Distribution of cytoskeletal elements in the ovarioles of Mutilla sp. (Insecta,Hymenoptera)

The ovaries of Mutilla sp., as those of other hymenopterans, consist of meroistic-polytrophic ovarioles. Within each ovariole, a terminal filament, a germarium, and a vitellarium can be distinguished. The germaria contain numerous dividing and/or differentiating groups (clusters) of germ cells. The vitellaria are composed of several, linearly arranged, ovarian follicles; each follicle consists of an oocyte and a group of nurse cells. Distribution of cytoskeletal elements (microfilaments and microtubules) throughout the ovarioles of Mutilla sp. has been studied on whole mount preparations stained with rhodamine-conjugated phalloidin and FITC-labelled anti-tubulin.     

The ovary ofCatajapyx aquilonaris (Insecta,Entognatha): ultrastructure of germarium and terminal filament

Summary Each of the two ovaries ofCatajapyx aquilonaris is composed of seven segmentally (metamerically) arranged ovarioles. The two lateral oviducts that join and bear ovarioles extend throughout the abdomen. In the ovariole three regions can be recognized: the terminal filament, the germarium and the vitellarium. The terminal filaments do not fuse with each other but attach separately (by means of muscle fibres) to the closest lobes of the fat body. Germ cells in the germarium are not joined by intercellular bridges and do not form clusters. Thus the ovarioles ofC. aquilonaris are interpreted as being primarily panoistic. The results obtained support the hypothesis that both dipluran subgroups (Campodeina and Japygina) do not form a monophyletic unit.     

Formation and structure of nutritive cords in telotrophic ovarioles of snake flies (Insecta: Raphidioptera)

Telotrophic ovariole of Raphidia spp. is composed of the anteriorly located terminal filament, tube-shaped tropharium, the vitellarium and the ovariole stalk. The tropharium consists of a central syncytial core surrounded by one cell thick layer of tapetum cells. Early previtellogenic oocytes differentiate at the base of tropharium. Both the oocytes and the tapetum cells are connected with the central syncytium by delicate intercellular bridges. At the onset of previtellogenic growth, the anterior parts of the oocytes become extended and form long cytoplasmic projections--nutritive cords. Each nutritive cord contains numerous microtubules that show no preferential orientation within the cord but diminishing anterior-posterior gradient of distribution. Irregular arrangement of microtubules indicates that this cytoskeletal scaffold does not play any role in directed transport within the ovariole but instead constitutes one of the elements of the structural framework of the nutritive cord. Besides microtubules, the stability of the nutritive cords in Raphidia ovarioles is maintained by the rim-shaped membrane foldings lined with microfilaments.     

Structure of the ovary and the differentiation of follicular epithelium in the pig louse, Haematopinus suis (Insecta: Phthiraptera)

The female reproductive system of the pig louse, Haematopinus suis (Insecta: Phthiraptera) is composed of paired ovaries, lateral oviducts, and a common oviduct that leads into a vagina. Clusters of mycetocytes (= cells filled with symbiotic organisms) are associated with lateral oviducts. Each ovary is composed of five loosely arranged ovarioles of the polytrophic-meroistic type. An individual ovariole is covered by a basal lamina and is composed of a terminal filament, germarium, and vitellarium. The terminal filament is composed of large, disc-shaped cells that are orientated perpendicularly to the long axis ofthe ovariole. The basal part of the terminal filament is separated from the germarium by a well-developed transverse septum. The germarium is short and filled with clusters of oogonial cells. In each cluster the cells arejoined by intercellular bridges, filled with fusomal material. Within the cluster, only one cell, the future oocyte, enters the prophase of the first meiotic division; the other cells differentiate into nurse cells. The basal part ofthe germarium is filled with the somatic prefollicular cells. The boundary between the germarium and the vitellarium is not distinct. The vitellarium contains linearly arranged ovarian follicles in subsequent stages of oogenesis (previtellogenesis, vitellogenesis and choriogenesis). Each follicle consists of an oocyte and 7 nurse cells and is surrounded by follicular cells. During oogenesis the follicular cells diversify, so that ultimately, five morphologically distinct subpopulations of these cells can be distinguished: (1) cells in contact with the nurse cells, (2) anterior cells, (3) mainbody cells, (4) posterior cells, and (5) interfollicular cells. Interestingly, the follicular cells associated with the anterior part of the oocyte, i.e. located in space at the oocyte/nurse cell border (fold cells) are mitotically active throughout previtellogenesis. It might be suggested, in this context, that the separation of the oocyte from the nurse cell compartment is brought about by mitotic divisions, consequent multiplication and centripetal migration of these cells.     

