spherical, membrane-bound, and Golgi derived, the granules differed in morphology and size between the two types of glands. Subventral gland extensions in preparasitic second-stage juveniles were packed with secretory granules which varied in diameter from 700–1,100 nm and had a finely granular matrix. Within the matrix of each subventral gland granule was an electron-transparent core that contained minute spherical vesicles. The size and position of the core varied within different granules. Few granules were present in the dorsal gland extension in preparasitic juveniles. The matrix of dorsal gland secretory granules formed during parasitism was homogeneous and more electron-dense than the matrix of subventral gland granules. Subventral gland secretory granules of parasitic juveniles and adult females appeared degenerate.