Stanniocalcin-like immunoreactivity in the corpuscles of Stannius of the bowfin Amia calva L.

Summary We used an antiserum against salmon stanniocalcin in an immunohistochemical, immunocytochemical, and Western blot analysis of bowfin (Amia calva) corpuscles of stannius. The bowfin is one of two extant holostean species with ancient ancestral links to modern-day bony fishes. The corpuscles of Stannius (white corpuscles) of the bowfin were scattered throughout much of the kidney among the adrenocortical homolog (yellow corpuscles) but could be distinguished from the adrenocortical homolog by their positive staining with both the periodic acid-Schiff reaction and a salmon stanniocalcin antiserum. Immunoreactivity was confined to cytoplamic granules and was absent when the antiserum was blocked with salmon stanniocalcin or with a crude extract of bowfin corpuscles of Stannius. When bowfin corpuscle-of-Stannius extracts were subjected to sodium dodecyl sulphate electrophoresis and Western blot analysis, two closely spaced bands were evident (43–45 kDa). Staining of both bands was abolished by pre-absorption of the antiserum with salmon stanniocalcin. In comparison to salmon stanniocalcin, the reputed bowfin hormone migrated faster in gels, suggesting a smaller apparent size. The purification of bowfin stanniocalcin should yield important new information regarding the evolution of this unique calcium-regulating hormone.     

Ontogenetic development of neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system of the dogfish

This immunocytochemical study was carried out to elucidate the ontogenetic development of neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system of the cloudy dogfish, Scyliorhinus torazame. Immunostained cells first appeared in the pancreas of the embryo at the 15-mm stage, and were also detected in the vitellointestinal duct of the yolk stalk at the 20-mm stage. These cells were polymorphic, with occasional processes that were sometimes directed toward the vascular wall or into the cavity of the vitellointestinal duct. At the 34-mm stage, immunostained cells could also be found in the proximal part of the spiral intestine and, by the 74-mm stage, immunopositive cells were present in the gastric mucosa. In the gut and pancreas, the cells gradually increased in number with development, whereas in the vitellointestinal duct and internal yolk sac, they decreased and seemed to disappear following hatching. Thus, in juveniles, the distribution of the neuropeptide Y-like-immunoreactive cells in the gastroenteropancreatic endocrine system had attained that of adults. Electron-microscopic immunocytochemistry demonstrated that, in the labeled cells of the vitellointestinal duct, the neuropeptide Y-like antigen was located in cytoplasmic granules, as in the cells of the gut and pancreas.This paper is dedicated to Professor Yoshiharu Honma, on the occasion of his retirement and inauguration as Emeritus Professor     

Ultrastructure of endocrine pancreatic granules during pancreatic differentiation in the grass snake,Natrix natrix L. (Lepidosauria,Serpentes)

We used transmission electron microscopy to study the pancreatic main endocrine cell types in the embryos of the grass snake Natrix natrix L. with focus on the morphology of their secretory granules. The embryonic endocrine part of the pancreas in the grass snake contains four main types of cells (A, B, D, and PP), which is similar to other vertebrates. The B granules contained a moderately electron‐dense crystalline‐like core that was polygonal in shape and an electron‐dense outer zone. The A granules had a spherical electron‐dense eccentrically located core and a moderately electron‐dense outer zone. The D granules were filled with a moderately electron‐dense non‐homogeneous content. The PP granules had a spherical electron‐dense core with an electron translucent outer zone. Within the main types of granules (A, B, D, PP), different morphological subtypes were recognized that indicated their maturity, which may be related to the different content of these granules during the process of maturation. The sequence of pancreatic endocrine cell differentiation in grass snake embryos differs from that in many vertebrates. In the grass snake embryos, the B and D cells differentiated earlier than A and PP cells. The different sequence of endocrine cell differentiation in snakes and other vertebrates has been related to phylogenetic position and nutrition during early developmental stages.     

