Rearrangement of the tubulin and actin cytoskeleton during programmed cell death in Drosophila salivary glands

During larva-to-pupa metamorphosis Drosophila salivary glands undergo programmed cell death by autophagocytosis. Although ultrastructure of Drosophila salivary glands has been extensively studied in the past, little is known about mechanism of programmed cell death, especially the role of the cytoskeleton. In this paper we describe changes in microtubule and actin filament network compared to the progress of DNA fragmentation and redistribution of acid phosphatase. In feeding and wandering larvae microtubules and actin filaments form regular networks localized mostly along the plasma membrane. The first major rearrangement of microtubules and actin filaments occurred when larvae everted spiracles and the glands shifted their secretion from saliva to mucoprotein glue (stage L1). Microtubule cytoskeleton became denser and actin filaments concentrated along cell boundaries. At the same time nuclei flattened and migrated into the microtubule-rich layer near the basal membrane. In late prepupae (8-10 h after P1) the microtubule network became fainter, and actin filaments appeared frequently deeper in cytoplasm, gradually concentrating around nuclei. Simultaneously large patches of acid phosphatase activity surrounded nuclei and shortly thereafter chromosomal DNA began to fragment. During the final collapse of the gland (early pupae, 13.5 h after formation of white puparium) cellular fragments and autophagic vacuoles contained a continuous F-actin lining and the microtubule network displayed signs of extensive degradation. The results are consistent with the hypothesis that, in Drosophila salivary glands, extensive autophagic activities target nuclei for degradation; that this process occurs late in the course of programmed cell death; and that it directly involves cytoskeletal structures which are altered far earlier during the course of cell death.     

Salivary gland function and chromosomal puffing patterns in Drosophila hydei

Summary The salivary glands of D. hydei larvae show differences between the cells in the distal (posterior) part and those of the proximal (anterior) part during the third instar. The first sign of these differences is an increase in cellular and nuclear volume in the distal cells of the gland, beginning at 103 hours after oviposition. After 125 hours the cytoplasm of the extreme distal cells acquires a reticulated structure, and at 130 hours these cells contain large granules or droplets of mucoprotein. From this moment up to puparium formation the number of cells containing these granules increases and the boundary of this type of cells shows a shift in the proximal direction. Just before puparium formation the granules disappear from the cells and a glue substance is secreted by the larvae. At this moment only a few cells in the extreme proximal part still lack granules. Electron-microscopical observations indicate that these cells were active in secretion, whereas all cells containing large granules are inactive in this respect during most of the third instar.During the early third instar a change in cell function occurs, i.e. from synthesis of substances presumed to be digestive enzymes which are secreted, to a synthesis of a glue substance which is stored. This change begins in the extreme distal cells of the gland.Investigation of the chromosomal puffing pattern revealed that a total number of 148 puffs were present during some period of the third instar, prepupal, and early pupal stages. The activity of 110 puffs was evaluated during a series of successive time intervals. Changes in the puffing pattern during puparium formation were compared with those observed during pupation.Proximal and distal nuclei differ in the activity level of a number of puffs, but only puff 47 B is restricted in activity to the distal cells. This puff becomes active at 119 hours and disappears 4 hours before puparium formation (156 hours). Determination of nuclear diameter and DNA in nuclei of both parts of the gland revealed a correlation between a particular DNA content and the function of the cell. Distal cells show higher nuclear diameters than proximal cells after the onset of granule production. The first differences in nuclear diameter can be seen at 103 hours. Cells in the transitional part of the gland, located between distal granulecontaining and proximal granule-negative cells, always show the same DNA content. These cells are found at different locations within the gland during the third instar. This zone of cells shows a shift in proximal direction during the third instar, identical to that of the neighbouring granule-containing cells.The possible interrelation between nuclear DNA content, the activity of puff 47 B, and the production of the glue substance were discussed.     

Biochemical and ontogenetic aspects of glycoprotein synthesis in Drosophila virilis salivary glands

After SDS-polyacrylamide gel electrophoresis two glycosylated glue proteins are found in the salivary glands of Drosophila virilis late third instar larvae. Synthesis of larval glue protein 1 occurs in three successive steps: at first a precursor protein with a molecular weight of about 138,000 daltons is formed. This is modified by two subsequent steps of glycosylation, the first one involving hexosamine, the second one hexoses. Studies with tunicamycin and β-hydroxynorvaline suggest that glycosylation occurs at threonine residues. Larval glue protein 2 has a molecular weight of approximately 15,000 daltons and is weakly glycosylated. The synthesis of glue proteins is stage specific. It starts at about 120 hr after oviposition and attains its maximal rate about 20 hr later. At this time the larvae leave the food. Between ecdysone release and puparium formation (146–151 hr) larval glue protein synthesis is terminated. Throughout the prepupal stage a different set of glycoproteins is synthesized. Thus, the larval-prepupal transition is accompanied by the reprogramming of glycoprotein synthesis in salivary glands. The secretion products formed during the two developmental stages seem to possess different biological functions.     

