RNA TRANSPORT FROM NUCLEUS TO CYTOPLASM IN CHIRONOMUS SALIVARY GLANDS

The fine structure and cytochemistry of the extremely large RNA puffs, or Balbiani rings, in salivary gland nuclei of midge, Chironomus thummi, larvae have been investigated. The Balbiani rings are composed of a diffuse mass of electron-opaque 400 to 500 A granules, short threads about 180 to 220 A in diameter and associated fine chromatin fibrils. These components appear to be organized into brushlike elements which form the ring. Electron microscope cytochemistry has shown that the granules and short threads contain RNA. After ribonuclease digestion, only 50 to 100 A chromatin fibrils were apparent in the Balbiani ring, and the granules were no longer demonstrable. Deoxyribonuclease digestion had no apparent effect on these structures. Observations indicate that the granules are formed from the short threads and released into the nucleoplasm in which they are evenly distributed. At the nuclear envelope, many granules have been observed partially or completely within the nuclear pores. These granules become elongated and are shown to penetrate the center of the pore in a rodlike form, about 200 A in diameter. The Balbiani ring granules are not normally visible within the cytoplasm adjacent to the nuclear envelope, but have been rarely found in this region. It is suggested that the granules represent the product of the Balbiani ring, possibly a messenger RNA bound to protein, and that they regularly pass into the cytoplasm through a narrow central channel in the pores of the nuclear envelope.     

Spermatodesm reorganization in the spermatophore and in the spermatheca of the bushcricket Tylopsis liliifolia (Fabricius) (Orthoptera,Tettigoniidae)

Spermatozoa of Tettigoniidae are usually transferred to the female by means of a spermatophore which is also the site of feather-shaped spermatodesm formation. These spermatodesms are then transferred to a spermatheca, composed of a spermathecal duct and of a seminal receptacle, involved in storing spermatozoa. In order to extend the knowledge about sperm transfer and spermatodesms reorganization in the Tettigoniidae, a morpho-structural investigation was carried out on spermatophore and spermatheca of Tylopsis liliifolia and on the reorganization of the gametes from the spermatophore. Our results show that the spermatodesms undergo disorganization in the spermatophore; unlike other Tettigoniidae, however, feather-shaped spermatodesms are never found. The epithelium of the spermatheca consists of two cell types, the cuticle-forming and the gland cells, with secretory features. The gland cells, absent in the distal tract of the seminal receptacle, release their secretion in a “reservoir” where an efferent duct opens. In the distal tract of the spermathecal duct, adjacent epithelial cells show diversified ultrastructural characteristics whose probable role is discussed. A particular feature of T. liliifolia is the genesis of the feather-shaped spermatodesms in the seminal receptacle. This feature and the peculiar organization of the feather-shaped spermatodesm are a possible autapomorphy of T. liliifolia.     

Fine structure of supporting elements of the small pit organ in the far eastern catfish,silurus asotus

The small pit organ of the catfish,Silurus asotus, was examined by electron microscopy. On the basis of their fine structures and positions in the organ, five types of cells were distinguished: 1) receptor cell (RC), 2) granular (supporting) cell (GC), 3) non-granular (supporting) cell (NGC), 4) mantle cell (MC), and 5) channel cell (CC). Both GCs and NGCs were located between the RCs in the sensory epithelium and had similar fine structures except for striking differences in their apical cytoplasm. The GC contained numerous secretory granules and scattered tonofilaments. By contrast, the NGC was characterized by abundant tonofilaments in their apical cytoplasm where only a small number of secretory granules were counted. The NGC always occurred in direct contact with RCs, whereas the GC never did so. The MCs, forming the outermost part of the organ, were characterized by numerous tonofilaments in the cytoplasm and resembled the ordinary epidermal cells in their fine structure. The CCs lined the duct of the organ, and contained electron dense cytoplasm and rather degenerative organelles. Presumably the GCs contribute to secretion of the mucous substance to the ampullary lumen, whereas the NGCs deal with nutrition and insulation for the RCs.     

