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The gland cells of Lyonet's gland, which is accessory to the silk gland in the silkworm larva, is characterized by the presence of complicated canaliculi bearing microvilli on their inner surface, large numbers of mitochondria and remarkably convoluted basal plasma membrane. On the other hand, the cell lacks the well-developed cytoplasmic membrane system such as rough- and smooth-surfaced endoplasmic reticula and Golgi bodies, though free ribosomes are numerous. Secretory vesicles are absent, and the canaliculi contain no dense material. From such ultrastructural observations, it was suggested that a possible role of the gland may be the exchange of the small molecules such as water and ions, rather than the hitherto supposed secretory role of a cementing sunstance of silk proteins. The lumen of the proximal part of the glandular duct contains a kind of proteinaceous substance which can be demonstrated histochemically and is regarded as similar to one of the silk proteins in the silk gland, not to the real product of the Lyonet's gland.

The incorporation of [3H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [3H]fucose and autoradiographs analyzed quantitatively. At 5 min after [3H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [3H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [3H]fucose. The incorporation of [3H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.

Golgi bodies of immature carposporangia ofPolysiphonia sp. are composed of a polarized stack of six to ten curved cisternae. The cisternae are surrounded by 50–200 nm diameter slightly granular vesicles. Hypertrophied, fibrillar Golgi cisternae occur in mature carposporangia. Secretory vesicles originate from ends of cisternae and by complete vesiculation of terminal cisternae; 0.6–1.2 μm diameter, fibrous vesicles, many with electron dense nucleoids are abundant throughout the cytoplasm of mature sporangia. Vesicles expand, fuse with each other and cluster around starch granules. Some vesicles secrete their content into the spore wall. Morphological analyses of starch granules as well as topographical relations between vesicles, starch granules and the adjacent cytoplasm suggest that these Golgi vesicles function like lysosomes. The significance of these observations is discussed in relation to the composition of plant cell walls and cellular expansion.

Experiments in which glycine-(3)H has been introduced into excised neurointermediate lobes of Xenopus laevis incubated in a modified Krebs-Ringer bicarbonate medium have shown that approximately 50% of the incorporated radioactivity is present in small peptides which have an electrophoretic mobility characteristic of the melanocyte-stimulating (MSH) peptides shown to be elaborated within the tissue. Based on these results and the demonstration that a discrete approximately 7 min pulse of the label can be introduced into the tissue, electron microscope radioautography has been employed to follow the subcellular events concerned with the synthesis, intracellular transport, and packaging of the labeled secretory product. Together, these studies indicate that the newly synthesized material arises in peptide form, rather than as part of a larger prohormone molecule, on the ribosomes of the rough endoplasmic reticulum within the parenchymal cells of the intermediate portion of the lobe. A proportion is then incorporated into and remains for an extended period within the intracisternal granules which are a feature of the rough endoplasmic reticulum within these cells in vitro Most ( approximately 60%) of the labeled secretory product, however, is transferred to the Golgi complex within 30 min and, within a further 10 min, becomes packaged into small ( approximately 200 mmicro) electron-opaque secretory granules. It is probable that under the conditions employed these granules represent the final intracellular location of secretory product before it is released

Our earlier work demonstrated that the rate of protein synthesis in the exocrine cells of the rat pancreas is constant in different physiological states, including prolonged fasting. In this study we have followed the fate of the protein in the pancreatic cells of the fasting animal in vivo as well as in vitro. The data were obtained by quantitative radioautography and by biochemical determinations. In nonanesthesized, fasting rats, without cannulated pancreatic duct, some 80% of the proteins synthesized at a given time leaves the cell within 12 hr by way of secretion, intracellular breakdown not being important. Two mechanisms of fasting secretion exist. The first, starting at a slow rate after 20 min, is inferred to result from fortuitous contacts of young secretory granules with the apical cell membrane. The rate of secretion is the same in vivo as in vitro, at least during the first 4 hr after pulse labeling. Within 7 hr about 20% of the total amount of newly synthesized protein has left the cell. The second mechanism consists of an orderly movement of the mass of secretory granules towards the apical cell membrane as caused by the continuous assembly of new granules. The granules that come into contact with the cell membrane are discharged. It takes about 7–12 hr for secretory protein transported in this way to reach the cell membrane. The addition of new secretory granules to those present is essential for the second mechanism, for the blockade of protein synthesis by cycloheximide decreases the rate of this phase of secretion without interfering with the secretory process proper. Atropin does not inhibit the fasting secretion in vitro, nor does extensive washing of the tissue slices, excluding possible secretagogues as important factors in fasting secretion.

Insect cuticle forms as a result of overlapping sequences of two kinds of process, those involving vesicles of the Golgi complex, and those related to transport through and/or assembly at the apical plasma membrane. The ecdysial droplets are the last layer of old cuticle to be deposited before ecdysis and form from the contents of secretory vesicles from Golgi complexes. Ecdysial droplets and secretory vesicles both stain with PTA and react with silver hexamine after oxidation with periodic acid. The vesicles discharge in localized apical areas devoid of microvilli where they accumulate as droplets measuring about 3 [ x 1 [ . The. droplets span the last few lamellae of the endocuticle which becomes the ecdysial membrane. They dissolve to leave the ecdysial membrane full of holes at the time that the rest of the old cuticle is digested.

