CHANGES IN PANCREATIC ACINAR CELLS DURING PROTEIN DEPRIVATION

After 10 days of a protein-free diet the acinar cells of the rat pancreas showed a coarsening of nuclear matrix, depletion of zymogen granules, some loss of ribosomes, and a widening of the spaces between ergastoplasmic membranes. In addition, there could be found, but rarely, a lesion of the ergastoplasm consisting of vacuoles of agranular, disoriented membranes, which was similar to a lesion produced by ethionine. Thereafter, a return toward normal structure occurred which was characterized by beginning increase in the size of the Golgi apparatus at 12 days, appearance of zymogen granules at 18 days, and a relatively normal appearing but smaller cell at 28 days. After 10 to 12 days of protein deprivation a reversal of many of the morphologic effects of protein deprivation was accompanied by a return toward normal of some pancreatic enzyme activities. Possibly this spontaneous return toward normal levels represented a raiding of protein stores, or it may have been an adaptive phenomenon.     

THE ULTRASTRUCTURE OF AMELOBLASTS DURING MATRIX FORMATION AND THE MATURATION OF ENAMEL

Ameloblasts from different regions of upper incisors of rats were examined with the electron microscope. During matrix formation, the cells resemble secretory cells. They are extremely long, tightly packed, and show considerable polarity. Nuclei are at the basal end of the cell. Mitochondria are proximal and the Golgi apparatus distal to the nucleus. Ergastoplasm is found in all levels but mainly in the distal end. A terminal bar apparatus separates the distal end of the cell from Tomes's process. Next to this is soft enamel. The next incisal region is a transitional zone in which the ameloblasts separate easily from the enamel. Endoplasmic reticulum is dilated and very obviously in communication with the perinuclear space. Mitochondria are present not only proximal, but also distal, to the nucleus. The next incisal zone consists of cells related to the maturation of enamel. They no longer resemble secretory cells, but now have more characteristics of transporting cells. Processes from the distal end of the cell are present with mitochondria closely applied to the base of the processes. A considerable amount of intercellular space exists with microvilli projecting into the space. Iron granules appear in these cells, and the ergastoplasmic cisternae are dilated. In the incisal end of this zone, the iron granules form aggregates. The iron finally leaves the cells to enter the enamel. Free RNP particles and fibrils become more evident after the iron leaves the cells. In the most incisal region, the ameloblasts are further reduced in height. Distal processes are no longer present and fibrils are more conspicuous.     

THE DEGENERATIVE CHANGES IN PANCREATIC ACINAR CELLS CAUSED BY DL-ETHIONINE

Degeneration of pancreatic acinar cells in rats injected with ethionine was studied by electron microscopy. The most conspicuous morphologic lesions occurred in the ergastoplasm. There was a widening of the endoplasmic reticulum, a decrease in number of membrane-associated ribosomes, and a development of fine and coarse vacuoles containing agranular disoriented membranes. Cytoplasmic ribosomes unassociated with membranes were less numerous. Nuclear changes consisted of a coarsening and clumping of the nuclear chromatin, chromatin margination, and increased osmiophilia and vacuolation of the nucleolus. Eight to ten days after the beginning of ethionine injections, changes in zymogen granules, mitochondria, and the Golgi apparatus appeared, but only after extensive damage to the acinar cell. The effects were consistent with ethionine's known interference with protein metabolism but also suggest disturbance in ribonucleic acid metabolism. The ergastoplasmic changes after ethionine were similar in some respects to the early lesions produced in liver parenchymal cells by fasting, to the changes occurring in animals on protein-free diets, or to some of the liver changes produced by azo dye carcinogens. The ribosomal and ergastoplasmic changes represent early morphologic expressions of the biochemical effect of ethionine.     