Ultrastructure of the reproductive system of the black swamp snake (Seminatrix pygaea): Part I. Evidence for oviducal sperm storage

Oviducal sperm storage in the viviparous (lecithotrophic) colubrid snake Seminatrix pygaea was studied by light and electron microscopy. Out of 17 adult snakes examined from May–October, sperm were found in the oviducts of only two specimens. In a preovulatory female sacrificed 14 May, sperm were found in the oviducal lumen and sperm storage tubules (SSTs) of the posterior infundibulum. In a nonvitellogenic female sacrificed 9 June, sperm were found in the lumen and glands of the posterior uterus and anterior vagina, indicating a recent mating. The glands in the posterior infundibulum and vagina were simple or compound tubular, whereas glands in the uterus always were simple tubular. The epithelium of the sperm storage glands was not modified from that lining the rest of the oviduct. The cuboidal or columnar epithelium consisted of alternating ciliated and secretory areas. The secretory product released into the lumen by a merocrine process contained mucoprotein. Lipid droplets also were numerous in the epithelium. Portions of sperm sometimes were embedded in the apical cytoplasm or in secretory material. A carrier matrix containing a mucoid substance, desquamated epithelium, lipids, membranous structures, and possibly phagocytes was found around sperm in the posterior uterus. J. Morphol. 241:1–18, 1999. © 1999 Wiley‐Liss, Inc.     

Noncoordinate expression of Drosophila glue genes: Sgs-4 is expressed at many stages and in two different tissues.

The glue genes of Drosophila melanogaster comprise a family of genes expressed at high levels in the salivary glands of late third instar larvae in response to the insect hormone ecdysone. We present evidence that, in contrast to the other glue genes, Sgs-4 is turned on throughout Drosophila development and is not expressed exclusively in the larval salivary glands. Larvae transformed with an Sgs-4/Adh (alcohol dehydrogenase) hybrid gene exhibit Sgs-4-directed Adh expression in the larval proventriculus as well as in the salivary glands as early as the first instar. Sgs-4-specific RNA can be detected at very low levels during all stages of development. During late third instar, levels of Sgs-4 RNA in the salivary glands increase several-thousand-fold, thereby accounting for the large amounts of Sgs-4 protein present in the glue produced by the salivary glands. This pattern of expression is unique to the Sgs-4 gene. While expression of several of the other glue genes can be detected in embryos and early larvae, they appear to be expressed neither throughout development nor in the larval proventriculus. Appearance of the glue gene RNAs in mid third instar salivary glands is noncoordinate, even for the chromosomally clustered genes Sgs-3, Sgs-7, and Sgs-8.     

日本血吸虫尾蚴人工方法转变的童虫超微结构的观察

本文报道日本血吸虫尾蚴经注射器推压和血清孵育两种人工方法转变的童虫与载体皮肤型童虫的透射及扫描电镜的观察结果。描述了三种童虫在转变后3小时至12小时其糖膜、外质膜、体被内包含体及腺体的超微结构的变化。     

Peptidergic regulation of secretory activity in amphibian olfactory mucosa: Immunohistochemistry,neural stimulation,and pharmacology

Summary The role of substance P in the regulation of secretion from sustentacular cells, Bowman's glands and deep glands in the amphibian olfactory mucosa was investigated using immunohistochemical, electrophysiological, and pharmacological methods. Substance P-like immunoreactive varicose fibers extended through the olfactory epithelium, terminating at or near the surface. In addition, immunoreactive varicose fibers innervated Bowman's glands, deep glands, and blood vessels in the lamina propria. Innervation of Bowman's gland was sparse, with fibers terminating on basal acinar cell membranes; deep gland innervation was abundant, with fibers often extending between acinar cells almost to the lumen. Stimulation of the ophthalmic branch of the trigeminal nerve resulted in slow potentials recorded at the surface of the olfactory epithelium. When the olfactory mucosae from trigeminal-stimulated animals were examined histologically, morphological signs of secretory activity were observed, suggesting that substance P was released from the trigeminal nerve terminals. Topical application of 10^-5 to 10^-3 mol substance P resulted in morphological signs of secretion that were very similar to those seen as a result of trigeminal stimulation. Thus, substance P released from trigeminal fibers may modulate secretory activity within the olfactory mucosa.     