The pineal body of the mouse was studied by electron microscopy especially as to whether this organ is secretory in nature. The cytoplasmic processes of the pinealocytes were frequently observed together with the axons around the blood capillaries. The pinealocyte processes contain variable amounts of vesicles of various sizes and appearances. Large vesicles are of two kinds: granular (cored) and agranular (clear). The granular vesicles measure a mean diameter of 1160A. Small vesicles contain no electron dense core, resembling those in axon terminals in size and shape. It is most likely that the large granular vesicles are formed in the Golgi apparatus in the cell bodies and transported to the cell processes. By a morphological analogy to the secretory vesicles of the neurosecretory cells the large granular vesicles would be considered as the secretory material of the pinealocytes. The nerve axons in the perivascular spaces are loaded with numerous small, granular and agranular vesicles which measure approximately 600A in diameter. They are considered sympathetic and adrenergic in nature.The endothelial cells of the capillaries which are surrounded by the tissue spaces are attenuated and possess numerous fenestrations with an average diameter of 650A. The occurrence of the fenestrations may also suggest the possible secretory nature of the pineal body.

A fractiion rich is membranes of the Golgi apparatus was isolated from rat liver by discontinuous density gradient centrifugation. Electron microscopic analysis of the fraction revealed the presence of structures very similar to those of the Golgi apparatus in intact cells, namely stacked cisternae, secretory vesicles, and tubular elements. The Golgi-rich fraction contained over 90% of the UDP-galactose; N- acetylglucosamine galactosyltransferase, about 2% of the glucose-6-phosphatase and 12% of the AMP phosphohydrolase present in the post-nuclear supernatant of liver homogenates.

A structure for a generalized insect epidermal cell during the formation of the epicuticle is proposed, based on studies of several different epidermal cell types. The protein epicuticle is defined as the dense homogeneous layer below the cuticulin. The formation of the protein epicuticle involves secretory vesicles arising in Golgi complexes, and marks an interlude in the involvement in cuticle formation of plasma membrane plaques. The plaques are concerned in cuticulin formation before and in fibrous cuticle formation after the deposition of the protein epicuticle. The epidermis is characterized by the possession of a cytoskeleton of microtubules and a matrix of microfibers. In the elongated cells forming bristles and spines, the microfibers are often oriented in bundles with an axial banding which repeats every 120 A. The microtubules are also arranged in columns with a trigonal packing and center to center spacing of about 800 A. These cytoskeletal structures separate the other organelles into channels which may restrict the pathways open for the movement of secretory and pinocytotic vesicles. The protein epicuticle arises from the secretory vesicles which discharge at the apical surface. The contents disperse and reaggregate below the cuticulin. The Golgi complexes in the basal and central regions have many secretory vesicles and a small saccular component, differing from those nearer the apex which are smaller and have fenestrated saccules. The small coated vesicles (800 A in diameter) associated with both sorts of complex, probably move to the apical and basal faces of the cell where they may give rise to the large coated vesicles (2000 A in diameter) inserted in the plasma membrane. Pinocytosis occurs from both apical and basal faces but most lytic activity is in the apical region. Plant peroxidase injected into the haemocoel is taken up basally and transported to the apical MVBs. The large coated vesicles on the apical face may be concerned in the control of the extracellular subcuticular environment. They appear to fill up and detach, fusing to become the apical MVBs.

A partially purified secretory granule fraction, isolated from rat islets of Langerhans by differential centrifugation, was used for investigating the stability of the beta granules during incubation in various conditions. Effects of pH, temperature, and time were studied; the granules possessed optimal stability at 4° and pH 6.0, and could be solubilized at pH 4.0 or 8.5, or in the presence of sodium deoxycholate, but not by phospholipase c, ouabain, or alloxan. Incubation with glucose or some of its metabolites, or with tolbutamide, ATP, or cyclic 3',5'-AMP did not alter the stability of the beta granules Exogenous insulin-131I was not bound by the isolated granules under the conditions used; no specific insulin-degrading activity could be detected in subcellular fractions of the islets. These findings indicate that intracellular solubilization of the granules with subsequent diffusion of the insulin into the extracellular space is not a likely mode of insulin secretion in vivo, and suggest that a crystalline zinc-insulin complex may exist in the matrix of the beta granules.

The distribution of phenoloxidase and polyphenols have been studied during cuticle formation at the 4th to 5th molt in Colpodes ethius. Cuticular phenoloxidases arise in the epidermis in cisternae of the rough endoplasmic reticulum, pass through the Golgi complex and are transported to the apical face in secretory vesicles. From the cuticular environment some enzyme is pinocytosed and broken down in the apical multivesicular bodies. Phenoloxidase and polyphenols are present during the formation of the cuticulin layer which also reacts as if it were at least partly composed of a phenoloxidase. The rest of the epicuticle incorporates phenoloxidase as it is deposited, particularly that over the dorsal tubercles which later melanize. Polyphenols do not appear until shortly before ecdysis. They are associated with the epicuticular filaments in both epicuticle and presumptive epocuticle. It is proposed that the epicuticular filaments may arise as liquid crystals with a protein component which becomes stabilized like the rest of the cuticle. These structures could provide a channel for the movement of both lipids and quinones to the surface. Phenoloxidases may pass through fibrous cuticle to be deposited as part of the epicuticle but are incorporated in fibrous cuticle scheduled for sclerotization. The time of stabilization is determined by the availability of polyphenols.