Morphology of the kidney of adult bowfin, Amia calva, with emphasis on "renal chloride cells" in the tubule

The nephron of adult bowfin, Amia calva, was described using light and electron microscopic techniques. The kidney of the bowfin possesses an abundant supply of renal corpuscles with each consisting of a glomerulus and a Bowman's capsule of visceral (podocyte) and parietal layers. No juxtaglomerular apparatus is present. The epithelium of the tubule is continuous with the parietal epithelium and is divisible in descending order into neck, first proximal, second proximal, first distal, second distal, and collecting segments. The tubules drain into a complex system of collecting ducts that ultimately unite with the main excretory duct, the archinephric duct. Mucous cells are the dominant cell throughout the entire ductular system. Nephrostomes are dispersed along the kidney capsule. The neck segment has a ciliated epithelium, and while both proximal segments possess a prominent brush border, the fine structure of the first implies involvement in protein absorption and the second in the transport and reabsorption of solutes. The cells of the first distal segment are characterized by deep infolding of the plasma membrane and a rich supply of mitochondria suggesting the presence of a mechanism for ion transport. The second distal segment is composed of cells resembling the chloride cells of fishes and these cells are present in progressively decreasing numbers in the collecting segment and duct system so that only a few are present in the epithelium of the archinephric duct. The "renal chloride cells" possess an abundant network of smooth tubules and numerous mitochondria with a rich supply of cristae. Glycogen is also a conspicuous component of these cells. The presence of "renal chloride cells" in this freshwater holostean, in other relatively primitive freshwater teleosts, and in larval and adult lampreys is discussed with reference to both phylogeny and the need for a special mechanism for renal ion conservation through absorption.     

Morphology and vascular anatomy of the gills of a primitive air-breathing fish,the bowfin (Amia calva)

Summary The morphology of the gills of a primitive air breather (Amia calva) was examined by light microscopy of semithin sections of gill filaments, and gill perfusion pathways were identified by scanning-electron microscopic analysis of corrosion replicas prepared by intravascular injection of methyl methacrylate. The arrangement of gill filaments and respiratory lamellae is similar to that of teleosts with the exception of an interfilamental support bar that is fused to the outer margins of lamellae on adjacent filaments. The prebranchial vasculature is also similar to that of teleosts, whereas the postbranchial circulation of arches III and IV is modified to permit selective perfusion of the air bladder. Gill filaments contain three distinct vascular systems: (1) the respiratory circulation which receives the entire cardiac output and perfuses the secondary lamellae; (2) a nutrient system that arises from the postlamellar circulation and perfuses filamental tissues; (3) a network of unknown function consisting of subepithelial sinusoids surrounding afferent and efferent margins of the filament and traversing the filament beneath the interlamellar epithelium. Prelamellar arteriovenous anastomoses (AVAs) are rare, postlamellar AVAs are common especially at the base of the filament where they form a dense network of small tortuous vessels before coalescing into a large filamental nutrient artery. Unlike in most teleosts, the outer vascular margins of the lamellae are embedded in the interfilamental support bar and become the sole vasculature of this tissue. Arterial-arterial lamellar bypass vessels were not observed. Previously observed decreases in oxygen transfer across the gills during air breathing can be explained only by redistribution of blood flow between or within the respiratory lamellae.Supported by NSF Grant No. PCM 79-23073The author wishes to thank Miss K. Drajus and D. Kullman for their excellent technical assistance and Dr. W. Gingerich, Mr. J. Crowther and D. Zurn for help in obtaining bowfin     

The endocrine pancreas in the adult gray short-tailed opossum, Monodelphis domestica (Marsupialia)

Monodelphis domestica, a South American marsupial proposed as a laboratory animal, presents some characteristics that are favourable for research on the development of pancreatic tissue. Therefore, it is important to know if Monodelphis domestica is similar to other mammals concerning the cellular composition of the pancreas and if the different regions of the organ share similar morphologic features. In the present study the pancreas of ten adult male or female Monodelphis domestica were examined: serial paraffin sections were stained immunohistochemically using polyclonal antibodies against insulin or glucagon. The size of the immunoreactive area was measured with a computer-based image-analysing system differentiating lobus dexter, corpus pancreatis, and lobus sinister.

The islets' profiles varied in size and shape. The insulin-positive cells were mainly located in the centre of the islets, while the glucagon-positive cells were arranged at the islets' periphery. However, some glucagon-positive cells also occurred in the centre of most islets. On average, 0.66% of the entire pancreatic section area was insulin-positive, while 0.23% was glucagon-positive. Thus, the ratio between glucagon-positive and insulin-positive area was 1:2.9. Differences between the three pancreatic parts, lobus dexter, corpus pancreatis, and lobus sinister, were revealed. In general, the findings compared well with those of species that have a history as laboratory animals, i.e. mice, rats, cats, and dogs.     

