Cytodifferentiation in the accessory glands of Tenebrio molitor. III. Fine structure of the spermathecal accessory gland in the pupa

To establish indices for studying the hormonal control of differentiation of the accessory reproductive glands of insects, the ultrastructural development of the spermathecal accessory gland (SAG) of female mealworm beetles has been analyzed. Over the 9 days between adult and pupal ecdysis, the SAG transforms from a stubby sac of columnar epithelium into an elongate cylindrical gland, lined with cuticle, and containing several distinct types of differentiated cells. The first phase of pupal differentiation is one of cell division and overall gland morphogenesis which lasts 3--4 days; at its close, two populations of cells can be distinguished. One of these populations will produce the cuticular ductules while the other will yield the three-cell secretory units or organules. In the second phase which lasts 2 days, the three cells of each organule become wrapped around one another and then the innermost puts out a pseudocilium and retracts within the next ensheathing cell. In the third phase which lasts 4 days, the cuticles of the axial duct, of the efferent ductule, of the vestibule upon which the ductules converge, and of the end apparatus, are deposited. The ciliary process degenerates, and after ecdysis, the secretory cells undergo peak differentiation.     

The hairpencils of the flour moth Ephestia kuehniella

The hairpencils of the Mediterranean flour moth, Ephestia kuehniella Zeller, are tufts of modified scales between the seventh and eighth abdominal segments of the adult males. The fine structure and development of a hairpencil organule is described. A process of the trichogen cell secretes the modified scale and then withdraws, and the cell invaginates forming a microvillous lumen into which pheromone is probably secreted. This lumen communicates with the outside via rows of pores in the hollow scale. The opening of these pores when the moth emerges from the pupal cuticle may be a result of the drying out of the membrane that covered them. The socket of the hairpencil scale is secreted by a tormogen cell.
The hairpencils of male Lepidoptera differ in position from family to family, but the organules that compose them seem to share a common structure. Epidermal glands and organules are classified in a scheme that takes account of their mode of development.     

Further studies on the development of the of Drosophila melanogaster. II. The interommatidial bristles

An electron microscope examination of the minute bristle organules in the Drosophila eye revealed an organisation characteristic of insect hair sensilla. They were derived from four concentrically arranged cells which were active at the mid-pupal stage in producing the bristle, socket and receptor structures. Two of the cells degenerated towards the end of pupation, the mature organule consisting of a bipolar sense cell and an accessory cell. Growth in length of the bristle was accompanied by a proliferation of longitudinally oriented microtubules which gradually disappeared after maximum growth had been achieved. The breakdown of microfibrillar aggregates, which were also present as transient structures in the developing bristle and showed some correspondence with the longitudinal ridges formed at the surface, may similarly be related to the establishment of cell shape by the deposition of the cuticle which occurred at the same time.     

Morphology of the aedeagal gland of the male mealworm beetle (Tenebrio molitor L.)

The aedeagal gland of male Tenebrio molitor consists of numerous acini containing several secretory units (organules) of three epithelial cells in series. The distal cortical cell and intermediate cell are secretory cells. Secretory products are passed into microvilli-lined extracellular reservoirs. From these storage areas products flow through minute canaliculi and into the efferent ductule. Canaliculi, cuticular trabeculae, and fibrillar material are characteristic features of the efferent ductules within the extracellular reservoirs of secretory cells. After passing from the secretory cells, the efferent ductule penetrates the basal ductule cell. The thin epicuticle that comprises the wall of the ductule is confluent with the epicuticle of the cuticular sheath forming the wall of the genital pocket. Secretory products flow from the cortical cell ductule into the intermediate cell and eventually empty into the genital pocket. A chemical reaction apparently takes place in the intermediate cell ductule, resulting in a frothy secretion product. When released from the ductule, this frothy product forms a foam-like layer that coats the inner wall of the genital pocket. Ultrastructural and probable functional aspects of this gland are described and discussed.     