RESTITUTION OF PANCREATIC ACINAR CELLS FOLLOWING ETHIONINE

The regeneration of the pancreatic acinar cell was studied at four time periods after ethionine had destroyed most of the acinar cells. Within 2 days of the last ethionine injection, small basophilic cells (pre-acinar cells) with whorls of ergastoplasm or nebenkern were present. These cells also contained a decreased amount of Golgi substance, small zymogen granules, and a fine granularity of the nuclear matrix. They showed persistence of the characteristic ergastoplasm lesion produced by ethionine. Eight days after the last ethionine injection, the nebenkern was replaced by approximately normal appearing ergastoplasm and the nucleoli and Golgi bodies were enlarged. Zymogen granules were less dense but more abundant. Mitochondria were considerably enlarged. Most cells showed no ethionine lesions or only small foci of damage. Eighteen days after the cessation of ethionine, a good approximation of the normal acinar cell was present. The whorls of ergastoplasm appeared at a time (day 12) when basophilia was pronounced. Other studies showed that nucleic acid and protein precursors began to show an increased concentration in acinar cells at this time. The appearance of nebenkern during a phase of cellular recovery and its absence during a phase of replication when mitotic indices were high suggest that its presence is more indicative of ergastoplasmic synthesis than of cell multiplication as such. Possibly the increased density of zymogen granules was a reflection of this increased protein synthesis. The increase in size of Golgi apparatus occurred prior to the replenishment of zymogen granules and thus satisfied a precursor relationship for a possible role in the formation of these secretory structures. Evidence suggests that some injured acinar cells recover from the ethionine and protein-free regimen and give rise to most of the new acinar cells formed. It is possible that, under the severe conditions which prevailed, the centroacinar ductule cells may also have given rise to some acinar cells.     

ELECTRON MICROSCOPY OF THE OXYNTIC CELL IN THE GASTRIC GLANDS OF THE BULLFROG (RANA CATESBIANA) : I. The Non-Acid-Secreting Gastric Mucosa

The fine structure of the oxyntic cell from the gastric glands of the bullfrog was studied in lead hydroxide—stained sections of gastric mucosa fixed in buffered osmium tetroxide and embedded in n-butyl methacrylate. The oxyntic cell in non-acid-secreting stomachs (gastric juice pH, 7.4–7.8) is characterized by: (a) numerous closely packed smooth surfaced vesicular and tubular profiles disposed randomly in the cell; some of these elements show interconnections making it possible to identify this component with smooth surfaced endoplasmic reticula of certain other cell types, (b) a small percentage of rough surfaced profiles characteristic of endoplasmic reticula possessing RNP particles on the outer membrane surfaces, (c) a Golgi complex consisting of multiple isolated non-polarized arrays of smooth surfaced parallel elongated profiles and associated vesicular elements, (d) a sparse granular component (140 A) scattered freely in the cytoplasmic matrix, (e) numerous mitochondria with a dense matrix and containing an unusually large number of closely approximated cristae, (f) a number of zymogen granules consisting of either a dense body limited by a membrane or surrounded by a halo of less dense material which is in turn limited by a membrane, and (g) a number of granules (~260 A) containing several smaller granules (~80 A) identified presumably as glycogen. Intracellular canaliculi were not observed. Instead the free surface of the oxyntic cell facing the lumen of the gastric gland shows a complicated plication of the plasma membrane. Intercellular canaliculi are seen frequently between adjacent oxyntic cells. The walls of these canaliculi are made up of folded and ruffled cell membranes. The basal surface of the cell also exhibited this type of configuration. Occasional smooth surfaced profiles are seen communicating with the free surface, the wall of an intercellular canaliculus, or the basal surface of the cell. Although nerve endings were not found in association with oxyntic cells, unmyelinated nerves were observed in the vicinity of the gastric glands.     

SPECULATIONS BASED ON THE MORPHOLOGY OF THE GOLGI SYSTEMS IN SEVERAL TYPES OF PROTEIN-SECRETING CELLS

Electron microscopical observations on the relationship of the Golgi region to other intracellular organelles in certain protein-secreting cells have substantiated and extended existing hypotheses. In micrographs of several cell types, the juxtanuclear Golgi regions were observed to be closely associated with nuclear "pores." The "transition elements" of the ergastoplasmic membranes possess "blebs" which may represent a transport process facilitating the movement of intracisternal contents into the Golgi zone. A "blebbing" process of this nature may be one source of the small variety of Golgi vesicles. Zymogen granules of different densities were observed and their significance was postulated. Light Golgi vacuoles were observed. It is suggested that these vacuoles represent accumulations of relatively fluid material segregated from the secretory product in these cell types. These hypotheses from inferential evidence are discussed and extended.     