The nature and content of lytic bodies and the localization of acid phosphatase (AcPase) activity were investigated in mammotrophic hormone-producing cells (MT) from rat anterior pituitary glands. MT were examined from lactating rats in which secretion of MTH1 was high and from postlactating rats in which MTH secretion was suppressed by removing the suckling young. MT from lactating animals contained abundant stacks of rough-surfaced ER, a large Golgi complex with many forming secretory granules, and a few lytic bodies, primarily multivesicular bodies and dense bodies. MT from postlactating animals, sacrificed at selected intervals up to 96 hr after separation from their suckling young, showed (a) progressive involution of the protein synthetic apparatus with sequestration of ER and ribosomes in autophagic vacuoles, and (b) incorporation of secretory granules into multivesicular and dense bodies. The content of mature granules typically was incorporated into dense bodies whereas that of immature granules found its way preferentially into multivesicular bodies. The secretory granules and cytoplasmic constituents segregated within lytic bodies were progressively degraded over a period of 24 to 72 hr to yield a common residual body, the vacuolated dense body. In MT from lactating animals, AcPase reaction product was found in lytic bodies, and in several other sites not usually considered to be lysosomal in nature, i.e., inner Golgi cisterna and associated vesicles, and around most of the immature, and some of the mature secretory granules. In MT from postlactating animals, AcPase was concentrated in lytic bodies; reaction product and incorporated secretory granules were frequently recognizable within the same multivesicular or dense body which could therefore be identified as "autolysosomes" connected with the digestion of endogenous materials. Several possible explanations for the occurrence of AcPase in nonlysosomal sites are discussed. From the findings it is concluded that, in secretory cells, lysosomes function in the regulation of the secretory process by providing a mechanism which takes care of overproduction of secretory products.

A fine structural analysis of the esophagus of the tardigrade Milnesium tardigradum Doyere is presented. This foregut structure exhibits pronounced secretory activity with discharge of accumulated material by fusion of secretory vesicles with luminal and lateral surfaces of the esophageal cells. The possible function of this secretory material as well as the organization of esophageal cells and structure of the cuticular lining are discussed.

Replicas of fractured chromaffin cells are indicative of a range of activities thought to characterize exocytosis, including attachment of secretory vesicles to the plasma membrane, fusion, extrusion of contents, and membrane retrieval. Exocytosis sites are abundant on stimulated cells but are infrequent when calcium is omitted from the system.

Pygidial defence glands, which are universal in Caraboidea, are paired, cuticular invaginations of the body wall. In Pterostichus madidus F. the glands contain a nauseous, corrosive secretion that can be discharged forcibly to deter attack by predators. The cells producing the secretion are of one type only. Electronmicroscopy shows that the ducts which drain these cells are not intracellular but are separated from the cell contents by a plasma membrane. The cuticular walls of the duct appear to effectively screen the cell from the disruptive effect of its final products of secretion. The duct-carrying epidermal cells which are modifications of the epidermis covering the rest of the gland support the ducts from the secretory vesicles to their entry into the central lumen of each secretory lobe. There is a single duct-carrying cell around each duct.

The Reuber hepatoma H-35 and Morris hepatoma 5123 have been studied by electron microscopy and by cytochemical staining methods for a number of phosphatases. These studies emphasize the resemblances of the two tumors to rat liver, but they also indicate distinctive features in each of the three tissues. Secretory product accumulates within the cisternae of the Golgi apparatus that dilate to form the Golgi vacuoles. The vacuoles apparently separate, and secretory material undergoes further condensation within them. These "secretory vacuoles" possess acid phosphatase activity and may thus be considered lysosomes. The membranes of the Golgi apparatus are without acid phosphatase activity but show high levels of thiaminepyrophosphatase activity. The endoplasmic reticulum also hydrolyzes thiaminepyrophosphate but at a lower rate; it hydrolyzes the diphosphates of uridine, guanosine, and inosine rapidly. These observations and the electron microscopic images are consistent with the view that the cytomembranes are in a dynamic state of flux, movement, and transformation in the living cell, and that smooth surfaced derivatives of the endoplasmic reticulum become refashioned into the Golgi membranes as the Golgi membranes are being refashioned into those that delimit secretory vacuoles. The variations encountered in the two hepatomas are described. The electron microscope literature dealing with the relations of the Golgi apparatus to secretory granules, on the one hand, and the endoplasmic reticulum, on the other, is reviewed briefly.

Secretory vesicles containing polysaccharide were isolated from germinated pollen of Lilium longiflorum and characterized by biochemical and ultrastructural investigation. Pollen tubes exhibit a secretory pathway in which the vesicles concentrated in the tube apex are produced by the Golgi apparatus and contributed to the cell wall at the apex upon fusion of the vesicle membrane with the plasma membrane. Secretory vesicles were isolated by a method involving the size discrimination of cytoplasmic components using Millipore filters. Cells were disrupted under conditions which minimized membrane vesiculation. Identification was made by electron-microscopic comparison of the periodic acid-silver hexamine (PASH) reactivities of in situ and isolated secretory vesicles. The secretory vesicles contained polysaccharides which were high in galacturonic acid and similar in sugar composition to those of the hot-water-soluble fraction of pollen tube cell wall. A hot-water-insoluble, non-cellulosic glucan was the major component of the cell wall. Less than 7 % of the wall was cellulosic. Chitin was absent. Similarities in the ultrastructure and PASH staining of apical secretory vesicles and an amorphous component of the cell wall support a precursor-product relationship between these 2 cell components. Ultrastructural investigations revealed complexes of the endoplasmic reticulum (ER) associated with electron-translucent regions of cytoplasm, suggesting a possible function of the ER in cell wall formation. Additionally, patterns of PASH staining show that changes in polysaccharides occur in secretory vesicles after vesicles have been formed by dictyosomes. Therefore, secretory vesicles may have a role in polysaccharide synthesis as well as in membrane and product transport.