Immunocytochemical localisation of GABA in endocrine cells of the rat entero-pancreatic system

The occurence of GABA-containing cells in the rat entero-pancreatic system was investigated by using anti-GABA-glutaraldehyde antibodies at the light and electron microscope level. In the pancreas, the B cells showed intense immunoreactivity, contrary to non-B and exocrine cells. Moreover, post-embedding immunogold staining was localised mostly in mitochondria, close to rough endoplasmic reticulum and in the nucleus. The insulin granules appeared non-significantly stained, which suggests the lack of cosecretion of GABA together with insulin. In the duodenum, GABA immunoreactivity was detected in certain endocrine cell types, suggesting a possible interaction with this amino acid. The well established GABAergic innervation in the enteric system was also confirmed by immunolabelling.     

Intranuclear filamentous inclusions in the gastro-entero-pancreatic (GEP) endocrine cells of birds

Summary Intranuclear filamentous inclusions were found in the normal endocrine cells of the avian stomach and pancreas. These inclusions were composed of a bundle of closely packed filaments (6–8 nm in diameter), being ultrastructurally similar to those found in the nucleus of various neurons. Most of them appeared as single rod- or spindle-shaped bodies; aggregations of two or more inclusions were rarely seen within a single nucleus. Cells with an intranuclear inclusion often contained a cytoplasmic fibrillar bundle similar to the intranuclear inclusion.     

Effect of air breathing on acid-base and ion regulation after exhaustive exercise and during low pH exposure in the bowfin, Amia calva.

To explore a potential conflict between air breathing and acid-base regulation in the bowfin (Amia calva), we examined how individuals with access to air differed from fish without air access in their response to acidosis. After exhaustive exercise, bowfin with access to air recovered significantly more slowly from the acidosis than fish without air access. While arterial blood pH (pH(a)) of fish without air access recovered to resting levels by 8 h, pH(a) was still significantly depressed in fish having access to air. In addition, Pco(2) was slightly more elevated in fish having air access than those without it. Fish with access to air still had a significant metabolic acid load after 8-h recovery, while those without air access completely cleared the load within 4 h. These results suggest that bowfin with access to air were breathing air and, consequently, were less able to excrete CO(2) and H(+) and experienced a delayed recovery. In contrast, during exposure to low pH, air breathing seemed to have a protective effect on acid-base status in bowfin. During exposure to low pH water, bowfin with access to air developed a much milder acidosis than bowfin without air access. The more severe acidosis in fish without air access was caused by an increased rate of lactic acid production. It appears that enhanced O(2) delivery allowed air-breathing bowfin to avoid acidosis-induced anaerobic metabolism and lactic acid production. In addition, during low pH exposure, plasma Na(+) and Cl(-) concentrations of fish without air access fell slightly more rapidly than those in fish with air access, indicating that the branchial ventilatory changes associated with air breathing limited, to some degree, ion losses associated with low pH exposure.     

Culture of endocrine pancreatic cells in protein-free,chemically defined media

Summary Cell suspensions prepared by collagenase digestion of pancreata obtained from 21.5-d-old rat fetuses were preincubated in RPMI medium containing 10% fetal bovine serum (FBS), to ensure cell adhesion. Twenty hours later, this medium was replaced by a chemically defined medium. Dulbecco's modified Eagle's (DME)-F12 was used alone or supplemented with various combinations of transferrin, sodium selenite, or Ultroser G. The evolution of the culture and the islet ultrastructure were similar in defined and serum-containing media. However, in the defined medium, the neoformed islets seemed less numerous, and the fibroblast layer less dense, when compared to the RPMI+10% FBS control medium. At Day 7, in defined media, the total insulin content per dish was half that of control cultures. None of the tested additives improved the yield of the cultures. The fractional insulin release per day was elevated in defined media. In subsequent incubations, glucose and leucine stimulated insulin release in a way characteristic of these cells of fetal origin. The labeling index of islet cells cultured in DME-F12 reached 10.7%, which is not far from that observed in RPMI+10% FBS. Such a defined medium is useful to study B cell physiology, avoiding the possible interaction of serum components with substances to be tested. The support of the Fonds National de la Recherche Scientifique and the ‘Loterie Nationale’ of Belgium is also acknowledged.     