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 fine structure of the terminal sensilla on the maxillary palps of Schistocerca gregaria (Forskål) (Orthoptera,Acrididae)

Summary The fine structure of the terminal sensilla on the maxillary palps of Schistocerca gregaria has been investigated. Most organules include six neurons with dendrites extending to the tip of the cuticular peg, the opening of which is controlled so that the dendrites are not always exposed. The neurons are isolated from each other by a neurilemma cell and two other glial cells, while typical epidermal cells containing dense bundles of microtubules support the whole group of cells. At the poles of the neurons are specialised areas in which the cytoplasm is differentiated from that elsewhere. It contains a large number of mitochondria and small helical structures, while close to it are characteristic spheres of membranes, termed onion bodies, in various stages of development.It is suggested that the fluid bathing the distal parts of the dendrites and exuding from the tip of the peg has a number of specialised functions. It is probably concerned in forcing open the tip of the peg by hydrostatic pressure, it prevents the exposed tips of the dendrites from desiccating and it acts as a transmitter in which chemicals on the surfaces touched by the sensillum must dissolve before reaching the dendrites. This fluid may be produced by the neurilemma cell or by the neurons themselves. Closure of the pegs does not seem to produce any material reduction in the overall loss of water by the insect.Each neuron sends an axon to the brain; there is no peripheral fusion of axons. Possibly one neuron has a mechanoreceptor function, although no specialised terminal at the base of the peg has been observed. The concentration of mitochondria at either end of the neuron may be concerned in the production of action potentials, while the cavity of the peg and tormogen cell perhaps has a role in the conduction of the receptor potential to the perikaryon. Intercellular connections are such as to give mechanical stability to the cells of the organule and permit transport between the cells. Extracellular tubules extending from the wall of the peg into the cell complex may serve to anchor the peg during the moulting process.We are grateful to the Royal Society for a grant for the purchase of an ultramicrotome, to the Anti-Locust Research Centre for supplying the locusts, and to various colleagues for assistance and advice. We are also indebted to the Cambridge Instrument Company Ltd. for permission to publish Figs. 2 and 3.     

T lymphocytic subpopulations of normal human peripheral blood defined by monoclonal antibodies: an ultrastructural study using immunogold staining method

Submicroscopic findings of T lymphocytic subpopulations of normal human peripheral blood defined by OKT monoclonal antibodies were studied using ultrastructural immunocytochemistry (immunogold staining). The results show that OKT4-and OKT8-positive lymphocytic subpopulations have a distinct morphological pattern, although some variations in the ultrastructural details of cells in each subset are evident. The most representative features of OKT8-positive cells are regular or slightly indented nucleus with condensed chromatin, abundant cytoplasm, prominent Golgi apparatus, several granules frequently localized in the Golgi area, and numerous mitochondria, often in cluster disposition. OKT4-positive cells show generally less condensed chromatin, scanty cytoplasm and poor organule pattern. Furthermore, positive lymphocytes with prominent nuclear irregularities are found in both lymphocytic subpopulations. Ultrastructural similarities between the T cell subsets phenotypically characterized by monoclonal antibodies and other immunological criteria are also discussed.     

Origin and early development of female germ cells in Eoleptestheria ticinensis Balsamo-Crivelli, 1859 (Crustacea, Branchiopoda, Conchostraca)

We have studied the early stages of the development of the female germ cells in the Conchostraca Eoleptestheria ticinensis Balsamo-Crivelli, 1859. The gametes originate in a scattered way throughout the tubular units of the gonad, with no development gradient recognizable. The female germ cells arise from successive karyokineses not followed by cytokinesis, within an unorganised area in which a sort of plasmodium is formed. Each primordial nucleus of this germarium develops and then forms an individual plasmic membrane. Subsequently, the usual organules differentiate in the cytoplasm. The presence of synaptinemal complexes and the beginning of the endogenous vitellogenesis by the rough endoplasmic reticulum and the Golgi apparatus qualify the previtellogenesis. The general characteristics of this early development phase of the gametes, as well as several substantial differences in the gametogenesis with respect to the other Phyllopoda studied, lead us to suggest the systematic positioning of the Leptestheriidae.     

Morphology and ultrastructure of a secretory region enclosed by the venom reservoir in social wasps (Insecta,Hymenoptera)

The morphology and ultrastructure of the convoluted gland inside the venom reservoir of four species of social Vespidae are described. The cells of the venom gland (including the convoluted gland) can be divided into six groups: (1) epithelial cells, (2) glandular cells with the end apparatus secreting into the tubule inside the convoluted gland (internal or embedded tubule), (3) a continuous arrangement of glandular cells with the end apparatus secreting directly into the venom reservoir, (4) glandular cells that are loosely dispersed along the tubule lumen between the free tubules and the embedded tubule of the convoluted gland, (5) secretory cells of the free tubules and (6) duct cells. One kind of secretory cell, hitherto unknown and described in this paper (group 3), is characterized by the presence of a well-developed end apparatus, usually with enlarged extracellular spaces, but lacking the normally associated duct cells. The secretory cells contain several stacks of granular endoplasmic reticulum, but these are mainly concentrated in the middle of the cell. The basal half of the cells contains many lipid droplets. Although the function of the convoluted gland is not yet understood, an hypothesis is related to what is known of the function of reservoir secretory cells in solitary wasps. All wasp species studied showed the same organization of the convoluted gland, which clearly distinguishes their venom gland from that of Sphecidae.     