A comparative study has been made of the fine structure of the two neurosecretory nuclei of the diencephalon. In the neurons of both nuclei we find a similar arrangement of the various structural features. Two zones may be distinguished in the cytoplasm: a marginal one nearly devoid of all organelles and secretory droplets, but with ergastoplasmic structures (Nissl substance) of varying appearance and importance; and a central one with a well developed endoplasmic reticulum of vesicular type. Numerous mitochondria are found in this zone. The ribonucleoprotein granules abound everywhere between the membranes and on their outer surfaces. The Golgi complex consists of several groups of agranular membranes. Vacuoles, cisternae, and small vesicular elements are seen in each group. Numerous osmiophilic secretory droplets are grouped around the zone of Golgi. The nucleus has a very irregular shape. The cytoplasmic invaginations are enveloped by nuclear lobes. No true nuclear inclusions have been found. The secretory granules arise in the Golgi system of both the nucleus preopticus (NPO) and the nucleus lateralis tuberis (NLT). A comparison between the preoptic neurosecretory granules and those of the nucleus lateralis tuberis of Salmo irideus shows that the mean maximum diameter of the latter is approximately 30% smaller. Droplets from the NLT are not as osmiophilic in sections as are those from preoptic neurons. The diameters of the secretory droplets do not change during their intraaxonal transport toward the axon endings in the neurohypophysis. Because of this, the hypothesis of a progressive synthesis of the neurosecretory material in the secretory vesicles during transport in the axon cannot be maintained. The neurosecretory neurons of both preoptic and tuber nuclei are in relation with other parts of the nervous system. The presence of typical synaptic structures on these secretory neurons indicates that nerve impulses may intervene in the control of the secretory activity, or that these neurons may be activated and produce nerve impulses. It is also possible that such impulses may control the release of the neurosecretion from the axon endings.

The defensive glands of tenebrionid beetles produce a mixture of benzoquinones and hydrocarbons. These reactive toxicants are manufactured within a complex morphological system, consisting of secretory cells, secretory vesicles within these cells, and cuticular organelles and tubules which carry the products to the gland reservoir. Histochemical and thin-layer chromatographic techniques were employed to follow the process of toxicant production within these cells. Chromatographic evidence indicated the presence of both p -diphenols and their glucosides in extracts of the secretory cells, which suggested that quinones are generated from phenol glucosides by hydrolysis and subsequent oxidation. Histochemical tests demonstrated concentrations of phenolic materials in the cytoplasm of one cell, type 2a, and on the basis of their sensitivity to emulsin, it was concluded that these are phenol glucosides. Glucosidase activity, as shown by a modified bromonaphthol-glucoside technique, was associated with the margins of the vesicle in the same cell type. The cuticular organelle, within this vesicle, yielded positive reactions for a phenolase both with dopa and with N-phenyl- p -phenylene diamine as substrates. Along the efferent tubule which runs from this organelle to the gland reservoir, a haemoprotein peroxidase was detected by the benzidine peroxidase test. Each of these steps in quinone production was associated with a particular morphological compartment, and it is apparent that the cytoplasm of the secretory cell is never exposed to the toxic product of the secretory unit. Lipid reserves, which are possible precursors of the hydrocarbon components in the secretory end-products, were found in all secretory cells. However, the densest deposits were concentrated in the type 1 units, and in these units, a paraoxon-resistant carboxylic esterase was demonstrated with indoxyl substrate, along the margins of and within the secretory vesicle. Deposits of unsaturated lipids were detected in the cuticular organelle of this vesicle.

Theta cells reported previously as a new cell type in the anterior pituitary of the mouse were examined with the electron microscope. This type of cell is distinguished by the presence of pleomorphic secretory granules, a characteristic arrangement of the rough surfaced variety of endoplasmic reticulum, a well developed Golgi complex, and an eccentrically located nucleus. The secretory granules are seen at first as small granules of low density within the Golgi vesicles. While they are within the Golgi vesicles they become larger and denser. Simultaneously they move from the proximal to the distal part of the Golgi region and finally emerge from the Golgi area as mature granules in the cytoplasm. Thus, secretory granules are always enveloped by a limiting membrane which originates from the wall of the Golgi vesicle. At the stage of granule-extrusion, the cell membrane fuses with the limiting membrane of the granules and openings in the cell membrane appear at the place of extrusion. The granules then appear to lie within inpocketings of the cell membrane. They lose their density within these inpocketings or within the cytoplasm and occasionally show fragmentation. After complete loss of density, the granules are extruded as amorphous materials to the territory outside of the cell.

The normal parathyroids of six humans and a Virginia deer were studied by light and electron microscopy. The parenchyma of the deer parathyroid is composed of uniform chief cells, which contained 100 to 400 mµ electron-opaque, membrane-limited granules, presumed to be secretory granules, in addition to the usual cytoplasmic organelles. Desmosomes are present between adjacent cells, and rare cilia are observed protruding from the chief cells into the intercellular space. The human parathyroids contain chief cells in two phases—active and inactive—as well as oxyphil cells. Active chief cells have a large Golgi apparatus, sparse glycogen, numerous secretory granules, and rare cilia. Inactive chief cells contain a small Golgi apparatus, abundant glycogen, and few secretory granules. Both forms have the usual cytoplasmic organelles and, between adjacent cells, desmosomes. Oxyphil cell cytoplasm is composed of tightly packed mitochondria and glycogen granules, with rare secretory granules. Cells with cytoplasmic characteristics intermediate between chief and oxyphil cells, possibly representing transitional cells, have been observed. Secretory granules of both man and deer are composed of 100 to 200 A particles and short rods, and the granules develop from prosecretory granules in the Golgi region of the cell. The human secretory granules are smaller and more variable in shape than those of the deer. The granules are iron and chrome alum hematoxylin-positive, argyrophilic, and aldehyde fuchsin-positive, permitting light microscopic identification. They are also found in the capillary endothelial cells of the parathyroid and in its surrounding connective tissue. The secretory granules of the parathyroid cells can thus be followed from their formation in the Golgi apparatus almost to their extrusion into the blood stream.