Calbindin-D28K and the peptidergic neuroendocrine system in rat gut: an immunohistochemical study

Calbindin-D28K was immunohistochemically localized in myenteric and submucosal plexuses throughout the rat intestine. Calbindin-D28K immunoreactivity was found in about half of myenteric neurons and in more than 90% of submucosal neurons. Calbindin-D28K was also observed in nerve processes running inside ganglia, muscle layers and lamina propria. No correlation could be established between the presence of calbindin-D28K and the distribution of neuropeptides localized in this study (VIP, enkephalin, somatostatin and substance P). In addition, some endocrine-like cells of the ileum were calbindin-D28K-positive. Half of these endocrine cells also contained neurotensin but none of the other neuropeptides investigated.     

Interstitial Cajal-like cells in rat mesentery: an ultrastructural and immunohistochemical approach

Interstitial Cajal-like Cells (ICLC) were recently recognized in a plethora of non-digestive organs. Here, we describe a cell type of rat mesentery sharing ultrastructural and immunohistochemical features with ICLC. Mesenteric ICLC were demonstrated by transmission electron microscopy (TEM) and further tested by light microscope immunohistochemistry. The cell described here fulfils the TEM diagnostic criteria accepted for ICLC: location in the connective interstitium; close vicinity to nerves, capillaries and other interstitial cells; characteristic long, moniliform cell processes; specialized cell-to-cell junctions; caveolae; mitochondria at 5-10% of cytoplasmic volume; rough endoplasmic reticulum at about 1-2%; intermediate and thin filaments, microtubules; undetectable thick filaments. The processes of this mesenteric ICLC were particularly long, with a mean length of 24.91 microm (10.27-50.83 micorm), and a convolution index of 2.32 (1.37-3.63) was calculated in order to measure their potential length. Mean distances versus main target cells of ICLC-nerve bundles, vessels, adipocytes and macrophages-were 110.69, 115.80, 205.07 and 34.65 nm, respectively. We also tested the expression of CD117/c-kit, CD34, vimentin, alpha-smooth muscle actin, nestin, NK-1, tryptase and chymase and the antigenic profile of the mesenteric ICLC was comparable if not identical with that recently observed in ICLC from other extra-digestive tissues. Due to the peculiar aspect of the mesenteric ICLC processes it can be hypothesized that these cells form a three-dimensional network within the mesentery that is at the same time resistant and deformable following stretches consequent to intestine movements, mainly avoiding blood vessels closure or controlling blood vessels rheology. It remains, however, to be established if and how such cells are connected with the archetypal enteric ICC.     

Morphocytochemical, immunohistochemical and ultrastructural characterization of the head kidney of fat snook Centropomus parallelus

This study characterized the structure and the morphocytochemical, immunohistochemical and ultrastructural aspects of the head kidney (HK) of the fat snook Centropomus parallelus. The HK is enclosed by a thin capsule of connective tissue, from which fine trabeculae originate and branch into the interior of organ. In the parenchyma, there are aggregates of lymphoid cells containing populations of lymphocytes T immunopositive for CDRO45, in a nodular arrangement, around blood vessels and melano-macrophage centres. Among the cells that constituted these aggregates and surrounded them, were macrophages and monocytes, and their precursors, with strong immunopositivity for CD68, along with cells of the granulocytic lineage in various phases of maturation positive for lysozyme and PAS. Macrophages and chromaffin and interrenal cells are also present. Ultrastructurally, the HK comprises a reticulum-endothelial stroma consisting of endothelial cells, reticulocytes of the fibroblast type and macrophage type and a parenchyma with increased cellularity, principally blood cells of the erythrocytic, granulocytic, lymphocytic, monocytic and thrombocytic series.     

The amylase-secreting cells of the stomach of the scallop, Pecten maximus: ultrastructural, immunohistochemical and immunocytochemical characterizations

(1) alpha-amylase was extracted and purified from the stomach/digestive gland complex of the scallop Pecten maximus and an anti-serum was induced against the purified amylase by rabbit immunization. (2) The anti scallop amylase was used to localize the amylase-secreting cells in the stomach of Pecten maximus by immunofluorescence and immunogold labelling. The amylase-secreting cells are glandular cells particularly numerous in the main sorting area of the stomach. Their secretory granules were found strongly positive for anti-amylase. Three types of glandular cells were observed, actually corresponding to the three stages of the glandular-cell activity, synthesis, secretion and excretion. (3) The synthesizing cell shows the characteristic features of a protein-synthesizing cell: a conspicuous nucleolus and abundant granular endoplasmic reticulum. In the secretory cell, the secretory granules are formed by the Golgi apparatus and accumulate in the apical part of the cell. The secretory cell is filled with two types of secretory granules which are released in the stomach lumen by apocrine excretion. (4) The present study brings the first demonstration of the synthesis and extracellular release of amylase by glandular cells of the stomach epithelium of a bivalve.     