CHANGES IN FINE STRUCTURE DURING SILK PROTEIN PRODUCTION IN THE AMPULLATE GLAND OF THE SPIDER ARANEUS SERICATUS

The ampullate silk gland of the spider, Araneus sericatus, produces the silk fiber for the scaffolding of the web. The fine structure of the various parts of the gland is described. The distal portion of the duct consist of a tube of epithelial cells which appear to secrete a substance which forms the tunica intima of the duct wall. At the proximal end of the duct there is a region of secretory cells. The epithelium of the sac portion contains five morphologically distinct types of granules. The bulk of the synthesis of silk occurs in the tail of the gland, and in this region only a single type of secretory droplet is seen in the epithelium. Protein synthesis can be stimulated by the injection of 1 mg/kg acetylcholine into the body fluids. 10 min after injection, much of the protein stored in the cytoplasm of the epithelial cells has been secreted into the lumen. 20 min after stimulation, the ergastoplasmic sacs form large whorls in the cytoplasm. Protein, similar in electron-opacity to protein found in the lumen, begins to form in that portion of the cytoplasm which is enclosed by the whorls. The limiting membrane of these droplets is formed by ergastoplasmic membranes which lose their ribosomes. No Golgi material has been found in these cells. Protein appears to be manufactured in the cytoplasm of the tail cells in a form which is ready for secretion.     

FINE STRUCTURAL CHANGES IN RESPONSE TO HORMONAL STIMULATION OF THE PERFUSED CANINE PANCREAS

The dog pancreas isolated in situ was perfused with oxygenated dog blood and stimulated with pancreozymin, secretin, or both. There were no significant changes in the fine structure of the acinar, centroacinar, or duct cells attributable to the perfusion. Combined glutaraldehyde and osmium fixation gave good preservation of the secretory products of the acinar cell. Before stimulation, the lumen of the acini is filled with material similar in texture to the content of the zymogen granules, but of somewhat lower density. Release of secretion commonly takes place by coalescence of the limiting membrane of zymogen granules with the plasmalemma, but one granule opening at the surface may frequently be joined by others coalescing with its membrane and forming an interconnected series all with contents having the same texture as the released zymogen. Such a mechanism seems to permit a more rapid release of secretory product than discharge of individual granules. Pancreozymin stimulation caused marked depletion of zymogen granules, but no obvious changes in the Golgi apparatus. It is clear, therefore, that this hormone exerts its effect upon release of granules rather than upon their formation. Secretin stimulation of water and bicarbonate secretion caused a marked washing out of the luminal contents, but had little detectable effect on cellular structure.     

THE STRUCTURE AND FORMATION OF PROTEIN GRANULES IN THE FAT BODY OF AN INSECT

In the larva of the butterfly Calpodes ethlius, the fat body begins to store protein in the form of granules at about 30 to 35 hours before pupation, at a time when the endocuticle is being resorbed. At least two sorts of granule can be distinguished. The first granules to arise are those within vesicles of the Golgi complex. These may increase in size by incorporating material from microvesicles at their surface and by coalescence with one another. Later, at about 10 hours before pupation, another sort of granule arises by the isolation of regions of the endoplasmic reticulum (ER) within paired membranes derived from Golgi vesicles. Several of these ER isolation bodies coalesce, with fusion of their outer isolating membranes. The ribosomes and membranes may then disappear and the granules become indistinguishable from the protein granules formed from Golgi vesicles, or the ribosomes may remain and be embedded in dense crystalline protein, forming a storage body for both protein and RNA. Mitochondria are isolated within paired membranes in the same way as regions of the ER. The isolated mitochondria also coalesce in a similar manner. When the inner membranes are lost, the structure of a group of isolation bodies is indistinguishable from that of a cytolysome. Isolation within paired membranes, as described here, may be of general importance in segregating regions of massive lysis or massive sequestration.     