Germ tube apices ofGilbertella persicaria contain cytoplasmic vesicles, similar to the secretory vesicles found at the tips of vegetative hyphae. The vesicles are present at all stages of development, from the time of germ tube initiation to the establishment of branched hyphae. In contrast to the abundant vesicles at tips of established hyphae, the germ tubes have only a few apical vesicles in a layer next to the plasma membrane. When germinated spores are treated by washing and centrifuging prior to fixation, the cytoplasm is often disrupted near the apex, and the clusters of apical vesicles disappear. The findings indicate the delicate nature of hyphal tips and the necessity of avoiding prefixation stresses in order to preserve the apical apparatus of growing hyphae.

The postnatal ontogeny of the retinal interphotoreceptor space of mice and rats was studied by electron microscopy to elucidate the cytological developments in the surrounding cells relevant to the accumulation of extracellular glycosaminoglycans and glycoproteins constituting the interphotoreceptor matrix. This extracellular material at birth is principally the cell coat on all the immature cells that border the space at that time, but later additional weblike strands are seen in the space. The cells delimiting the space in the adult are the pigment epithelium (PE), the photoreceptors, and the glial cells of Muller. The Golgi complex of the PE is large at birth but involutes by day 15. Melanogenesis is the principal activity in this cell at birth but as the melanin granules mature, lysosomes gradually accumulate. Growth of the apical microvilli of the PE is most pronounced between day 5 and 15, which is also the time of rapid expansion of the interphotoreceptor space. The Golgi complex of the photoreceptor enlarges during this interval also, and remains voluminous thereafter. Muller's cells insert only slender apical processes lacking in secretory vesicles, into the interphotoreceptor space. All the adult cells have a cell coat on the surfaces facing the interphotoreceptor space. Secretory vesicles were not identified in any of the cells impinging on the space.

Golgi apparatus were released without fixatives from rat hepatocytes by gentle homogenization, concentrated by differential centrifugation, and purified by sucrose gradient centrifugation. Examination of sections of purified fractions by electron microscopy showed fields of morphologically intact units of Golgi apparatus consisting of stacks of parallel flattened cisternae, secretory vesicles, and small vesicular profiles. Negative staining of unfixed pellets revealed a complex network of anastomotic tubules continuous with platelike structures and secretory vesicles. These structures corresponded, respectively, to the small vesicular profiles and parallel flattened cisternae with attached secretory vesicles of sectioned material. Small fragments of granular endoplasmic reticulum were often closely associated with the peripheral tubules, suggesting sites of continuity in intact hepatocytes.

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.

The cytological modifications of the epidermal cells secreting sternal and pleural cuticle of adultTenebrio are studied, specially during the formation and evolution of the outer and inner epicuticle. Both layers are secreted by condensation of diffuse material just above the epidermal microvilli. There is no peculiar phase without microvilli when inner epicuticle is formed. During the secretion of pre-ecdysial cuticle, the epidermal cell is characterized by an heterolytic process which is demonstrated by peroxidase injection into the ecdysial space: horseradish peroxidase is absorbed by large pinocytic coated vesicles which discharge their content into multivesicular bodies, after the loss of their coat. The intensity of pinocytosis is studied by vesicles counts at different stages of the pupal life. These counts show a pinocytic peak when the inner epicuticle is formed. When the epicuticular barrier is completely secreted, exogenous peroxidase is no more absorbed, but morphological study and counts show that pinocytosis still occurs. This demonstrates that epidermis cannot absorb macromolecules arising from the digested old cuticle since digestion occurs after the completion of adult epicuticle. Locke's hypothesis about the “control of the extracellular subcuticular environment” by pinocytosis is confirmed. The Golgi complexes are involved in lytic process with their small coated vesicles and in secretion of cuticular material with their large dense secretory vesicles, but according to vesicle counts, the secretory vesicles are not concerned in epicuticle formation since their apparition is posterior to the deposition of the inner epicuticle. Les modifications cytologiques des cellules epidermiques secretant la cuticule sternale et pleurale de l'adulte deT. molitor sont etudiees pendant la formation et l'evolution de l'epicuticule externe et interne. L'epicuticule externe et interne sont secretees de la meme facon par condensation de materiel diffus au-dessus de l'apex des microvillosites. Un cycle heterolytique est mis en evidence par l'emploi de peroxydase injectee dans l'espace exuvial: la peroxydase est captee par de grosses vesicules revetues et incorporee dans des corps multivesiculaires apicaux. Par comptage des differentes populations de vesicules, des variations nettes dans l'intensite de la pinocytose sont mises en evidence. Ces variations semblent en rapport non pas avec la digestion de l'ancienne cuticule mais avec les differences de nature et de structure des composants cuticulaires. L'hypothese du controle de la secretion epidermique par les phenomenes de pinocytose enoncee par Locke (1969) est confirmee. Des vesicules d'origine golgiennes sont secretees surtout apres la formation de l'epicuticule et ne sont donc pas a l'origine du materiel epicuticulaire, mais il est possible qu'elles jouent un role dans l'evolution ulterieure des structures epicuticulaires.