Immunohistochemical localization of huntingtin-associated protein 1 in endocrine system of the rat.

Huntingtin-associated protein 1 (HAP1) was originally found to be localized in neurons and is thought to play an important role in neuronal vesicular trafficking and/or organelle transport. Based on functional similarity between neuron and endocrine cell in vesicular trafficking, we examined the expression and localization of HAP1 in the rat endocrine system using immunohistochemistry. HAP1-immunoreactive cells are widely distributed in the anterior lobe of the pituitary, scattered in the wall of the thyroid follicles, or clustered in the interfollicular space of the thyroid gland, exclusively but diffusely distributed in the medullae of adrenal glands, and selectively located in the pancreas islets. HAP1-containing cells were also found in the mucosa of stomach and small intestine with a distributive pattern similar to that of gastrointestinal endocrine cells. However, no HAP1-immunoreactive cell was found in the cortex of the adrenal gland, the testis, and the ovary. In the posterior lobe of the pituitary, HAP1-immunoreactive products were not detected in the cell bodies but in many stigmoid bodies, one kind of non-membrane-bound cytoplasmic organelle with a central or eccentric electron-lucent core. HAP1-immunoreactive stigmoid bodies were also found in the cytoplasm of endocrine cells in the thyroid gland, the medullae of adrenal gland, the pancreas islets, the stomach, and small intestine. The present study demonstrates that HAP1 is selectively expressed in part of the small peptide-, protein-, and amino-acid analog and derivative-secreting endocrine cells but not in steroid hormone-secreting cells, suggesting that HAP1 is also involved in intracellular trafficking in certain types of endocrine cells.     

The nervous system of the chicken proventriculus: an immunocytochemical and ultrastructural study

The proventriculus constitutes the glandular region of the chicken stomach. This organ is innervated by two parasympathetic networks, the myenteric and submucous plexus, and here we present a systematic study of this system by immunohistochemistry and electron microscopy. All the neurons and fibres were positive for the neural markers, protein gene product 9.5 and the amidating enzymes. Immunoreactivities for the constitutive neuronal isoform of the enzyme nitric oxide synthase and the vasoactive intestinal peptide were present in neuronal bodies suggesting an intrinsic origin for the similarly immunoreactive fibres found in the proventriculus. On the other hand, immunoreactivity to gastric inhibitory peptide was only found in varicose fibres making contact with the blood vessels and the glandular epithelium, but never in the neuronal somas, suggesting that this substance may be provided by an extrinsic nervous system whose neuronal bodies are located elsewhere. Electron microscopy revealed frequent neuromuscular and neuroepithelial connections in the muscle layers, the wall of the blood vessels and the epithelium. In addition, synapsis-like structures were identified in the proximity of cells belonging to the diffuse endocrine system, providing a new example of neuroendocrine contacts. No positivity was found for antibodies against other neural substances including somatostatin, peptide histidine–isoleucine, peptide tyrosine–tyrosine, neuropeptide tyrosine, bombesin, met-enkephalin, serotonin, substance P, galanin, calcitonin gene-related peptide and S-100 protein.     

Vasoactive intestinal polypeptidergic innervation and gastroenteropancreatic system: an immunocytochemical study in the hedgehog Erinaceus europaeus.

The pattern of the digestive vasoactive intestinal polypeptide (VIP)-ergic innervation is described immunohistochemically in the hedgehog Erinaceus europaeus. This animal is a small-sized, wild, nocturnal, lower eutherian mammal whose gastrointestinal tract shows some similarities with the avian gut. The myenteric plexus of the stomach, the mucosa of the small intestine and the circular muscle layer of the large intestine are the best VIP-innervated structures. The pattern of the positive innervation is similar to that described in other mammals and in some bird species. The widespread diffusion of the neuropeptide in the gut is probably due to the importance of its functions in the digestive physiology.     

