Ultrastructure of the copulatory stylet and accessory spines in Haplopharynx quadristimulus (Turbellaria)

The copulatory organ of Haplopharynx quadristimulus Ax, 1971 (Carolina form, Rieger, 1977) consists of a proximal prostatic vesicle and a distal stylet apparatus comprising a central tubular stylet and four to five peripheral accessory spines. By electron microscopy it could be seen that the stylet and spines were intracellular specializations. The copulatory organ can be interpreted as a specialization of an epithelial canal extending from the testes to the body wall. In the complex stylet apparatus, the epithelium was differentiated into six cell types. The stylet, which was formed in a matrix syncytium next to the prostatic vesicle, extended into the lumen of the stylet canal. The interior of the stylet apparatus contained one group of cells that had thick ciliary rootlets and another that had rootlet-like ribbons.The cells that contain the rootlets enveloped bundles of longitudinally arranged muscles. The accessory spines were formed in cells which lay peripheral to the muscle bundles. The spines, stylet, rootlet-like ribbons, and rootlets had similar patterns of periodic cross striations. The similarity in striation patterns suggests that the accessory spines and stylet are composed of modified ciliary rootlets.     

An ultrastructural analysis of mature sperm from the crayfish Pacifastacus leniusculus,Dana

Sperm from the crayfish, Pacifastacus leniusculus, resemble other reptantian sperm in that they are composed of an acrosome, subacrosomal region, nucleus, membrane lamellar complex, and spikes which radiate from the nuclear compartment. The acrosome (PAS positive vesicle) can be subdivided into three regions: the apical cap, crystalline inner acrosomal material, and outer acrosomal material which is homogeneous except for a peripheral electron dense band. The nucleus contains uncondensed chromatin and bundles of microtubules which project into the spikes. The orientation of the microtubule bundles relative to the nuclear envelope near the base of the subacrosomal region suggests that the nuclear envelope may function in the organization of the spike microtubules.     

东方扁虾精子的超微结构

利用电镜研究了东方扁虾（Ｔｈｅｎｕｓ ｏｒｉｅｎｔａｌｉｓ）精子的形态和结构。精子由核、膜复合物区和顶体区３部分组成。核内含非浓缩的染色质、微管及细纤维丝,外被核膜;５～６条辐射臂自核部位伸出,臂内充满微管。膜复合物区位于核与顶体之间,由许多膜片层结构及其衍生的囊泡共同组成。顶体区由顶体囊和围顶体物质组成,顶体结构复杂,由顶体帽、内顶体物质和外顶体物质等构成;围顶体物质呈细颗粒状,主要分布于顶体囊     

Sperm structure and ultrastructure of the Melittobia hawaiiensis, Perkins and M. australica, Girault (chalcidoidea: eulophidae)

Spermatozoa morphology has, for some years, been used to help answer some phylogenetic questions for Hymenoptera. This is the second study describing spermatozoa morphology of an Eulophidae species in which important characteristics were observed. Melittobia spermatozoa are spiralled and measure approximately 270mum in length. The head contains a small acrosome, apparently formed only by an acrosomal vesicle, which, together with the initial nuclear region, is surrounded by an extracellular sheath, from which innumerable filaments irradiate. The nucleus is helicoidal and completely filled with compact chromatin. A centriolar adjunct is observed at the nucleus-flagellum transition; it associates laterally with the nucleus and exhibits two small expansions, which reach around the centriole. In the flagellum there are two mitochondrial derivatives, which in cross-sections are asymmetric. In the derivative with the larger diameter, two distinct regions are observed, a small one, near the axoneme, with a clear "fissure" inside, and a larger region where the cristae occur. Both derivatives initiate at the nuclear base, but the larger diameter derivative finishes first, before the flagellum extremity. At the end of the axoneme, the accessory microtubules are the first to finish.     

Microtubule dynamics during infection-related morphogenesis of Colletotrichum lagenarium.

Using a green fluorescent protein (GFP)-tubulin fusion protein, we have investigated the dynamic rearrangement of microtubules during appressorium formation of Colletotrichum lagenarium. Two alpha-tubulin genes of C. lagenarium were isolated, and GFP-alpha-tubulin protein was expressed in this fungus. The strain expressing the fusion protein formed fluorescent filaments that were disrupted by a microtubule-depolymerizing drug, benomyl, demonstrating successful visualization of microtubules. In preincubated conidia, GFP-labeled interphase microtubules, showing random orientation, were observed. At conidial germination, microtubules oriented toward a germination site. At nuclear division, when germ tubes had formed appressoria, mitotic spindles appeared inside conidia followed by disassembly of interphase microtubules. Remarkably, time-lapse views showed that interphase microtubules contact a microtubule-associated center at the cell cortex of conidia that is different from a nuclear spindle pole body (SPB) before their disassembly. Duplicated nuclear SPBs separately moved toward conidium and appressorium accompanied by astral microtubule formation. Benomyl treatment caused movement of both daughter nuclei into 70% of appressoria and affected appressorium morphogenesis. In conidia elongating hyphae without appressoria, microtubules showed polar elongation which is distinct from their random orientation inside appressoria.     