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.     

Glycoprotein synthesis in the adult rat pancreas: II. Characterization of Golgi-rich fractions

Golgi-rich fractions were prepared from homogenates of adult rat pancreas by discontinuous gradient centrifugation. These fractions were characterized by stacks of cisternae associated with large, irregular vesicles and were relatively free of rough microsomes, mitochondria, and zymogen granules. The Golgi-rich fractions contained 50% of the UDP-galactose: glycoprotein galactosyltransferase activity; the specific activity was 12-fold greater than the homogenate. Such fractions represented < 19% of thiamine pyrophosphatase, uridine diphosphatase, adenosine diphosphatase, and Mg²⁺-adenosine triphosphatase. Zymogen granules and the Golgi-rich fractions were extracted with 0.2 m NaHCO3, pH 8.2, and the membranes were isolated by centrifugation. The glycoprotein galactosyltransferase could not be detected in granule membranes, while the specific activity in Golgi membranes was 25-fold greater than the homogenate.At least 35 polypeptide species were detected in Golgi membranes by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. These ranged in molecular weight from 12,000 to <160,000. There were only minor differences between Golgi membranes and smooth microsomal membrane. In contrast, zymogen granule membranes contained fewer polypeptides. A major polypeptide, which represented 30–40% of the granule membrane profile, accounted for less than 3% of the polypeptides of Golgi membranes or smooth microsomal membranes.     

STUDIES ON THE FINE STRUCTURE OF ULTRACENTRIFUGED SPINAL GANGLION CELLS

The following structures were observed in electron micrographs of the mouse spinal ganglion cells: Nissl bodies composed of both aggregated rough-type, largely oriented, membranes of the endoplasmic reticulum and discrete particles; short rodlike mitochondria with well-developed transverse, obliquely or longitudinally arranged cristae, and a relatively typical Golgi complex. The components of ultracentrifuged ganglion cells (400,000 times gravity for 20 minutes) are stratified, the layers appearing in the order of their decreasing density as follows: (1) A microsomal or ergastoplasmic layer which may be further divided into three sublayers without sharp boundaries, namely, a discrete particle layer, a layer of discrete particles and highly distorted membranes of the endoplasmic reticulum, and a layer composed of relatively intact, but stretched membranes of the endoplasmic reticulum and discrete particles. (2) Mitochondria constitute a relatively broad layer. They are sometimes stretched; however, they retain most of their fine structure. The stratified nucleus is found within the mitochondrial layer. (3) A relatively wide layer of tightly packed vesicles. (4) At the centripetal end, resting against the cell membrane, are a few lipid vacuoles. A comparison is made between the ultrastructure of the stratified layers in situ and those described by others in differentially ultracentrifuged homogenates.     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF THIN SECTIONS: THE GOLGI ZONE AS A SITE OF PROTEIN CONCENTRATION IN PANCREATIC ACINAR CELLS

Electron microscopic radioautographs of guinea pig pancreatic exocrine cells were obtained by covering thin sections (~ 600 A) of OsO₄-fixed, methacrylate-embedded tissue with thin layers of Ilford K-5 nuclear research emulsion. After an exposure of 13 days at 4°C., the preparations were photographically processed, stained with uranyl acetate, and examined in an electron microscope. The label used was leucine-H³ injected intravenously 20 minutes before collection of the specimens. Conventional radioautographs of thicker sections (0.4 micron) were also examined in a phase contrast microscope. The advantages obtained from electron microscopic radioautography are: the higher radioautographic resolution (of the order of 0.3 micron) due to the thinness of the emulsion and the specimen, and a high optical resolution permitting a clear identification of the labeled structure. In the guinea pig pancreas this technique demonstrated that, at the time studied, newly synthesized proteins were concentrated in the structures of the Golgi complex and especially in large vacuoles partially filled with a dense material. The vacuoles are probably a precursor to the secretion granules (zymogen granules) in which the label becomes segregated at a later time. These observations demonstrate directly the role of the Golgi complex in the secretion process. They also illustrate the possibilities of this method for radioautography at the intracellular level.     