An autoradiographic and electron microscopic study of collagen synthesis in differentiating cartilage

Summary The synthesis of the proline-rich collagen component of cartilage matrix has been studied by autoradiography using both the light and electron microscope. Amblystoma maculatum larvae had their forelimbs amputated, were allowed to regenerate for 12–15 days, and then injected intraperitoneally with tritiated proline. The animals were fixed at various times (1 min. to 28 days) after the injection and sections of the developing limbs were coated for autoradiography by dipping in Ilford L 4 or Gevaert 3.07 emulsion. The sequential labeling of the organelles of the cartilage cell which occurred is illustrated in light and electron micrographs. Radioactive products first appeared in the ergastoplasm and were associated with the cisternae of the endoplasmic reticulum. Twenty to thirty minutes after the injection, labeled material began to appear in the Golgi zone. There, the newly synthesized protein accumulated within large vacuoles. The fibrillar material within the vacuoles may represent collagen and the more amorphous material, mucoprotein. The vacuoles subsequently (2 hrs. later) discharge their labeled contents into the extracellular space. The secreted protein is probably soluble collagen (tropocollagen) for it diffuses readily through the matrix to polymerize into striated collagen fibrils some distance from the cell. These findings contradict some widely held opinions that the fibrillar component of the matrix arises by excortication and appositional growth of fibrils originating from the ectoplasm of chondrocytes. It seems reasonable to conclude that the secretory pathway by which extracellular proteins are produced in cartilage is analogous to that suggested for epithelial gland cells.Supported by grants CA 05196-04S1 and GM-K3-13, 979-C1-A from the United States Public Health Service.The results reported in this paper were presented at the second annual meeting of the American Society for Cell Biology, November 6, 1962.     

Puffs and salivary gland function: The fine structure of the larval and prepupal salivary glands ofDrosophila melanogaster

Summary The salivary glands ofDrosophila melanogaster have been examined by electron microscopy for fine structural alterations occurring during larval and prepupal stages. The changes observed in the glands have been correlated with the puffing patterns of the polytene chromosomes at corresponding stages. In early third instar larvae, the lumen of the salivary gland appears empty, and no signs of secretory activity are visible in the glandular cytoplasm. From puff stages 1 to 6 the endoplasmic reticulum becomes reorganized and increases in volume. Electron dense material appears within its cisternae and subsequently within the Golgi saccules. Dense secretory granules then appear to be elaborated from the Golgi by terminal budding; these granules represent the glue for adhering the pupa to its substrate, and gradually increase in size and complexity. By puff stage 6 their contents have been liberated into the glandular lumen. Following puparium formation, those granules which are not extruded coalesce to form larger granules. Other dense bodies and autophagic vacuoles, considered to be lysosomes, appear, and the surplus secretory granules begin to display myelination at their peripheries; ultimately they are reduced to dense residual bodies. At puparium formation, the lumen is depleted of the glue and contains flocculent material. Histolysis commences after puff stage 11, and the cytoplasm becomes vacuolated and opaque; the nucleus becomes reduced in volume and crenated in outline. Nuclear blebbing occurs after puff stage 12, and material seemingly moves from the nucleus into the cytoplasm; the glandular lumen now becomes empty. An attempt has been made to ascertain how the chromosomal puffing activity relates to these cytoplasmic developments.     

A pair of rosette glands in the embryo and zoeal larva of an estuarine crab Sesarma haematocheir, and classification of the tegumental glands in the embryos of other crabs