Effect of attachment to a solid substrate on the structural organization of skeletal muscle symplasts

A study was made of the ultrastructural reorganization of myosymplasts passed from the primary suspension culture to the secondary monolayer culture. The microtubules in myoblasts and myosymplasts from suspension are very rare, the intermediate filaments form a perinuclear network, small bundles of myofilaments are arranged in disorder, often as whorls around nuclei. After attachment to the solid substrate the myosymplasts form pseudopodia to move as non-muscle fibroblast-like cells. On the leading end of moving symplasts some stress fiber-like structures are found. Numerous microtubules appear. The microtubules and intermediate filaments are arranged in parallel along the axis of a lengthening symplast. A stepwise reorganization of the non-muscle type cytoskeleton to sarcomeres of differentiated myotubes is observed later. The role of attachment and mechanical stress in myotube morphogenesis are discussed.     

Segregative cell division and the cytoskeleton in two species of the genus Struvea (Cladophorales,Ulvophyceae, Chlorophyta)

The detailed segregative cell division (SCD) processes and changes in the arrangement of cortical microtubules and actin filaments were examined in two species of Struvea. SCD was initiated by the appearance of annular constrictions along the lateral side of a mother cell. The constrictions decreased in diameter, became thin, tubular in shape, and pinched the protoplasm of the mother cell into several protoplasmic sections. The protoplasmic sections expanded and developed into daughter cells, which appressed each other, and were arranged in a single row. Lateral branches protruded from the upper parts of the daughter cells. The protoplasm of the lateral branches was divided by secondary SCDs and was distributed amongst the new daughter cells. SCD and lateral branch formation were essential for morphogenesis in Struvea. Cortical microtubules were arranged parallel and longitudinally to the cell axis before SCD. When SCD was initiated, there was considerable undulation of the cortical microtubules and several transverse bundles appeared in the cytoplasmic zone where annular constrictions occurred. A microtubule‐disrupting drug (amiprophos methyl) inhibited SCD. Actin filaments maintained reticulate patterns before and during SCD. These results demonstrated that SCD in Struvea species was quite distinct from that in Dictyosphaeria cavernosa reported previously.     

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element     

Structure and development of ovaries in the weevil, Anthonomus pomorum (Coleoptera, Polyphaga). I. Somatic tissues of the trophic chamber

In developing ovarioles of Anthonomus pomorum (Coleoptera, Polyphaga, Curculionidae) the trophic chambers (tropharia) are relatively large and consist of clusters (clones) of germ cells and various somatic tissues. Each ovariole is enclosed within an outer epithelial sheath (tunica externa). Throughout the pupal phase, the growth of this sheath is accelerated and precedes the development of the rest of the ovariole. As a result, the epithelial sheath proliferates anteriorly and forms an elongated "sleeve" that during the later stages of development becomes gradually filled by the growing tropharium. In the early pupal stage, a few terminal filament cells are observed in contact with the anterior end of the tropharium. These cells are separated from the rest of the trophic chamber by a transverse septum, which maintains continuity with the basal lamina. Beneath the basal lamina there is a layer of inner sheath cells, whereas inside the tropharium there are interstitial cells. These two types of cell differ morphologically in a mature ovary but they retain, until the end of the imago-B stage, a similar ultrastructure testifying to their common origin. At the posterior end of the tropharium, from the imago-B stage on, many young oocytes, surrounded by prefollicular cells, are observed. This is the so-called neck region of the tropharium. Extraction with Triton X-100 detergent showed that in a mature trophic chamber there are only individual microtubules arranged along the projections of interstitial cells. This indicates that the cytoskeleton elements (microfilaments and microtubules) participate only to a very limited extent in the spatial organisation of the tropharium in A. pomorum.     

Ultrastructural studies of the ovary of Palaeococcus fuscipennis (Burmeister) (Insecta, Hemiptera, Coccinea: Monophlebidae)

Ovaries of Palaeocoocus fuscipennis are composed of about 100 telotrophic ovarioles that are devoid of terminal filaments. In the ovariole a tropharium ( = trophic chamber) and vitellarium can be distinguished. The tropharium contains 7 trophocytes. A single oocyte develops in the vitellarium. The oocyte is surrounded by follicular cells that do not undergo diversification into subpopulations. The obtained results are discussed in a phylogenetic context.     