A deep-etching study of the guinea pig tracheal cilium with special reference to the ciliary transitional region

The fine structure of the cilium was examined by freeze-fracture-etch studies. In the interior of the transitional region, three types of plate structures were clearly observed. While the terminal plate contained fine fibrillar linkers suspending the central core plates from its peripheral doublet microtubules, two other types of plates had no suspending linkers. At the upper level of transitional region, one of the central microtubules elongated deeper than the other in the space surrounded by ring structure. Axosome-like structure was not observed in our replicas. Central vesicle of the basal body was also suspended by fine fibrillar linkers from peripheral triplets. Though membrane particles of ciliary necklace were recognized on protoplasmic and external fracture faces, and the external surface, particle arrays were not observed on protoplasmic surface. Instead, Y-shaped, cross bridges, one end of which attached to the doublet microtubules, merged in the circular ridge structure at opposite ends. This circular ridge structure at the necklace region may play a role as an anchoring site of both membrane particles of the necklace and cross bridges from peripheral doublet microtubules.     

Structural and ultrastructural studies of male reproductive tract and spermatozoa in Xylocopa frontalis (Hymenoptera,Apidae)

Fiorillo, B. S., Zama, U., Lino‐Neto, J. and Báo, S. N. 2010. Structural and ultrastructural studies of male reproductive tract and spermatozoa in Xylocopa frontalis (Hymenoptera, Apidae). —Acta Zoologica (Stockholm) 91 : 176–183. In Xylocopa frontalis the reproductive tract is composed of testes, deferent ducts, seminal vesicles, accessory glands and an ejaculatory duct. Each testis comprises four testicular tubules in which multiple cysts are present containing approximately 64 spermatozoa per cyst. The seminal vesicle consists of an epithelium, a thick basement lamina and a muscular external sheet. In the luminal region some vesicles can be observed; however, the epithelial cells of the seminal vesicle do not display morphological features associated with secretory functions. The spermatozoa, measuring approximately 260 µm long, are similar to the hymenopteran pattern. The head region consists of an acrosome with an inner perforatorium that penetrates an asymmetrical nuclear tip. The nucleus is linear, electron‐dense and its posterior tip projects into the beginning of the axoneme. The centriolar adjunct is asymmetric with many electron‐lucent lacunae interspersed throughout. The axoneme has the 9 + 9 + 2 pattern of microtubules and in the posterior region the central microtubules finish first, followed by the doublets and finally the accessory microtubules. The mitochondrial derivatives are asymmetric in both length and diameter with paracrystalline material present only in the larger one. These features may be useful characters for taxonomy and phylogenetic studies.     

Organization of the cytoskeleton in early Drosophila embryos

The cytoskeleton of early, non-cellularized Drosophila embryos has been examined by indirect immunofluorescence techniques, using whole mounts to visualize the cortical cytoplasm and sections to visualize the interior. Before the completion of outward nuclear migration at nuclear cycle 10, both actin filaments and microtubules are concentrated in a uniform surface layer a few micrometers deep, while a network of microtubules surrounds each of the nuclei in the embryo interior. These two filament-rich regions in the early embryo correspond to special regions of cytoplasm that tend to exclude cytoplasmic particles in light micrographs of histological sections. After the nuclei in the interior migrate to the cell surface and form the syncytial blastoderm, each nucleus is seen to be surrounded by its own domain of filament-rich cytoplasm, into which the cytoskeletal proteins of the original surface layer have presumably been incorporated. At interphase, the microtubules seem to be organized from the centrosome directly above each nucleus, extending to a depth of at least 40 microns throughout the cortical region of cytoplasm (the periplasm). During this stage of the cell cycle, there is also an actin "cap" underlying the plasma membrane immediately above each nucleus. As each nucleus enters mitosis, the centrosome splits and the microtubules are rearranged to form a mitotic spindle. The actin underlying the plasma membrane spreads out, and closely spaced adjacent spindles become separated by transient membrane furrows that are associated with a continuous actin filament-rich layer. Thus, each nucleus in the syncytial blastoderm is surrounded by its own individualized region of the cytoplasm, despite the fact that it shares a single cytoplasmic compartment with thousands of other nuclei.     

Somatic nuclear division in the sporidia of Ustilago violacea. IV. Microtubules and the spindle-pole body.