A pair of rosette glands (one of the tegumental glands in crustaceans) is present at the root of the dorsal spine of the thorax in mature embryos of the estuarine crab Sesarma haematocheir. Each rosette gland is spherical, 45-50 microm in diameter. This gland consists of three types of cells: 18-20 secretory cells, one central cell, and one canal cell. The secretory cells are further classified into two types on the basis of the morphology of secretory granules. There are 17-19 a cells, and only one b cell per rosette gland. An a cell contains spherical secretory granules of 2-3 microm in diameter. The granules are filled with highly electron-dense materials near the nucleus but have lower electron-density near the central cell. The secretory granules contained in the b cell have an irregular shape and are 1-1.5 microm in diameter. The density of the materials in the granules is uniform throughout the cytoplasm. The secretory granules contained in both the a and b cells are produced by the rough endoplasmic reticulum. Materials in the granules are exocytotically discharged into the secretory apparatus inside the secretory cell, sent to the extracellular channels in the central cell, and secreted through the canal cell. The rosette gland can be distinguished from the epidermal cells 2 weeks after egg-laying and the gland matures just before hatching. Materials produced by this gland are secreted after hatching and secretion continues through five stages of zoeal larvae. These rosette glands were never found in the megalopal larva. Rosette glands are found in the embryos of Sesarma spp. and Uca spp. In other crabs, tegumental glands are also found at the same position as in the embryo of S. haematocheir, but the fine structure of their glands is largely different from that of the rosette gland. On the basis of the morphology of secretory cells (a-g cell types), the tegumental glands of a variety of crab embryos can be classified into four types, including rosette glands (type I-IV). The function of these tegumental glands is not yet known, but different types of the gland seem to reflect the phylogeny of the crabs rather than differences of habitat.     

Specific repression of a puff in the salivary gland chromosomes of Drosophila virilis after injection of glucosamine

Five hours after the injection of glucosamine into the hemolymph of Drosophila virilis larvae, puff 55E in the salivary gland chromosomes is significantly reduced in its activity. This observation in connection with the circumstances under which the activity of puff 55E decreases during normal development led to the proposition that its activity is involved in mucoprotein synthesis in the salivary glands during the third larval instar. Factors that may regulate the activity of puff 55E are discussed.     

Male reproductive tract and accessory glands of a stomatopod,Squilla holoschista

In S. holoschista, the vas deferens is differentiated into 3 regions based on the functional morphology of the epithelial lining. The proximal part with a highly secretory epithelial layer may be responsible for the secretion which is rich in protein, and moderately so in carbohydrate and lipid substances. The mid-region serves for storage of the spermatozoa. The distal part has specialized epithelial cells of a secretory nature. In addition, there are 4 typhlosole-like ridges fringed with cilia which probably aid in the conduction of the sperm cord during copulation. Secretions of the accessory reproductive glands contain a large number of discrete granules rich in mucoprotein. The principal function of this secretion appears to be the digestion of the sperm cord in the oviduct.     

Anatomy and ultrastructure of the salivary (prosomal) glands in unfed water mite larvae Piona carnea (C.L. Koch, 1836) (Acariformes: Pionidae)

Anatomy and ultrastructure of prosomal salivary glands in the unfed water mite larvae Piona carnea (C.L. Koch, 1836) were examined using serial semi-thin sections and transmission electron microscopy. Three pairs of alveolar salivary glands shown are termed lateral, ventro-lateral and medial in accordance with their spatial position. These glands belong to the podocephalic system and are situated on the common salivary duct from back to forth in the above mentioned sequence. The arrangement of the medial glands is unusual because they are situated one after another on the medial (axial) body line, therefore they are termed anterior and posterior medial glands. The secretory duct of the anterior medial gland mostly turns right, and the duct of the posterior gland turns left. The salivary glands are located in the body cavity partly inside the gnathosoma and in the idiosoma in front of the brain (synganglion). Each gland is represented by a single acinus (alveolus) and is composed of several cone shaped secretory cells arranged around the large central (intra-acinar) cavity with the secretory duct base. The cells of all glands are filled with secretory vesicles of different electron density. The remaining cell volume is occupied by elements of rough endoplasmic reticulum, and the membrane enveloping vesicles may have ribosomes on its external surface. Large nuclei provided with large nucleoli occupy the basal cell zones. The pronounced development of the prosomal salivary glands indicates their important role in extra-oral digestion of water mite larvae.     

Silk formation mechanisms in the larval salivary glands of<Emphasis Type="Italic">Apis mellifera</Emphasis> (Hymenoptera: Apidae)

The mechanism of silk formation inApis mellifera salivary glands, during the 5th instar, was studied. Larval salivary glands were dissected and prepared for light and polarized light microscopy, as well as for scanning and transmission electron microscopy. The results showed that silk formation starts at the middle of the 5th instar and finishes at the end of the same instar. This process begins in the distal secretory portion of the gland, going towards the proximal secretory portion; and from the periphery to the center of the gland lumen. The silk proteins are released from the secretory cells as a homogeneous substance that polymerizes in the lumen to form compact birefringent tactoids. Secondly, the water absorption from the lumen secretion, carried out by secretory and duct cells, promotes aggregation of the tactoids that form a spiral-shape filament with a zigzag pattern. This pattern is also the results of the silk compression in the gland lumen and represents a high concentration of macromolecularly well-oriented silk proteins.     