Anatomical correlates of venom production in Conus californicus

Like all members of the genus, Conus californicus has a specialized venom apparatus, including a modified radular tooth, with which it injects paralyzing venom into its prey. In this paper the venom duct and its connection to the pharynx, along with the radular sac and teeth, were examined using light and transmission electron microscopy. The general anatomy of the venom apparatus resembles that in other members of the genus, but several features are described that have not been previously reported for other species. The proximal (posterior) quarter of the venom duct is composed of a complex epithelium that may be specialized for active transport rather than secretion. The distal portion of the duct is composed of a different type of epithelium, suggestive of holocrine secretion, and the cells display prominent intracellular granules of at least two types. Similar granules fill the lumen of the duct. The passageway between the lumen of the venom duct and pharynx is a flattened branching channel that narrows to a width of 10 micro m and is lined by a unique cell type of unknown function. Granular material similar to that in the venom duct was also found in the lumen of individual teeth within the radular sac. Mass spectrometry (MALDI-TOF) demonstrated the presence of putative peptides in material derived from the tooth lumen, and all of the more prominent species were also evident in the anterior venom duct. Radular teeth thus appear to be loaded with peptide toxins while they are still in the radular sac.     

Fine Structure of Female Accessory Reproductive Gland in the Mealworm Beetle, Tenebrio molitor

ABSTRACT The fine structure of female accessory reproductive gland (FARG) of the adult mealworm beetle, Tenebrio molitor is studied with light and electron microscopes. The FARG is a simple tubular organ that composed of two kinds of cells-secretory epithelial cells and duct forming cells. The lumen of FARG is lined with a thin cuticle and filled with secretory materials. Each secretory epithelial cell has its peculiar end apparatus in addition to well-developed rough endoplasmic reticulum (rER), mitochondria, and secretory vesicles. They are forming basal infolding along the plasma membrane. Along the inner surface of the plasma membrane, numerous secretory vesicles are seen. The glandular secretions of the epithelial secretory cells are synthesized via rER to Golgi apparatus, and are stored in the extracellular cavity in the epithelial cell. These secretions are drained to the lumen through the end apparatus and this type of glandular secretion in the insects is type III. Histochemical reactions reveal the major component of these glandular secretions is an acid mucopolysaccharide.     

SULFATE METABOLISM IN PANCREATIC ACINAR CELLS

The metabolism of inorganic sulfate in pancreatic acinar cells was studied by electron microscope radioautography in mice injected with sulfate-³⁵S. Labeled sulfate was concentrated in the Golgi complex at 10 min. Within 30 min, much of the radioactive material had been transferred to condensing vacuoles. These were subsequently transformed into zymogen granules. By 4 hr after injection, some of the zymogen granules with radioactive contents were undergoing secretion, and labeled material was present in the pancreatic duct system. The Golgi complex in pancreatic acinar cells is known to be responsible for concentrating and packaging digestive enzymes delivered to it from the endoplasmic reticulum. Our work demonstrates that the Golgi complex in these cells is also engaged in the manufacture of sulfated materials, probably sulfated mucopolysaccharides, which are packaged along with the enzymes in zymogen granules and released with them into the pancreatic secretion.     

Sulfated compounds in the zymogen granules of the guinea pig pancreas

Sulfate incorporation into the guinea pig pancreas was investigated by light (LM) and electron microscope (EM) autoradiography using a system of minilobules incubated in vitro for 60 min in Krebs-Ringer bicarbonate medium (KRB) containing 35SO4(-2). In acinar cells, examined by EM autoradiography, the label was found concentrated over Golgi elements (including condensing vacuoles) and zymogen granules. 35SO4(-2) was also incorporated by the epithelial cells of the entire pancreatic duct system, the incorporation being surprisingly high in the epithelium of the major ducts. In all ductal epithelia, autoradiographic grains appeared over the Golgi complex and the plasmalemma. Since a contribution of duct epithelium to the sulfated compounds found in the discharged secretion could not be ruled out, a purified zymogen granule fraction was used as a source material for the isolation of sulfated compounds of acinar origin. The presence of 35S- radioactivity in the zymogen granules and condensing vacuoles of this fraction was ascertained by autoradiography (of sectioned pellets). From a lysate of this zymogen granule fraction, a soluble sulfated compound of low isoelectric point and high molecular weight was isolated by gel filtration under conditions that allowed its satisfactory separation from the bulk of the secretory proteins.     