In unbudded cells of the anther smut fungus Ustilago violacea there is a dome-shaped spindle-pole body (SPB) consisting of a core 0.1 mum in diameter surrounded by a ribosome-free region 0.3-0.4 mum in diameter lying in a pocket of the nuclear membrane. After budding the nucleus moves towards the bud and begins to rotate rapidly. At about this stage the SPB divides into two parallel bars each about 0.1-0.15 mum in diameter and 0.3 mum long, separated by a distance of about 0.3 mum. Microtubules associated with the nuclear membrane but not with the SPB are present at the time of nuclear rotation. These microtubules disappear when rotation stops. Microtubules attached to the SPB are found during migration of the chromatinic portion of the nucleus into the bud cell. These microtubules disappear when migration stops and the nuclear membrand begins to break down. The twin SPB bars appear to move into the nucleus through a break in the membrane and begin to move apart forming a spindle about 1 mum long. Chromosomal microtubules (one per kinetochore) were found in several serial sections, and in addition there appeared to be several continuous microtubules present. The separation of the two chromatinic masses appeared to result from elongation of the continuous microtubules to about 3 mum long. Cytoplasmic microtubules and spindle microtubules were both found attached to the SPB as it elongated and one nucleus returned to the mother cell. The paper concludes with a discussion of the SPB as a multifuncitonal control center affecting nuclear migration, spindle formation, membrane breakdown and synthesis, karyogamy, conjugation, budding, chromosomal movement, replication, and disjunction.     

Meiotic maturation of mouse oocytes in vitro: inhibition of maturation at specific stages of nuclear progression.

In vitro studies of meiotic maturation of mouse oocytes have been carried out in the presence of several drugs. The individual steps of nuclear progression, including dissolution of the nuclear (germinal vesicle) membrane, condensation of dictyate chromatin into compact bivalents, formation of the first metaphase spindle, and extrusion of the first polar body, are each susceptible to one or more of these drugs. Germinal vesicle breakdown, the initial morphological feature characteristic of meiotic maturation, is inhibited by dibutyryl cyclic AMP. However, even in the presence of dibutyryl cyclic AMP, the nuclear membrane becomes extremely convoluted and condensation of chromatin is initiated but aborts at a stage short of compact bivalents. Germinal vesicle breakdown and chromatin condensation take place in an apparently normal manner in the presence of puromycin, Colcemid, or cytochalasin B. Nuclear progression is blocked at the circular bivalent stage when oocytes are cultured continuously in the presence of puromycin or Colcemid, whereas oocytes cultured in the presence of cytochalasin B proceed to the first meiotic metaphase, form an apparently normal spindle, and arrest. Emission of a polar body is inhibited by all of these drugs. The inhibitory effects of these drugs on meiotic maturation are reversible to varying degrees dependent upon the duration of exposure to the drug and upon the nature of the drug. These studies suggest that dissolution of the mouse oocyte's germinal vesicle and condensation of chromatin are not dependent upon concomitant protein synthesis or upon microtubules. On the other hand, the complete condensation of chromatin into compact bivalents apparently requires breakdown of the germinal vesicle. Failure of homologous chromosomes to separate after normal alignment on the meiotic spindle in the presence of cytochalasin B suggest that microfilaments may be involved in nuclear progression at this stage of maturation. Cytokinesis, in the form of polar body formation, is blocked when any one of the earlier events of maturation fails to take place.     

Microtubule configurations and post-translational alpha-tubulin modifications during mammalian spermatogenesis

The mechanisms underlying cell cycle progression and differentiation are tightly entwined with changes associated in the structure and composition of the cytoskeleton. Mammalian spermatogenesis is a highly intricate process that involves differentiation and polarization of the round spermatid. We found that pachytene spermatocytes and round spermatids have most of the microtubules randomly distributed in a cortical network without any apparent centrosome. The Golgi apparatus faces the acrosomal vesicle and some microtubules contact its surface. In round spermatids, at step 7, there is an increase in short microtubules around and over the nucleus. These microtubules are located between the rims of the acrosome and may be the very first sign in the formation of the manchette. This new microtubular configuration is correlated with the beginning of the migration of the Golgi apparatus from the acrosomal region towards the opposite pole of the cell. Next, the cortical microtubules form a bundle running around the nucleus perpendicular to the main axis of the cell. At later stages, the nuclear microtubules increase in size and a fully formed manchette appears at stage 9. On the other hand, acetylated tubulin is present in a few microtubules in pachytene spermatocytes and in the axial filament (precursor of the sperm tail) in round spermatids. Our results suggest that at step 7, the spermatid undergoes a major microtubular reordering that induces or allows organelle movement and prepares the cell for the formation of the manchette and further nuclear shaping. This new microtubular configuration is associated with an increase in short microtubules over the nucleus that may correspond to the initial step of the manchette formation. The new structure of the cytoskeleton may be associated with major migratory events occurring at this step of differentiation.     