FURTHER STUDIES ON THE THETA CELL OF THE MOUSE ANTERIOR PITUITARY AS REVEALED BY ELECTRON MICROSCOPY, WITH SPECIAL REFERENCE TO THE MODE OF SECRETION

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 origin of the zymogen granule membrane of the pancreatic acinar cell as examined by ultrastructural cytochemistry of acid phosphatase, thiamine pyrophosphatase, and ATP-diphosphohydrolase activities

Cytochemical distributions of acid phosphatase, thiamine pyrophosphatase, and ATP-diphosphohydrolase activities have been examined on thin sections of rat pancreas and on isolated zymogen-granule membranes. Acid phosphatase was found in the rigid lamellae separated from the Golgi stacked cisternae, in condensing vacuoles, and in the trans-saccules of Golgi apparatus; it was not detected in purified zymogen-granule membranes. Thiamine pyrophosphatase was detected in trans-saccules of the Golgi apparatus, in purified zymogen-granule membranes, and in the plasmalemma of the acinar cell. It was absent in condensing vacuoles. The ATP-diphosphohydrolase activity has a distribution similar to thiamine pyrophosphatase. These observations illustrate the similarity between the trans-saccules of the Golgi apparatus and the membrane of mature zymogen granules and the disparity between the latter membrane and the membrane of the condensing vacuole. They suggest that the condensing vacuole might not be the immediate precursor of the zymogen granule as commonly assumed. An alternative possibility would be that condensing vacuoles would fuse with the trans-saccule (transition) of the Golgi apparatus which in turn would form mature zymogen granules.     

RAPID RELEASE OF THE ZYMOGEN GRANULE PROTEIN BY OSMIUM TETROXIDE AND ITS RETENTION DURING FIXATION BY GLUTARALDEHYDE

Fixation by osmium tetroxide and glutaraldehyde of zymogen granules isolated from rat parotid and pancreas was investigated. Protein determinations showed that osmium tetroxide caused rapid release of most of the soluble protein of the granule during fixation in buffered isotonic sucrose. Such granules when examined in the electron microscope after shadow casting appeared quite flat, indicating that most of the contents had indeed been removed. Numerous damaged membranes of the granules were also observed. In contrast, zymogen granules fixed by glutaraldehyde and shadow cast essentially retained the spherical shape and the protein contents. The application of the shadow-casting technique in quantitative studies on the protein content of zymogen granules is discussed.     

Concentration of amylase along its secretory pathway in the pancreatic acinar cell as revealed by high resolution immunocytochemistry

Summary The modified protein A-gold immunocytochemical technique was applied to the localization of amylase in rat pancreatic acinar cells. Due to the good ultrastructural preservation of the cellular organelles obtained on glutaraldehyde-fixed, osmium tetroxide-postfixed tissue, the labelling was detected with high resolution over the cisternae of the rough endoplasmic reticulum (RER), the Golgi apparatus, the condensing vacuoles, the immature pre-zymogen granules, and the mature zymogen granules. Over the Golgi area, the labelling was present over the transitional elements of the endoplasmic reticulum, some of the smooth vesicular structures at thecis- andtrans-faces and all the different Golgi cisternae. The acid phosphatase-positive rigidtrans-cisternae as well as the coated vesicles were either negative or weakly labelled. Quantitative evaluations of the degree of labelling demonstrated an increasing intensity which progresses from the RER, through the Golgi, to the zymogen granules and have identified the sites where protein concentration occurs. The results obtained have thus demonstrated that amylase is processed through the conventional RER-Golgi-granule secretory pathway in the pancreatic acinar cells. In addition a concomitance has been found between some sites where protein concentration occurs: thetrans-most Golgi cisternae, the condensing vacuoles, the pre- and the mature zymogen granules, and the presence of actin at the level of the limiting membranes of these same organelles as reported previously (Bendayan, 1983). This suggests that beside their possible role in transport and release of secretory products, contractile proteins may also be involved in the process of protein concentration.     