Three-dimensional analysis demonstrates the presence of exocrine cytoplasmic vela between endocrine cells and basal lamina in the stomach of mammals

Three-dimensional analysis demonstrated the presence of cytoplasmic vela extending from exocrine cells into the space between endocrine cells and basal lamina in the gastrointestinal epithelium of the rabbit; these structures were also observed in various other mammals. The following techniques were used to determine the morphologic characteristics of these vela and to study their significance: preparation of semiserial thin sections, three-dimensional reconstruction in plexiglass and lanthanum staining of pericellular spaces. It was found that these fine vela, devoid of major differentiated cell-constituents, sometimes form a pseudocircular crown at the base of endocrine cells. If the zone of basal apposition of the plasma membrane is referred to as ZBA and the zones of lateral apposition as ZLA, the presence of this velum makes it possible to distinguish a zone of immediate apposition without interposition (ZIA) and a mediate zone of apposition with interposition (ZMA) within the ZBA. Exocrine cell processes can also penetrate within endocrine cells in invaginations, and the depth of these invaginations can be demonstrated by lanthanum staining. Adjacent to the membrane zones defined above, other cytoplasmic microdomains-M(ZLA) and M(ZBA), as well as M(ZIA) and M(ZMA) of different morphofunctional significance may also be envisaged.     

Endocrine cells in atresic chick embryo intestine: histochemical and immunohistochemical study

Intestinal motility disorders are an important problem in the postoperative management of patients with intestinal atresia. Intestinal motility could be initiated by luminal factors that activate intrinsic and extrinsic primary afferent nerves involved in the peristaltic reflex. Endocrine cells act as a key point, because they transfer information regarding the intestinal contents and intraluminal pressure to nerve fibers lying in close proximity to the basolateral surface of the epithelium. In chick embryo, experimental intestinal atresia is associated with disorders in the development of the enteric nervous system, related to the severity of intestinal dilation. Our aim was to investigate the distribution pattern of endocrine cells in the developing endocrine system of chick embryo small intestine with experimentally-induced atresia on day 12 and on day 16. Changes in enteroendocrine population were examined in gut specimens (excised proximal and distal to the atresia) from experimental embryos 19 days old and in control sham-operated chick embryos at the same age. Sections from proximal and distal bowel and control bowel were stained with Grimelius silver stain, a valuable histochemical method for detecting the argyrophil and argentophilic cells, and with an immunohistochemical procedure for detecting serotonin and neurotensin immunoreactive cells. In chick embryo proximal bowel, intestinal dilation differed in the various embryos. We found significantly higher enteroendocrine cell counts in proximal bowel than in distal and control bowel. The differences depended on the precociousness of surgery and the severity of dilation. Considering the major contribution of enteroendocrine cells to the peristaltic reflex, our data may help to explain the pathogenesis of motility disorders related to intestinal atresia.     

Previtellogenic development and vitellogenin synthesis in the fat body of a mosquito: an ultrastructural and immunocytochemical study

We describe two phases, previtellogenic and vitellogenic, in the activity of the trophocytes in the fat body of the mosquito Aedes aegypti. The previtellogenic phase, leading to trophocyte competence to synthesize vitellogenin (Vg), occurred during the first 3 days after eclosion. This phase was characterized by enlargement and activation of the nucleoli, proliferation of ribosomes and rough endoplasmic reticulum (RER), development of Golgi complexes, and extensive invaginations of the plasma membrane. During the vitellogenic phase, initiated by a blood meal, Vg was first detected, by immunofluorescence, 1 hr after feeding. The intensity of the immunoreaction increased for the next 24 hr, was declining at 30 hr, and had disappeared by 48 hr. Vg synthesis was characterized ultrastructurally by the enlargement of the RER and the formation of dense secretion granules in Golgi complexes. These secretion granules were two to three times larger at the peak of Vg synthesis than at the beginning. The granules discharged their contents by exocytosis. Two electron microscopical immunocytochemical methods, immunoferritin and peroxidase-antiperoxidase, confirmed this pathway of Vg processing. For the first 12 hr after feeding. Vg synthetic organelles proliferated and the active nucleoli were multilobed; thereafter, while Vg synthesis continued, the nucleoli began to regress into compact bodies. Termination of Vg synthesis was marked by autophagical degradation of Vg synthetic and processing organelles.