DNA Synthesis and pronucleus development in pig zygotes obtained in vivo: An autoradiographic and ultrastructural study

Porcine zygotes flushed from oviducts 48,52,56,60, or 64 hr after hCG were incubated 30 min in ³H-thymidine, transferred to nonradioactive medium for 2 hr, and incubated for 30 min with ¹⁴C-thymidine. After this procedure, ova were prepared (i.e., at 51,55,59,63, or 67 hr after hCG) for autoradiography and ultrastructural observations, respectively. The first autoradiographic labelling, i.e., DNA synthesis, was observed at 56–56.5 hr after hCG, while the latest labelling was seen at 60–60.5 hr. At 51 hr after hCG, formation of the pronuclear envelope was observed, while no nucieolus precursor bodies or prestages to these structures were found. At 55 hr a few clusters of small electron-dense granules were observed, together with condensed chromatin in the pronuclei. At 59 hr the apposed regions of both pronuclei contained nucleolus precursor bodies and condensed chromatin, in close contact with both clusters of small granules and clusters of an additional category of large granules and the nuclear envelope. Additionally, large accumulations of the small granules were found in the vicinity of similarly sized accumulations of the large granules without chromatin association. At 63 hr the spherical accumulations large granules on some occasions presented a central vacuole, and condensed chromatin and clusters of small granules were attached to its periphery. Within the vacuole, electrondense material was found. It is concluded that (1) the S-phase in porcine zygotes is initiated around 56 hr post-hCG injection and is of a duration of 4.5–7.5 hr and (2) the progress of the S-phase is paralleled by the appearance of and complex interaction between different granules in the nucleoplasm. © 1995 Wiley-Liss, Inc.     

Zur Cytologie der Rectaldrüsen von Knorpelfischen

The stratified epithelium of the central collecting duct of the elasmobranch(Scylliorhinus canicula, Galeorhinus galeus andRaja batis) rectal gland consists of 3 to 6 layers of cells: one superficial, and several basal cell layers. In the superficial layer normally three different types of cells can be distinguished (a) goblet cells, (b) cells with apical secretory granules and (c) flask-shaped cells. The superficial layer ofScylliorhinus canicula reveals a further cell type, so-called mitochondria-rich cells. The epithelial areas built by these cells are always single-layered. The goblet-cells are very similar to goblet cells found in the intestine of vertebrates. Their dominant structures are a well developed ergastoplasm, a large Golgi-apparatus and mucous granules compactly filling the apical cell region. The cells with apical secretory granules are columnar or dumbbell shaped. They contain a rough-surfaced endoplasmic reticulum and a well developed Golgi-apparatus. The secretory granules are loosely distributed within the Golgi-field and are arranged in one or more rows just below the cell apex. The flask shaped cells are characterized by a cytoplasm rich in small vesicles. They posses few dictyosomes and several small mitochondria. There is some evidence for endocytotic activity. The mitochondria-rich cells are characterized by lateral cell interdigitations, by a basal labyrinth and by numerous mitochondria. They are similar to the excretory cells of rectal gland parenchyma. The cells of the basal epithelium layers are differenciated only to a small extent. They are joined in a loose formation with white blood cells often found in the intercellular spaces. The function of the elasmobranch rectal gland is not restricted to the excretion of concentrated salt solutions. There is also a significant secretion of mucous substances. The tubule glands are primarily excretory, the epithelium cells of the central collecting duct mainly secretory in function.     

Ultrastructure et cycle annuel des glandes annexes de l'appareil genital male de Campodea remyi Denis (Apterygota : Diplura : Campodeidea)

The accessory glands of the male reproductive system of Campodea remyi (Apterygota : Diplura : Campodeidea) consist of similar glandular units, each of which is made of a long secretory cell with a sieve at the tip of its extracellular cavity and a cell forming the excretory canalicule. During the annual cycle of sexual activity, they show morphological differences in terms of their size and the number of secretory granules. When ecdysis occurs, the cuticle of the ejaculatory duct and canalicules, the sieves, and the apical part of the glandular cells disappear. The gland's secretory products make up the spermatophore stalks.     

On sperm ultrastructure,spermiogenesis and the spermatophore of Heterolaophonte minuta (Copepoda,Harpacticoida)

Summary The spermatophore, mature spermatozoon and spermiogenesis of Heterolaophonte minuta have been investigated by light and electron microscopy. The spermatophore contains three different secretions which are responsible for the discharge of the contents of the spermatophore, the formation of the fertilization tube and the storage of the spermatozoa. The spermatozoon represents a type new for the Copepoda. It is a filiform cell about 25 m in length, ellipsoid in transverse section and tapered at the posterior end. The elongated nucleus contains chromatin fibrils and does not possess a nuclear envelope. Posterior to the nucleus, six mitochondria are placed one after the other. The posterior part of the spermatozoon contains parallel pseudomembranes. The gamete is not helically twisted and is without a flagellum and centrioles. The most remarkable feature of the spermatozoon is an osmiophilic cap in front of the nucleus. This cap corresponds to the acrosome of the spermatozoon. Early stages of spermiogenesis take place in the testis, where the spermatids are incorporated into accessory cells. The origin of the chromatin fibrils and the glycocalyx, as well as the breakdown of the nuclear envelope and centrioles, represent the final steps of spermiogenesis which occur in the vas deferens.     