Two methods used for the electron microscopic detection of glycoproteins were applied to a variety of cell types in the rat; one involved successive treatment of sections with periodic acid, chromic acid, and silver methenamine; and the other, a brief treatment with a chromic acid-phosphotungstic acid mixture. The results obtained with the two methods were identical and, whenever the comparison was possible, similar to those obtained with the periodic acid-Schiff technique of light microscopy. In secretory as well as in nonsecretory cells, parts of the Golgi apparatus are stained. The last saccule on one side of each Golgi stack is strongly reactive (mature face), and the last saccule on the other side shows little or no reactivity (immature face); a gradient of reactivity occurs in between these saccules. The more likely explanation of the increase in staining intensity is that carbohydrate is synthesized and accumulates in saccules as they migrate toward the mature face. In many secretory cells, the mature face is associated with strongly stained secretory granules. Other structures stained are: (1) small vesicles, dense and multivesicular bodies, at least some of which are presumed to be lysosomal in nature; (2) cell coat; and (3) basement membrane. The evidence suggests that the Golgi saccules provide glycoproteins not only for secretion, but also for the needs of the lysosomal system as well as for incorporation into the cell coat and perhaps basement membrane.

The formation of the intradermal eccrine sweat duct lumen began in the 15-week-old embryo with a dissolution of the desmosomal attachment plaques, followed by the separation of apposing inner cells and subsequent formation of microvilli at the luminal surfaces. The luminal cells possessed numerous microvilli and crypts and a periluminal band of tonofilaments. In the secretory portion of older embryos (22 weeks old), columnar secretory cells extended from the basement membrane to the luminal border. The presence in these secretory cells of microvilli, Golgi elements, mitochondria, small secretory vesicles, dense secretory granules and abundant endoplasmic reticulum, implies that preparation for secretory functioning begins in embryonic life. The pyramidal myoepithelial cells differentiated from the basal cells seen in younger embryo, and rested upon a basement membrane. In the oldest embryo examined (22 weeks) there was as yet neither intercellular canaliculi nor sufficient difference in the amount of glycogen to enable one to distinguish between dark and light secretory cells. Isolated cilia of 9 + 0 type were occasionally found in the luminal cells of the duct and the secretory segment.

To provide further evidence for the identification of the cell type responsible for the production of adrenocorticotrophin (ACTH), the effects of acute and chronic administration of cortisol on the cells of the pituitaries of intact and adrenalectomized rats were studied by electron microscopy. Hypertrophied ACTH-cells, previously described as adrenalectomy-cells, were scarce in controls and became more numerous after adrenalectomy. Adrenalectomy-cells are characterized by their secretory granules, which are dense, sparse, and measure 200 μn or more in maximal diameter, and by the irregularity of their cell outlines, determined by their interior position in the cell cords and their tendency to send processes of cytoplasm around neighboring cells toward the capillaries. Normal ACTH-cells are identified by similar criteria, although they are less voluminous and their cell outlines tend to be less complex and varied. Acute administration of cortisol caused a striking accumulation of secretory granules in bot...

Electron microscopic studies of two cases of vulvar Paget's disease (1 invasive and 1 in situ) were undertaken in an attempt to elucidate further the histogenesis of the characteristic Paget cell. Secretory vesicles, mitochondria, ferritin-like granules, and membrane junctional structures are described which are typical of apocrine cells. These findings, plus the location of some cells against the basement membrane, support the hypothesis that the Paget cell is an apocrine cell differentiating within a more generalized epithelial neoplasm.

The elaboration of enamel matrix glycoprotein was investigated in secretory ameloblasts of incisor teeth in 30–40-g rats. To this end, the distribution of glycoprotein was examined histochemically by the use of phosphotungstic acid at low pH, while the formation of glycoprotein was traced radioautographically in animals sacrificed 2.5–30 min after galactose-3H injection. Histochemically, the presence of glycoprotein is observed in ameloblasts as well as in the enamel matrix; in ameloblasts glycoprotein occurs within the Golgi apparatus in amounts increasing from the outer to the inner face of the stacks of saccules, and is concentrated in condensing vacuoles and secretory granules; in the enamel matrix, glycoprotein is observed within linear subunits. Radioautographs at 2.5 min after injection demonstrate the uptake of galactose-3H label by Golgi saccules, indicating that galactose-3H is incorporated into glycoprotein within this organelle. After 5–10 min, the label collects in the condensing vacuoles and secretory granules of the Golgi region. By 20–30 min, the label appears in the secretory granules of the apical (Tomes') processes, as well as in the enamel matrix (next to the distal end of the apical processes, and at the tips of matrix prongs). In conclusion, galactose contributes to the formation of glycoprotein within the Golgi apparatus. The innermost saccules then distribute the completed glycoprotein to condensing vacuoles, which later evolve into secretory granules. These granules rapidly migrate to the apical processes, where they discharge their glycoprotein content to the developing enamel.

The fine structure of the ergastoplasm of the pancreatic exocrine cell of Swiss albino mice has been studied with the electron microscope. It was found that this material consists of sac-like structures, which may be called ergastoplasmic sacs, embedded in an amorphous granular ground substance, the cytoplasmic matrix. The membranous wall of the ergastoplasmic sac is a structure approximately 250 A wide. Except for its greater electron density and granular structure, the ergastoplasmic membrane is similar in appearance to the nuclear, plasma, and mitochondrial membranes. From data available in the literature, and from our own evidence, the conclusion can be drawn that the ergastoplasmic membrane contains ribonucleic acid. The mode of formation of the ergastoplasm and secretory granules was studied in animals which were first fasted and subsequently fed. It was found that ergastoplasm is formed within the cytoplasm, near the nuclear membrane, and possibly from the plasma membrane. The secretory granules were observed to arise by accumulation of materials within small ergastoplasmic sacs.

Secretory granules have been seen within components of the Golgi bodies of rat pituitary acidophils and mouse pancreatic acinar cells. The fact that secretory granules are much more frequently encountered within Golgi components under conditions of increased secretory activity suggests that granule formation may occur within the Golgi apparatus in these two types of cells.