Glycoprotein synthesis in the adult rat pancreas: IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5–10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50–100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and $\text{N-}\text{acetylglucosamine}$. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes < smooth microsomes < zymogen granules.Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptide was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [³H]glucosamine or [³H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules.Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB³H₄. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Topographical and planar distribution of Helix pomatia lectin-binding glycoconjugates in secretory granules and plasma membrane of pancreatic exocrine acinar cells of the rat: demonstration of membrane heterogeneity

The lectin-gold technique was used to detect Helix pomatia lectin (HPL) binding sites directly on thin sections of rat pancreas embedded in Lowicryl K4M and on freeze-fractured preparations of rat pancreas submitted to fracture label. On thin sections of acinar cells, whereas the content of zymogen granules was negative or weakly labeled, the limiting membrane displayed a high degree of labeling. In the Golgi complex, labeling by HPL was localized on the trans saccules and the limiting membrane of the condensing vacuoles. The latter appeared to be more intensely labeled than the membrane of the zymogen granules. Intense labeling by HPL was also observed along the microvilli and the plasma membrane. In contrast to the weak labeling of the zymogen-granule content, labeling of the acinar lumen was intense. Fracture-label preparations revealed preferential partition of HPL-binding sites to the exoplasmic half of the zymogen-granule and plasma membranes. The population of zymogen granules was, however, heterogeneous with respect to labeling intensity; the exoplasmic fracture-face of the plasma membrane was intensely and uniformly labeled, while the protoplasmic membrane halves were only weakly labeled. These observations were further confirmed and extended by the thin-section fracture-label approach. In addition, favorable profiles of thin sections of freeze-fractured zymogen granules showed that the labeling was not associated with the external surface of the limiting membrane, but rather localized over the exoplasmic fracture-face. We conclude that 1) zymogen granules contain little HPL-binding glycoconjugates, 2) HPL-binding sites are preferentially associated with the exoplasmic half of the zymogen-granule and plasma membranes, and 3) the limiting membrane of the immature condensing vacuoles carries a greater number of HPL-binding sites than that of the mature zymogen granules. These last, in turn, constitute a heterogenous population with respect to labeling density. These results support the current view that glycoconjugates are directed toward the lumen in secretory granules but become external to the cell surface after fusion of the secretory-granule membrane with the plasma membrane. Also, the results reflect membrane modifications during the maturation process of secretory granules in the exocrine pancreas in which glycoproteins are removed from the limiting membrane of the granule to become soluble and secreted with the content.     

Fine structural observations on magnum mucosa in quail and hen oviducts

Summary Previous investigators have reported that albuminous material in the albumin-secreting (tubular gland) cells of the magnum of hen oviduct accumulates in the ergastoplasmic cisternae and is released directly into the glandular lumen without being first concentrated into secretory granules in the Golgi region (Zeigel and Dalton, 1962). Present fine structural studies on the tubular gland cells in oviducts from actively laying wild-type Japanese quail and in an oviduct from an actively laying hen indicate that the Golgi apparatus is directly involved in the formation of secretory granules. At least three types of granules can be observed in the tubular gland cells at various times, and all types seem to be associated initially with the Golgi apparatus.In actively laying quail, the distribution of electron opaque, intermediate, and light granules within the superficial and deep regions of the glandular epithelium varies, depending on the presence of an egg in a particular region of the oviduct. Secretion of the product is merocrine, involving fusion of granule membranes with the plasmalemma of the cell surface.Granules first appear in the tubular gland cells of quail oviducts at about 4 1/2 weeks of age. The granules are of the electron opaque type and probably represent secretion in concentrated storage form. At this age, a few of the tubular gland cells exhibit distended ergastoplasmic cisternae containing material of low electron density. The appearance of these light cells, which occur with greater frequency in oviducts from older quail, probably reflects an increased level of secretory activity initiated by changes in hormonal levels. From 5 weeks of age on, intermediate and light (less concentrated) granules, as well as dark granules, are present.Supported by the National and Medical Research Councils of Canada.