Ultrastructural study of spermiogenesis in a rare desert amphisbaenian Diplometopon zarudnyi

Spermiogenesis, in particular the head differentiation of Diplometopon zarudnyi, was studied at the ultrastructural level by Transmission Electron Microscope (TEM). The process includes acrosomal vesicle development, nuclear elongation, chromatin condensation and exclusion of excess cytoplasm. In stage I, the proacrosomal vesicle occurs next to a shallow fossa of the nucleus, and a dense acrosomal granule forms beneath it. This step commences with an acrosome vesicle forming from Golgi transport vesicles; simultaneously, the nucleus begins to move eccentrically. In stage II, the round proacrosomal vesicle is flattened by projection of the nuclear fossa, and the dense acrosomal granule diffuses into the vesicle as the fibrous layer forms the subacrosomal cone. Circular manchettes surrounded by mitochondria develop around the nucleus, and the chromatin coagulates into small granules. The movement of the nucleus causes rearrangement of the cytoplasm. The nucleus has uniform diffuse chromatin with small indices of heterochromatin. The subacrosome space develops early, enlarges during elongation, and accumulates a thick layer of dark staining granules. In stage III, the front of the elongating nucleus protrudes out of the spermatid and is covered by the flat acrosome; coarse granules replace the small ones within the nucleus. One endonuclear canal is present where the perforatorium resides. In stage IV, the chromatin concentrates to dense homogeneous phase. The circular manchette is reorganized longitudinally. The Sertoli process covers the acrosome and the residues of the cytoplasmic lobes are removed. In stage V, the sperm head matures.     

STRUCTURAL FEATURES OF MESOSOMES (CHONDRIOIDS) OF BACILLUS SUBTILIS AFTER FREEZE-ETCHING

Freeze-etched cells of Bacillus subtilis have been studied with the electron microscope. The outer surface of the plasma membrane, i.e. the side facing the cell wall, is covered with numerous granules and short strands, each measuring approximately 50 A in diameter. These strands are occasionally seen to enter the cell wall. The inner surface of the plasma membrane, i.e. the side facing the cytoplasm, appears to be sparsely dotted with small particles measuring about 50 A. The envelope of mesosomes differs from the plasma membrane. Blunt protrusions arise from its outer surface; the inner surface appears smooth. Stalked particles, as described by other investigators after negative staining with phosphotungstic acid, were not observed on any membrane surface in our material. Preparations were also made of specimens prefixed in osmium tetroxide prior to freeze-etching. Under these conditions the bacterial membranes appeared to be surprisingly well preserved. In contrast to directly frozen, unfixed cells, some osmium tetroxide-fixed preparations showed a differentiation in cytoplasm and nucleoplasm, which made it possible to observe the close association of the mesosome with the latter.     

Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.     

Nucleus-associated actin in Amoeba proteus

The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.     

The formation of the cyst wael of the metacercaria of Cloacitrema narrabeenensis (Bowell & Bearup, 1967) (digenea: Philophthalmidae)

Dixon K.E. and Colton M. 1978. The formation of the cyst wall of the metacercaria of Cloacitrema narrabeenensis (Howell &; Bearup, 1967) (Digenea: Philophthalmidae). International Journal for Parasitology8: 491–499. The cercaria of Cloacitrema narrabeenensis contains six different types of cystogenic cells which were distinguished on the basis of their position, ultrastructure and chemical composition. Four of the different types secrete carbohydrate-protein complexes and the other two protein granules. Early in development, the cercaria is bounded by a flattened, cellular envelope which contains a few mitochondria but lacks cytoplasmic ground substance and a nucleus. Later in development, this envelope is lost, shortly after a new cellular covering forms at the surface. The nuclei are later lost from this layer and it is suggested that they sink inwards, thus forming a cercarial tegument. The products of the cystogenic cells are gradually discharged through pseudopodial-like connections with the surface layer of the cercarial tegument to form the metacercarial cyst wall. The sequence of secretion is controlled so that the separate layers of the cyst wall are formed in their correct order.     