SECRETORY vesicles (‘trichocysts’) of Paramecium contain proteins1 which are discharged by exocytosis. In P. aurelia (mating type VIII), trichocysts are firmly attached to the cell membrane before the onset of exocytosis, and membrane-intercalated granules occur at these contact sites (Fig. 1); these granules are arranged in a regular pattern and have been described previously as ‘b-’ and ‘c-type granules’2. They are surrounded by a ring of ‘a-type granules’ which connect the cell membrane to the membrane of the alveolar sacs in the region where the latter are penetrated by the trichocysts2,3. When the trichocysts are expelled, the cell membrane is penetrated within the ring of a-type granules and the trichocyst membrane fuses temporarily with the cell membrane2. The present study takes advantage of several circumstances: (i) cells could be frozen without prefixation or cryoprotectants; (ii) exocytosis triggering involves only the final step of membrane fusion; (iii) the regular ‘landmarks’ in the plasmalemma serve to pinpoint intramembraneous changes occurring during exocytosis.

The formation of secretory vesicles is progressive across the stack of dictyosome cisternae from one of its faces to the other . As a cisterna matures, the secretion vesicles increase in size, and their contents change in staining characteristics, suggesting the accumulation or transformation of product. Vesicles are ultimately released at the maturing face of the dictyosome, apparently by a pinching off of the tubules which connect the secretory vesicles to the flattened, nonsecretory portions of the cisternae. The secretory vesicles migrate to the cell surface where they discharge their product to the cell exterior . These steps are well documented, but there is no information on the fate of the cisterna or cisternal fragments left behind. From the concept of the maturing face and forming face, we know that entire cisternae must be lost from the maturing face as new cisternae are produced at the opposite or forming face. This is emphasized by information that the number of dictyosome cisternae remain relatively constant during periods of active cisternal formation at the immature face and active secretory vesicle production at the mature face ; i.e.,the processes of cisternal formation and fragmentation are balanced . The morphological observations on maize root tips reported here suggest that nonsecretory portions of mature cisternae are sloughed from the dictyosomes at or about the same time that secretory vesicles are released into the cytoplasm (Figs . I and 2). These sloughed cisternae are frequently found near the mature faces of dictyosomes and are observed in various stages of decomposition . Their lifetime in the cytoplasm is probably short since recognizable cisternal fragments never accumulate. Morphological stages in the release and breakdown of cisternae at the distal face of the

Smooth endoplasmic reticulum is more plentiful in the secretory cells of both chin and brown inguinal glands than rough endoplasmic reticulum. The smooth reticulum has a relatively dense content in chin glands while in inguinal glands it often appears empty and swollen, resulting in a characteristic cytoplasmic meshwork. Stacks of Golgi cisternae, scattered in the supranuclear region of all cells, are most numerous in male chin glands, while lysosomes are more frequent in female chin glands. Rounded secretory granules in inguinal glands and irregularly shaped secretory lakes in chin glands occur primarily in the supranuclear region. Deformed mitochondria are seen only in close association with the secretory lakes in the chin glands of bucks with normal libido and of a doe treated with depotestosterone. Secretory lakes are numerous and widespread in the chin glands of a buck with poor libido and in a buck castrated 2 weeks previously. At 1 month following castration the male chin gland closely resembles those of does. No cytologic effect of castration or depotestosterone treatment was seen in the inguinal glands. Both chin and brown inguinal secretory cells apparently release their secretions by exocytosis of juxtaluminal secretory vesicles rather than by an “apocrine” mechanism.

Although it has been shown thathypothalamic neurohormones controlling adenohypophysealactivity are concentrated in the medianeminence, precise identification of the intracellularstructures containing hypophysiotropic hormoneshas not been achieved. Using an immunohistochemicaltechnique at the electron microscopiclevel, we have observed that luteinizinghormone-releasing hormone (LH-RH), the neurohormonestimulating the release of both FSHand LH, is contained in the secretory granules of somenerve endings located in the palisade layer of themedian eminence. This is the first demonstration atthe ultrastructural level that an hypothalamicreleasinghormone is stored in secretory granules ofthe median eminence. (Endocrinology 95:314, 1974)

The elaboration of dentin collagen precursors by the odontoblasts in the incisor teeth of 30–40-g rats was investigated by electron microscopy, histochemistry, and radioautography after intravenous injection of tritium-labeled proline. At 2 min after injection, when the labeling of blood proline was high, radioactivity was restricted to the rough endoplasmic reticulum, indicating that it is the site of synthesis of the polypeptide precursors of collagen, the pro-alpha chains. At 10 min, when the labeling of blood proline had already declined, radioactivity was observed in spherical portions of Golgi saccules containing entangled threads, and, at 20 min, radioactivity appeared in cylindrical portions containing aggregates of parallel threads. The parallel threads measured 280–350 nm in length and stained with the low pH-phosphotungstic acid technique for carbohydrate and with the silver methenamine technique for aldehydes (as did extracellular collagen fibrils). The passage of label from spherical to cylindrical Golgi portions is associated with the reorganization of entangled into parallel threads, which is interpreted as the packing of procollagen molecules. Between 20 and 30 min, prosecretory and secretory granules respectively became labeled. These results indicate that the cylindrical portions of Golgi saccules transform into prosecretory and subsequently into secretory granules. Within these granules, the parallel threads, believed to be procollagen molecules, are transported to the odontoblast process. At 90 min and 4 h after injection, label was present in predentin, indicating that the labeled content of secretory granules had been released into predentin. This occurred by exocytosis as evidenced by the presence of secretory granules in fusion with the plasmalemma of the odontoblast process. It is proposed that pro-alpha chains give rise to procollagen molecules which assemble into parallel aggregates in the Golgi apparatus. Procollagen molecules are then transported within secretory granules to the odontoblast process and released by exocytosis. In predentin procollagen molecules would give rise to tropocollagen molecules, which would then polymerize into collagen fibrils.