Control of prothoracic gland activity in larvae of Galleria mellonella

Prothoracic glands of last instar wax moth larvae maintain spontaneous secretory activity both in decapitated larvae and in isolated abdomens into which they have been transplanted, as judged by their ability to induce secretion of a new cuticle. Their activity is hormonally stimulated by the brain and inhibited by the prothoracic and mesothoracic ganglia. The subesophageal ganglion seems to suppress the inhibitory influence of the thoracic ganglia. The prothoracic glands of larvae decapitated at different times during the last instar all respond to brain implantation, and this response does not change when brains are implanted at increasing intervals after decapitation. The prothoracotropic activity of the isolated brain is highest in brains of pupae and adults but is relatively and consistently low in brains of last instar larvae. The results demonstrate that the control of prothoracic glands is a complex process governed by the nervous integration of various stimuli.     

Histological and histochemical study of rat salivary glands during their postnatal development

The postnatal development of the three major salivary glands (parotid, submaxillary and sublingual) was comparatively followed up from the histological viewpoint and in relation with some histochemical reactions. The sublingual gland presented a well developed cytomorphological structure at birth, whereas the parotid and the submaxillary one, immature at birth, gradually reached the overall appearance of adult glands, the former at 5 - 6 weeks, the latter at 8 weeks. In relation with the product secreted, it is already from birth that the parotid and the submaxillary glands presented negative reactions for mucosubstances and positive ones for revealing the protein-bound groups. The sublingual gland exhibited from the first postnatal 24 hrs positive reactions for revealing mucosubstances at the level of glandular secretory glands.     

Detection of Hepatoid Glands and Distinctive Features of the Hepatoid Acinus

In the 1920s–1930s, skin glands of a new type, hepatoid glands, were described in 13 mammal species (Rodentia, Canidae, and Bovidae). The hepatoid glands resemble sebaceous glands in their morphology, bur radically differ from them in specific structure of the acinus and another type of secretion. Later, these data either could not be confirmed or were considered insignificant and the hepatoid glands were described as modified sebaceous glands, glands with uncertain function, or modifications of epidermis. Based on the studies of various hepatoid glands in 22 species of Carniviora and Artiodactyla, the authors described in detail the characteristic features of the hepatoid acinus, which allow a precise discrimination of hepatoid and sebaceous glands. Extracellular secretory canaliculi have been described in the hepatoid glands, as well as the richness of hepatoid glands in protein, distribution of hydrophobic lipids in certain hepatoid glands, and formation of excretory ducts and cysts. The hepatoid glands are a source of great amounts of protein secreted in the merocrine way; the secretory substance of some of these glands has a strong odor.     

Glue proteins inDrosophila nasuta

Larval glue protein fractions ofDrosophila nasuta nasuta were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Seven major and at least four minor glue protein fractions were recognized. Six of the major fractions are glycosylated. They migrate as three prominent doublets (>100, 43, and 30/28 kd). The synthesis of traceable amounts of these major fractions begins already during the second as well as during the early stages of the third larval instar. The 43-kd and the 30/28-kd fractions are coded by X-chromosomal genes. They are probably clustered within the huge puff of division 10, which is the most prominent X-chromosomal puff in the polytene chromosomes of the third larval instar. Complex posttranslational modification of all but one major glue protein fraction (14 kd) leads to the formation of about 15 different protein fractions in the final glue product. The amount of glue protein produced byD. n. nasuta larvae (in relation to the total saliva proteins) is nearly twice the amount produced byD. melanogaster larvae (ca. 55 and 32%, respectively). This work was supported by the University Grants Commission, New Delhi, India, the Deutscher Akademischer Austauschdienst, FR Germany (to S.R.R.), and the Deutsche Forschungsgemeinschaft (Ka 309/9-1).     

Parasympathetic NANC-secretion of saliva in the mink, and effects of substance P and VIP.

In both parotid and submandibular glands a parasympathetic non-adrenergic, non-cholinergic (NANC) nerve-evoked secretion of saliva was demonstrated. Saliva evoked by exogenous substance P was poor in protein, while saliva evoked by VIP was protein-rich. In a subthreshold dose for fluid secretion VIP released protein and potentiated the responses elicited by substance P, particularly regarding the output of protein. The two neuropeptides may contribute to the parasympathetic NANC secretion of saliva in the mink. Further, agonists responsible for the secretory NANC response are also likely to contribute to the secretory response of the glands to parasympathetic stimulation in the absence of autonomic receptor blockade in this species.