The ultrastructure of the ejaculatory duct in the springtail Orchesella villosa (Geoffroy) (Hexapoda, Collembola) and the formation of the spermatophore

The initial part of the ejaculatory duct of Orchesella villosa contains a “valve” and a “sorter” avoiding respectively the reflow and allowing the separation of the secretion for the spermatophore stalk from the sperm fluid. For most of its length, the ejaculatory duct lumen is divided into two parts: in the dorsal part the sperm fluid flows while in the ventral district the secretion for the stalk occurs. Laterally, on both sides of the duct, longitudinal muscle fibers are present. The epithelium of the dorsal region consists of two types of long secretory cells; the most peculiar of them are those provided with extracellular cisterns flowing directly into the duct lumen as it occurs in 1st type of epidermal cells. These cells could be involved in the control of the viscosity of the sperm fluid. The second type of cells produce a secretion probably involved in the formation of the outer coat of the apical sperm droplet. The ventral epithelium consists of short cells contributing to the enrichment of the secretion for the spermatophore stalk and perhaps also to the viscosity of the secretion flowing in the lumen. In the distal part of the ejaculatory duct, the ventral district is provided with a thick layer of muscle fibers and with 3 + 3 cuticular laminae dividing the lumen into a series of slits through which the secretion of the stalk is squeezed out into filaments. This organization allows the twisting and hardening of these filaments. A drop of sperm fluid is laid on top of the long and rigid spermatophore stalk.     

EVIDENCE FROM ELECTRON MICROGRAPHS FOR THE PASSAGE OF MATERIAL THROUGH PORES OF THE NUCLEAR MEMBRANE

1. The nurse cells of Rhodnius possess nucleoli that stain with Heidenhain's hematoxylin but give a negative Feulgen reaction. In localized positions adjacent to the nuclear membrane are seen masses of material both within the nucleus and the adjoining cytoplasm that stain with Heidenhain's hematoxylin, but, like the nucleolus, give a negative Feulgen reaction. 2. Electron micrographs of the nurse cells of Rhodnius reveal the nuclear membrane to contain pores approximately 400 A in diameter. 3. In electron micrographs the nucleolus is seen to be composed of a reticulum containing tightly packed granules. Between the centrally located nucleolus and the nuclear membrane are observed relatively small bunches of granules of the same relative size as those occurring in the nucleolus. Aggregated at certain positions adjacent to the nuclear membrane both within the nucleus and in the adjoining cytoplasm are irregularly shaped masses of granules. Certain of these masses within the nucleus are seen to be continuous with those in the cytoplasm through narrow isthmuses of material extending through pores of the nuclear membrane. Other masses of granules show evidence of preparing to enter the pores by projecting tongues of material toward and into them. In the adjacent cytoplasm pear-shaped masses of granules are seen in front of and in contact with the pores which suggests that they were fixed in the process of or just after completing passage through the pores.     

ELCS in ice: cryo-electron microscopy of nuclear envelope-limited chromatin sheets

Nuclear envelope-limited chromatin sheets (ELCS) form during excessive interphase nuclear envelope growth in a variety of cells. ELCS appear as extended sheets within the cytoplasm connecting distant nuclear lobes. Cross-section stained images of ELCS, viewed by transmission electron microscopy, resemble a sandwich of apposed nuclear envelopes separated by ～30 nm, containing a layer of parallel chromatin fibers. In this study, the ultrastructure of ELCS was compared by three different methods: (1) aldehyde fixation/dehydration/plastic embedding/sectioning and staining, (2) high-pressure freezing/freeze substitution into plastic/sectioning and staining, and (3) high-pressure freezing/cryo-sectioning/cryo-electron microscopy. ELCS could be clearly visualized by all three methods and, consequently, must exist in vivo and are not fixation artifacts. The ～30-nm chromatin fibers could only be observed following aldehyde fixation; none were seen in cryo-sections. Electron microscopic tomography tangential views of aldehyde-fixed ELCS suggested an ordering of the separate chromatin fibers adjacent to the nuclear envelope. Possible mechanisms of this chromatin ordering are discussed.     

Morphology and ultrastructure of the abdominal integumentary glands of Acanthoscelides obtectus Say (Coleoptera : Bruchidae)

The morphology of the abdominal integumentary glands of the bean weevil Acanthoscelides obtectus (Coleoptera : Bruchidae) is described. There are 2 types: glands with long secretory ducts and ampullate glands with short ducts. The former are distributed throughout the integument and have 2 secretory cells, one of which is connected to the reservoir in the distal receptor zone, and the other to the duct. The duct, which is 100 μm long, has an epicuticular structure and its evacuation pore is 0.5 μm in diameter. It is surrounded by cytoplasm rich in microtubules. The epicuticular structure of the duct is resolved into fine filaments in the distal region. The ampullate glands exist only on the abdominal tergites and the pygidium. They are composed of an epicuticular receptor ampoule and a short duct connected to one secretory cell. The 2 types of glands can be placed in class 3 according to the nomenclature of Noirot and Quennedey (1974. Annu. Rev. Entomol. 19: 61–80).     