It is shown that in the cytoplasm of the fast growing cells of Avena coleoptiles, large sacs filled with vesicles are formed. The membranes of these sacs seem to have Golgi nature because they can fuse with the plasmalemma to form anastomoses with the space outside the plasmalemma. When subsequently the boundary membrane of the sacs flattens out and becomes continuous with the plasmalemma, the contents of the sacs come to lie outside the plasmalemma so as to form part of the outer cell wall. The sacs contain at least two types of vesicles, a majority which lose their membranes and furnish the mucopolysaccharide matrix of the wall, and numerous other vesicles which stay membrane-bounded and are incorporated into the structureless, electron transparent matrix, and which may even persist in the older portions of the wall. It is thought that this latter type of vesicle represents lysosomes. This multivesicular mode of exocytosis differs from the modes described so far in which only single, individual vesicles play a part.

In an electron microsope study, the morphology of mature Paneth cells from the small intestine of adult rats is compared with that of differentiating Paneth cells from young rats 2 to 4 weeks old. All mature cells exhibit a marked polarity similar to that of other exocrine gland cells and contain a well developed endoplasmic reticulum, an elaborate Golgi complex, and numerous large secretory granules; they also possess an abundance of lysosomes. The most conspicuous occurrence in the process of differentiation is the development of the endoplasmic reticulum. The most immature Paneth cells possess an endoplasmic reticulum of the vesicular type, which, during maturation, is replaced by the characteristic lamellated ergastoplasm of the mature cell. At a certain stage of differentiation the cavities of the developing cisternae show numerous communications with the perinuclear space, suggesting an outgrowth of the ergastoplasm from the nuclear envelope. Furthermore, the cavities and the perinuclear space at this particular stage contain a material which shows a remarkable intrinsic periodicity. An identical periodicity was exhibited by material contained in Golgi cisternae and secretory granules. Lysosomes are also present in the differentiating cells.

Adrenalectomy cells, previously demonstrated by autoradiography in the light microscope as the site of ACTH production in the adrenalectomized rat, were identified in the electron microscope by their peculiar morphology. The adrenalectomy cells were found to have certain characteristic fine structural features which aid in identifying and differentiating them from the other cell types. Their secretory granules, with a maximal diameter of about 200 mμ, are larger than thyrotroph granules, smaller than acidophil granules, and correspond in size roughly to the gonadotroph granules. Gonadotrophs are readily differentiated from adrenalectomy cells, however, by their typically rounded shape. (Endocrinology 76: 70, 1965)

Virtually nothing is known about the initial intracellular events that lead to the formation of a nascent lipoprotein particle. However, recent technical advances in electron microscopy, cell frationation, and the biochemistry of apolipoproteins are providing subcellular probes for identifying and characterizing both the plasma lipoprotein secretory mechanisms and the newly made secretory products. Evidence for an extremely close relationship between nascent plasma lipoproteins and the membrane lipoproteins constituting the secretory mechanism is beginning to emerge.

Aleurone cells of barley (Hordeum vulgare L.) contain microbodies as determined by histochemical localization with diaminobenzidine. These microbodies can be isolated from both water and gibberellic acid (GA3) treated cells and identified on sucrose density gradients as glyoxysomes on the basis of their buoyant densities (1.25 g cm(-3)) and their enzyme complement. Fractionation of aleurone layer homogenates by differential centrifugation after varying times of exposure to GA3, however, does not indicate the presence of a discrete secretory vesicle containing either α-amylase or β-1,3-glucanase. Cytological evidence also suggests that at least β-1,3-glucanase is not released from these cells by means of a discrete secretory vesicle.

Long-term im administration of porcine calcitonin to rabbits in large doses (100 MRC U/kg body weight as total dose) induces a striking proliferation of thyroid cells. Clusters of interfollicular cells disrupting the follicular pattern and predominance of small follicles lined by high columnar cells with infoldings are evident. Ultrastructurally, most of the thyroid follicular cells are in an apparent hypersecretory state, showing an increase in number and size of microvilli, colloid droplets and heterogeneous dense granules as well as a hypertrophy of Golgi complex. A hyperplasia and hypertrophy of parafollicular (C) cells are also observed. These cells display a large population of small dense granules, free ribosomes and secretory vesicles. These findings suggest that chronic calcitonin administration exerts a goitrogenic effect on rabbit thyroids and also inhibits the release of calcitonin from the parafollicular cells. (Endocrinology 90: 1046, 1972)

l -Fucose was used as a radioactive precursor to study the synthesis of glycoproteins in the Golgi complex isolated from rat liver using both biochemical and au toradiographic techniques. Protein-bound fucose appeared in the Golgi complex of rat liver initially in the perchloric acid-insoluble fraction (maximum specific activity at 10 min after fucose injection) of the isolated Golgi comples and then in the perchloric acid-soluble fraction (maximum at 15 min) before being released in the perchloric acid-soluble fraction of serum. The results of biochemical studies correlated well with electron microscope autradiographic studies on sections of rat liver and isolated Golgi fractions labelled with l -[ 3 H]fucose. Within 2 min, fucose was incorporated into the Golgi complex with the maximum number of silver grains over the Golgi complex observed at approximately 10 min, while the maximum number over the plasma membrane and associated vesicles appeared at 30 min. In isolated Golgi membranes, autoradiographic studies showed most radioactivity localized in the cisternae (maximum at 2 min) of the Golgi complex and then in the tubules (maximum at 10 min) befor appearing in the secretory vesicles (maximum at 20 min).