Effects of pancreatic duct ligation on pancreatic response to bombesin

To examine mechanisms that might be related to biliary pancreatitis, we examined the effects of pancreatic duct ligation (PDL) with pancreatic stimulation in vivo. PDL alone caused no increase in pancreatic levels of trypsinogen activation peptide (TAP), trypsin, or chymotrypsin and did not initiate pancreatitis. Although bombesin caused zymogen activation within the pancreas, the increases were slight and it did not cause pancreatitis. However, the combination of PDL with bombesin resulted in prominent increases in pancreatic TAP, trypsin, chymotrypsin, and the appearance of TAP in acinar cells and caused pancreatitis. Disruption of the apical actin network in the acinar cell was observed when PDL was combined with bombesin but not with PDL or bombesin alone. These studies suggest that when PDL is combined with pancreatic acinar cell stimulation, it can promote zymogen activation, the retention of active enzymes in acinar cells, and the development of acute pancreatitis.     

Quantitation of ultrastructural changes in mouse pancreatic acinar cells caused by foreign serum

Quantitative changes in the pancreatic acinar cell organelles were studied in BALB/c mice injected with 1.0 ml fresh rabbit serum intraperitoneally. Groups of 5 mice were killed at 0, 1, 3, 6 and 12 h after the serum injection. Pancreatic tissue was processed for electron microscopy by glutaraldehyde and osmium tetroxide fixation and Epon embedding. The proportions of acinar cell cytoplasm (volume fractions) occupied by zymogen granules, granular endoplasmic reticulum, Golgi apparatus, mitochondria and lysosomes (including autophagosomes) were determined by the point counting method from electron micrographs. The volume fraction of lysosomes increased during the first 3 h and remained markedly elevated up to 12 h. The volume fractions of zymogen granules increased from 12 to 28% in 12 h. It was concluded that the secretory mechanism of pancreatic acinar cells was injured by the foreign serum. The injury caused accumulation of zymogen granules and increased autophagic activity in the acinar cells.     

Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical cell surface and ATP-dependent and flow-induced calcium signals

Renal epithelial cells release ATP constitutively under basal conditions and release higher quantities of purine nucleotide in response to stimuli. ATP filtered at the glomerulus, secreted by epithelial cells along the nephron, and released serosally by macula densa cells for feedback signaling to afferent arterioles within the glomerulus has important physiological signaling roles within kidneys. In autosomal recessive polycystic kidney disease (ARPKD) mice and humans, collecting duct epithelial cells lack an apical central cilium or express dysfunctional proteins within that monocilium. Collecting duct principal cells derived from an Oak Ridge polycystic kidney (orpk ( Tg737 ) ) mouse model of ARPKD lack a well-formed apical central cilium, thought to be a sensory organelle. We compared these cells grown as polarized cell monolayers on permeable supports to the same cells where the apical monocilium was genetically rescued with the wild-type Tg737 gene that encodes Polaris, a protein essential to cilia formation. Constitutive ATP release under basal conditions was low and not different in mutant versus rescued monolayers. However, genetically rescued principal cell monolayers released ATP three- to fivefold more robustly in response to ionomycin. Principal cell monolayers with fully formed apical monocilia responded three- to fivefold greater to hypotonicity than mutant monolayers lacking monocilia. In support of the idea that monocilia are sensory organelles, intentionally harsh pipetting of medium directly onto the center of the monolayer induced ATP release in genetically rescued monolayers that possessed apical monocilia. Mechanical stimulation was much less effective, however, on mutant orpk collecting duct principal cell monolayers that lacked apical central monocilia. Our data also show that an increase in cytosolic free Ca(2+) primes the ATP pool that is released in response to mechanical stimuli. It also appears that hypotonic cell swelling and mechanical pipetting stimuli trigger release of a common ATP pool. Cilium-competent monolayers responded to flow with an increase in cell Ca(2+) derived from both extracellular and intracellular stores. This flow-induced Ca(2+) signal was less robust in cilium-deficient monolayers. Flow-induced Ca(2+) signals in both preparations were attenuated by extracellular gadolinium and by extracellular apyrase, an ATPase/ADPase. Taken together, these data suggest that apical monocilia are sensory organelles and that their presence in the apical membrane facilitates the formation of a mature ATP secretion apparatus responsive to chemical, osmotic, and mechanical stimuli. The cilium and autocrine ATP signaling appear to work in concert to control cell Ca(2+). Loss of a cilium-dedicated autocrine purinergic signaling system may be a critical underlying etiology for ARPKD and may lead to disinhibition and/or upregulation of multiple sodium (Na(+)) absorptive mechanisms and a resultant severe hypertensive phenotype in ARPKD and, possibly, other diseases.     