Dirofilaria immitis: ultrastructural aspects of oocyte development and zygote formation.

Ultrastructural observations of the ovary and uterus of Dirofilaria immitis reveal some characteristics of oogonia, oocytes, and uterine sperm. Oogonia are confined to the distal portion of the ovary including a blind tip, where a morphologically distinct terminal cap cell was not observed. These cells contain a nucleus with a nucleolus, numerous dense bodies, scanty ribosomes, lipid droplets, and an occasional mitochondrion. Endoplasmic reticulum is lacking and Golgi complexes were observed only in fully grown oogonia. Primary oocytes located in the middle portion of the ovary are large, elongate, and have a complete set of organelles including many small mitochondria, fragmentary endoplasmic reticulum, ribosomes, Golgi complexes, and very few dense bodies. These cells are arranged into many rosettes about central cytoplasmic masses, the rachises, to which they maintain cytoplasmic continuity by pseudopodlike processes. The rachises contain no organelle except a few dense granules and are bound by winding membranes. Oocytes from the proximal portion differ from those of the middle portion of the ovary in their larger size, round shape, absence of many organelles, presence of small dense granules, and lacking a rachis. Dense bodies are specific to the oogonia and exhibit DNase susceptibility and a positive reaction for a mitochondrial enzyme. These findings together with their decreased number and a concomitant increase of mitochondria in the oocytes suggest a relationship between these bodies and mitochondria.Uterine sperm of D. immitis are of the amoeboid type and contain several chromatin masses without a nuclear envelope, many mitochondria, and specialized membranous organelles referred to as mesosomelike vesicles. The vesicles are probably originated from the sperm plasma membrane. Upon fertilization, the entire spermatozoon penetrates the oocyte and its contents are gradually dissolved in the ooplasm with a simultaneous appearance of large numbers of ribosomes at the site of dissolution. Ribosomes were later found in the nucleus. A pronucleus was not observed. These findings are basically in agreement with those described for Ascaris but differ in the morphologic features and number of rachises, presence of dense bodies, absence of refringent granules in the oocytes and the absence of a refringent body and presence of several chromatin masses in the sperm.     

Schistosoma mansoni: Development of acetabular glands of cercaria at ultrastructural level

Cercariae of Schistosoma mansoni in daughter sporocysts in Biomphalaria glabrata were studied with the electron microscope to observe the maturing process of their acetabular glands. The undifferentiated acetabular gland displays its enlarged basal area (fundus) and extended narrow process (duct) before other organ systems are recognized. Its fundus contains a prominent nucleus and subcellular organelles typical of active secretory cells.The secretory granules of the postacetabular glands are formed in a milieu of dilated rough endoplasmic reticulum and Golgi. Two morphologically different secretory granules are produced: (1) homogeneously granular ones, and (2) other granular ones with electron dense bodies in their matrices. Mostly, the homogeneously granular ones are produced first in the fundus and are forced into the ducts as the other type is formed.The secretory granules of preacetabular glands are formed from translucent vacuoles which arise from an environment of endoplasmic reticulum and Golgi. Two morphologically different secretory granules are produced: (1) one type has a homogeneous dense matrix, and (2) the other type has a less dense matrix containing electron-lucid bodies.The duct of an undifferentiated acetabular gland has either filamentous material or microtubules dispersed in its cytoplasm. Once microtubules are formed, they persist during the life of the cercaria. The microtubules are believed to have possibly two functions: (1) to support the long duct, and (2) to assist the movement of the secretory granules into the channels of the ducts where they remain until released during host penetration.Few of the subcellular organelles associated with secretory granules formation are seen in the duct except the area in close proximity to the fundus; thus, the few secretory granules produced in the duct are in this region.     

Binding of Germanium to Pseudomonas putida Cells

The binding of germanium to Pseudomonas putida ATCC 33015 was investigated by using whole intact cells grown in a medium supplemented with GeO₂ and catechol or acetate. Electron-microscopic examination of the control and metal-loaded samples revealed that germanium was bound within the cell envelope. A certain number of small electron-dense deposits of the bound element were found in the cytoplasm when the cells were grown in the presence of GeO₂ and catechol. The study of germanium distribution in cellular fractions revealed that catechol facilitated the intracellular accumulation of this element.