The roles of cell size and cell number in determining ovariole number in Drosophila

All insect ovaries are composed of functional units called ovarioles, which contain sequentially developing egg chambers. The number of ovarioles varies between and within species. Ovariole number is an important determinant of fecundity and thus affects individual fitness. Although Drosophila oogenesis has been intensively studied, the genetic and cellular basis for determination of ovariole number remains unknown. Ovariole formation begins during larval development with the morphogenesis of terminal filament cells (TFCs) into stacks called terminal filaments (TFs). We induced changes in ovariole number in Drosophila melanogaster by genetically altering cell size and cell number in the TFC population, and analyzed TF morphogenesis in these ovaries to understand the cellular basis for the changes in ovariole number. Increasing TFC size contributed to higher ovariole number by increasing TF number. Similarly, increasing total TFC number led to higher ovariole number via an increase in TF number. By analyzing ovarian morphogenesis in another Drosophila species we showed that TFC number regulation is a target of evolutionary change that affects ovariole number. In contrast, temperature-dependent plasticity in ovariole number was due to changes in cell-cell sorting during TF morphogenesis, rather than changes in cell size or cell number. We have thus identified two distinct developmental processes that regulate ovariole number: establishment of total TFC number, and TFC sorting during TF morphogenesis. Our data suggest that the genetic changes underlying species-specific ovariole number may alter the total number of TFCs available to contribute to TF formation. This work provides for the first time specific and quantitative developmental tools to investigate the evolution of a highly conserved reproductive structure.     

Morphological evidence for cyclic AMP-induced reverse transformation in vole cells infected with avian sarcoma virus.

Normal fibroblasts of the vole displayed moderately spread or flattened, spindle-shaped, or polygonal morphologies and attached firmly to a substrate. Topographic features of these cells included sparse microvilli, ruffles, and filopodia. Microfilament bundles, intermediate filaments, and long microtubules generally parallel to each other, and the long axis of the cell or its extensions were present in the cytoplasm. Fibronectin was abundant, and fibronectin fibrils often formed junctions at the cell membrane with microfilament bundles. Transformation with avian sarcoma virus converted 90% of the cells to spheres 5 to 10 microns in diameter. In contrast to the normal vole cells, microfilament bundles were absent, microtubules were short and randomly arranged, and fibronectin was no longer visible. Exposure to dibutyryl cyclic AMP and testololactone caused a majority of the spherical cells to stretch and flatten, a process referred to as reverse transformation. Microtubules radiated out to the cell periphery and became parallel in cell extensions, while long microfilament bundles appeared in the cytoplasm. Parallel intermediate filaments were arranged throughout the cell. This ultrastructural analysis of reverse transformation in avian sarcoma virus-transformed vole cells detailed the status of the cytoskeletal system and showed agreement with earlier findings (Puck et al., J. Cell. Physiol. 107:399-412, 1981) using indirect immunofluorescence.     

ELECTRON MICROSCOPIC OBSERVATIONS ON TUBULAR STRUCTURES INVOLVED IN CRYSTAL FORMATION IN THE RED ALGA PLEONOSPORIUM SQUARRULOSUM (HARV.) ABBOTT1

Hexagonal or angular crystalline inclusions in Pleonosporium (Naeg.) Hauck vegetative cells were examined using electron microscopy. Ultrastructural analysis reveals that the inclusions initially contain tubular elements resembling microtubules but, with continued differentiation, are transformed into rod containing crystals. The tubular structures initially measure 25 nm in diameter. Scattered tubules become arranged in a parallel and alternate pattern and undergo subsequent enlargement to approximately 29 nm. Following enlargement, each tubule apparently disaggregates into rods that form a crystal having hexagonally arranged rod-like subunits. It is suggested that these tubules may represent microtubules and the resultant crystals are composed of tubulin.     

The ovaries of Mecoptera: basic similarities and one exception to the rule

Two entirely different types of ovaries (ovarioles) have been described in mecopterans. In the representatives of Meropeidae, Bittacidae, Panorpodidae and Panorpidae the ovarioles are of the polytrophic-meroistic type. Four regions: a terminal filament, germarium, vitellarium and ovariole stalk can be distinguished in the ovarioles. The germaria house numerous germ cell clusters. Each cluster arises as a result of 2 consecutive mitoses of a cystoblast and consists of 4 sibling cells. The oocyte always differentiates from one of the central cells of the cluster, whereas the remaining 3 cells develop into large, polyploid nurse cells. The vitellaria contain 7-12 growing egg chambers (= oocyte-nurse cell complexes). In contrast, the ovaries of the snow flea, Boreus hyemalis, are devoid of nurse cells and therefore panoistic (secondary panoistic). The ovarioles are composed of terminal filaments, vitellaria and ovariole stalks only; in adult females functional germaria are absent. Histochemical tests suggest that amplification of rDNA takes place in the oocyte nuclei. Resulting dense nucleolar masses undergo fragmentation into multiple polymorphic nucleoli. The classification of extant mecopterans as well as the phylogenetic relationships between Mecoptera and Siphonaptera are discussed in the context of presented data.     