Secretory apparatus assessed by analysis of pancreatic secretory stress protein expression in a rat model of chronic pancreatitis

Secretory stress proteins (SSP) are a family of proteins including isoforms of pancreatitis-associated protein (PAP) and pancreatic stone protein (PSP/reg). In vitro exposure to trypsin results in the formation of insoluble fibrillar structures. SSP are constitutively secreted into pancreatic juice at low levels. The WBN/Kob rat is a model for chronic pancreatitis, displaying focal inflammation, destruction of the parenchyma and changes in the architecture of the acinar cell; the synthesis and secretion of SSP are also increased. We have investigated the secretory apparatus by SSP immunohistochemistry at the light- and electron-microscopical (EM) levels. Immunocytochemistry of PSP/reg in Wistar control rats reveals low levels, with individual acinar cells exhibiting high immunoreactivity in zymogen granules. PAP is not detectable. In the WBN/Kob rat, PSP/reg and PAP immunoreactivity is markedly increased. Double immunofluorescence for PSP/reg and PAP I or II demonstrates that these proteins colocalize to the same cell. Acinar cells change their secretory architecture by fusion of zymogen granules and elongation of the fused organelles. The immunogold technique has demonstrated an increase of SSP in zymogen granules in WBN/Kob rats. PSP/reg-positive zymogen granules fuse to form elongated structures with fibrillar contents. An extensive PSP/reg-positive fibrillar network is established in the cytosol. Extracellular fibrils have been observed in several ductules. Thus, SSP-derived fibrils form concomitantly with acinar damage in the WBN/Kob rat. Based on the known tryptic cleavage site of SSP, the in vivo generation of fibrils is presumably the result of premature trypsin activation.     

Cytoplasmic dynein and dynactin as likely candidates for microtubule-dependent apical targeting of pancreatic zymogen granules.

The critical role of microtubules in vectorial delivery of post-Golgi carrier vesicles to the apical cell surface has been established for various polarized epithelial cell types. In the present study we used secretory granules of the rat and chicken pancreas, termed zymogen granules, as model system for apically bound post-Golgi carrier vesicles that underlie the regulated exocytotic pathway. We found that targeting of zymogen granules to the apical cell surface requires an intact microtubule system which contains its colchicine-resistant organizing center and, thus, the microtubular minus ends close to the apical membrane domain. Purified zymogen granules and their membranes were found to be associated with cytoplasmic dynein intermediate and heavy chain and to contain the major components of the dynein activator complex, dynactin, i.e. p150Glued, p62, p50, Arp1, and beta-actin. Kinesin heavy chain and the kinesin receptor, 160 kD kinectin, were not detected as components of zymogen granules. Immunofluorescence staining showed a zymogen granule-like distribution for dynein and dynactin (p150Glued, p62, p50, Arpl) in the apical cytoplasm, whereas kinesin and kinectin were largely concentrated in the basal half of the cells in a pattern similar to the distribution of calreticulin, a component of the endoplasmic reticulum. Secretory granules of non-polarized chromaffin cells of the bovine adrenal medulla, that are assumed to underlie microtubular plus end targeting from the Golgi apparatus to the cell periphery, were not found to be associated with dynein or dynactin. To our knowledge, this is the first demonstration of major components of the dynein-dynactin complex associated with the membrane of a biochemically and functionally well-defined organelle which is considered to underlie a vectorial minus end-driven microtubular transport critically involved in precise delivery of digestive enzymes to the apically located acinar lumen.     

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.