Involvement of microtubules in rhizoid differentiation of Spirogyra species

Summary.  Some species of Spirogyra form rosette-shaped or rod-shaped rhizoids in the terminal cell of the filaments. In the present study, we analyzed an involvement of microtubules (MTs) in rhizoid differentiation. Before rhizoid differentiation, cortical MTs were arranged transversely to the long axis of cylindrical cells, reflecting the diffuse growth. At the beginning of rhizoid differentiation, MTs were absent from the extreme tip of the terminal cell. In the other area of the cell, however, MTs were arranged transversely to the long axis of the cell. In the fully differentiated rosette-shaped rhizoid, MTs were randomly organized. However, at a younger stage of rosette-shaped rhizoids, MTs were sometimes arranged almost transversely in the lobes of the rosette. In the rod-shaped rhizoid, MTs were arranged almost transversely. MT-destabilizing drugs (oryzalin and propyzamide) induced swelling of rhizoids, and neither rosette-shaped nor rod-shaped rhizoids were formed. The role of MTs in rhizoid differentiation was discussed. Received June 17, 2002; accepted November 11, 2002; published online April 8, 2003 RID="*" ID="*" Correspondence and reprints: Department of Life Science, Graduate School of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo 678-1297, Japan.     

Cytoskeletal arrays in the cells of soybean root nodules: The role of actin microfilaments in the organisation of symbiosomes

Summary Within the infected cells of root nodules there is evidence of stratification and organisation of symbiosomes and other organelles. This organisation is likely to be important for the efficient exchange of nutrients and metabolites during functioning of the nodules. Using immunocytochemical labelling and confocal microscopy we have determined the organisation of cytoskeletal elements, micro tubules and actin microfilaments in soybean nodule cells, with a view to assessing their possible role in organelle distribution. Most microtubule arrays occurred in the cell cortex where they formed disorganised arrays in both uninfected and infected cells from mature nodules. In infected cells from developing nodules, parallel arrays of microtubules, transverse to the long axis of the cell, were observed. In incipient nodules, before release of rhizobia into the plant cells, the cells also had an array of microtubules which radiated from the nucleus into the cytoplasm. Three actin arrays were identified in the infected cells of mature nodules: an aster-like array which emanated from the surface of the nucleus, a cortical array which had an arrangement similar to that of the cortical microtubules, and, throughout the cytoplasm, an array of fine filaments which had a honeycomb arrangement consistent with a distribution between adjacent symbiosomes. Uninfected cells from mature nodules had only a random cortical array of actin filaments. In incipient nodules, the density of actin microfilaments associated with the nucleus and radiating through the cytoplasm was much less than that seen in mature infected cells. The cortical array of actin also differed, being composed of swirling configurations of filaments. After invasion of nodule cells by the rhizobia, the number of actin filaments emanating from the nucleus increased markedly and formed a network through the cytoplasm. Conversely, the cytoplasmic array in uninfected cells of developing nodules was identical to that in the cells of incipient nodules. The cytoplasmic network in infected cells of developing nodules is likely to be the precursor of the honeycomb array seen in mature nodule cells. We propose that this actin array plays a role in the spatial organisation of symbiosomes and that the microtubules are involved in the localisation of mitochondria and plastids at the cell periphery in the infected cells of root nodules.     

The ovaries of aphids (Hemiptera,Sternorrhyncha, Aphidoidea): morphology and phylogenetic implications

The ovaries of aphids belonging to the families Eriosomatidae, Anoeciidae, Drepanosiphidae, Thelaxidae, Aphididae, and Lachnidae were examined at the ultrastructural level. The ovaries of these aphids are composed of several telotrophic ovarioles. The individual ovariole is differentiated into a terminal filament, tropharium, vitellarium, and pedicel (ovariolar stalk). Terminal filaments of all ovarioles join together into the suspensory ligament, which attaches the ovary to the lobe of the fat body. The tropharium houses individual trophocytes and early previtellogenic oocytes termed arrested oocytes. Trophocytes are connected with the central part of the tropharium, the trophic core, by means of broad cytoplasmic processes. One or more oocytes develop in the vitellarium. Oocytes are surrounded by a single layer of follicular cells, which do not diversify into distinct subpopulations. The general organization of the ovaries in oviparous females is similar to that of the ovaries in viviparous females, but there are significant differences in their functioning: (1) in viviparous females, all ovarioles develop, whereas in oviparous females, some of them degenerate; (2) the number of germ cells per ovariole is usually greater in females of the oviparous generation than in females of viviparous generations; (3) in oviparous females, oocytes in the vitellarium develop through three stages (previtellogenesis, vitellogenesis, and choriogenesis), whereas in viviparous females, the development of oocytes stops after previtellogenesis; and (4) in the oocyte cytoplasm of oviparous females, lipid droplets and yolk granules accumulate, whereas in viviparous females, oocytes accrue only lipid droplets. Our results indicate that a large number of germ cells per ovariole represent the ancestral state within aphids. This trait may be helpful in inferring the phylogeny of Aphidoidea.     

The fine structure of the sperm bundles of the coccid,Aspidiotus perniciosus Cumst., and sperm movement

The mature sperm of A. perniciosus are organized into bundles, about 350 μm long by 9–10 μm wide. Each bundle contains 32 sperm enclosed by a common sheath. The sperm contains an elongated ‘central core’, representing nuclear material, surrounded by a spiral microtubular sheath and cytoplasm. The electron-dense nuclear material is localized in the more pointed half of the sperm. The spiral microtubular sheath is composed of 30— 100 microtubules (depending on the cross-sectional level), situated parallel to the longitudinal axis of the sperm. On the basis of this ultrastructural organization, the motility of the sperm and sperm bundle as a whole is discussed. The sperm of A. perniciosus provide strong evidence that the microtubules arranged asymmetrically represent the elements directly involved in sperm motility.     

Fine structure of bovine lens epithelial cells in vitro in relation to modifications induced by a retinal extract (EDGF)

Cultured bovine lens epithelial cells are polygonal in shape. In confluent and multilayer cultures they exhibit elaborate arrays of 6 nm filaments, bundles of intermediate-sized filaments, and a fibrous meshwork of subcellular and intercellular material. Cells grown in the presence of a retinal extract (RE) have a higher growth rate, and are smaller and more regular in shape. In them the 6 nm filaments are mostly aligned in sheets, the intermediate-sized filaments form a fine network, and the cells are closely apposed to the plastic substratum. Some homogeneous material is formed intercellularly in older cultures. Cellular elongation, induced in the former cultures by the addition of RE, is accompanied by an alignment of cytoskeletal elements, including microtubules, parallel to the long axis. Other structural features are similar in all cell types. The response to RE is discussed in terms of shape modulations associated with the restricted expression of structural characteristics acquired in vitro.     

The Fine Structure of Photoreceptor Terminals in the Compound Eye of Pandalus borealis (Crustacea)

Seven of the photoreceptor axons of each ommatidium in the compound eye of the prawn Pandalus borealis end in two layers in the optic lamina. They have expanded terminals in the optic cartridges; four distally and three proximally in each cartridge. All seven receptor terminals are presynaptic to one lamina monopolar neuron (M₂) of the cartridge. This monopolar neuron is situated centrally in the cartridge and has a thick axis fibre with radially arranged branches, and its axon has a terminal in medulla externa. At the synapses, an arrowlike presynaptic bar is found facing three postsynaptic profiles. The receptor terminals have several characteristics. Their cytoplasm is filled with empty and coated vesicles, and contains numeorus large mitochondria and clusters of tubular elements. There is a longitudinally arranged fascicle of filaments partly surrounded by electron-dense amorphous material in the terminals. Centrally towards M₂, numerous neural spines invaginate into the terminal. Along the entire terminal periphery, there are invaginations from the glial cells. The terminals also form small knoblike protrusions extending into the surrounding glial cells.     

Different aspects of tubulin polymerization in spermatids of cyprinodontidae (fish,teleost)

Our study concerns 10 genera and 26 species of cyprinodontid fish. In the cytoplasm of spermatids tubulin polymerizes in various forms according to species. We have demonstrated the presence of classic microtubules with a diameter of 24 nm and also of tubules of smaller diameter (15 run) and greater diameter (30 to 50 nm). Microtubules are very numerous in the cytoplasm of certain species. They may be arranged without any order or form bundles which may contain several hundred parallel elements. They never form a manchette. In two species (Aplocheilichthys normani and Epiplatys fasciolatus) only spermatids that degenerate show this peculiarity. The microtubules present two kinds of decorations. The first type are small elements composed of MAP which enable two or more microtubules to link up. The second type are curved tubulin elements that give the microtubule that bear them the appearance of an incomplete doublet. Doublets and triplets may also be formed. Cyprinodontidae spermatocytes and spermatids probably synthesize a very large quantity of tubulin which polymerizes in certain species.