Morphogenetic processes inAttheya decora (Bacillariophyceae,Biddulphiineae)

The valva of the diatomAttheya decora is formed within a silica deposition vesicle which enlarges centrifugally by the fusion of small vesicles. The silica deposition vesicle can already be seen when the spindle has not yet disappeared completely. Valva formation seems to begin with the shaping of an organic matrix within a silica deposition vesicle. Later, this material silicifies. The complicated shape of the labiate process is preformed by the silica deposition vesicle, the inner membrane of which is associated with electron dense material on both faces. The horns are formed when the expanding silica deposition vesicle has reached the cell corners. They are elaborated without participation of microtubules. Swelling of local depositions of polysaccharides seems to provide the forces that spread the silicified horns during daughter cell separation and to cause the local spontaneous plasmolyses under the valva and along the cell flanks in the region of the intercalary bands. The inner organic wall layers and the organic continuations of the intercalary bands are formed on the surface of the plasmalemma; each of the continuations is produced simultaneously with the intercalary band belonging to it and becomes attached to the latter when the silica deposition vesicle opens.     

Cyclic assembly-disassembly of cortical microtubules during maturation and early development of starfish oocytes

An extensive array of cortical microtubules in oocytes of the starfish Pisaster ochraceus undergoes multiple cycles of disappearance and reappearance during maturation and early development. These events were studied in isolated fragments of the oocyte cortex stained with antitubulin antibodies for indirect immunofluorescence. The meshwork of long microtubules is present in the cortex (a) of immature oocytes, i.e., before treatment with the maturation-inducing hormone 1-methyladenine, (b) for 10-20 min after treatment with 1-methyladenine, (c) after formation of the second polar body (in reduced numbers in unfertilized oocytes), and (d) in the intermitotic period between first and second cleavage divisions. The array of cortical microtubules is absent in oocytes (a) undergoing germinal vesicle breakdown, (b) during the two meiotic divisions (polar body divisions), and (c) during mitosis of the first and, perhaps, subsequent cleavage divisions. The cycle of assembly-disassembly of cortical microtubules is synchronized to the cycle of nuclear envelope breakdown and reformation and to the mitotic cycle; specifically, cortical microtubules are present when a nucleus is intact (germinal vesicle, female pronucleus, zygote nucleus, blastomere nucleus) and are absent whenever a meiotic or mitotic spindle is present. These findings are discussed in terms of microtubule organizing centers in eggs, possible triggers for microtubule assembly and disassembly, the eccentric location of the germinal vesicle, and the regulation of oocyte maturation and cell division.     

Spermiogenesis and sperm ultrastructure in the monogenean parasite Acanthocotyle lobianchi

The ultrastructural events of spermiogenesis and the ultrastructure of the mature spermatozoon of an acanthocotylid monogenean, Acanthocotyle lobianchi, are described. The early zone of differentiation (ZD) contains two roughly perpendicular centrioles which become parallel and produce two free flagella, although these later become incorporated into the same body of cytoplasm. No cortical microtubules were found supporting the ZD at any stage of spermiogenesis. Much of the length of the thread-like sperm contains two axonemes of the 9 + '1' pattern together with a nuclear and mitochondrial profile but the 'posterior' region is occupied only by a single axoneme and the nucleus. A laterally situated electron-lucent vesicle with specialization of the adjacent surface membrane is found in the 'anterior' region of the sperm. The phylogenetic implications of these observations are discussed.     

Aggregation of microtubule initiation sites preceding neurite outgrowth in mouse neuroblastoma cells.

By examining microtubule regrowth using immunofluorescence with antibody to tubulin, we have studied the structure and intracellular localization of microtubule initiation sites in undifferentiated and differentiated mouse neuroblastoma cells. The undifferentiated cells are round and lack cell processes. They contain an average of 12 initiation sites per cell. Each of these sites, which are located near the cell nucleus, initiates the growth of several microtubules in a radial formation. In contrast to the undifferentiated cells, neuroblastoma cells stimulated to differentiate by serum deprivation are asymmetrical, containing one or two very long neurites. These cells have a single, large microtubule initiation center which can be visualized not only by immunofluorescence but by phase-contrast and differential interference microscopy as well. The initiation site measures 3-4 mu in diameter and is located in the cell body along a line defined by the neurite. During cell differentiation, the large initiation, the large initiation center seems to be formed by the aggregation of many smaller sites. This process procedes neurite extension by about 24 hr. The growth of microtubules from this center appears to be highly oriented, since most microtubules initially grow into the neurite processes rather than into the cell interior. Thus major changes in the structure and location of microtubule initiation sites occur during the differentiation of neuroblastoma cells. Similar changes are likely to be involved in alterations in the morphology of other cell types.     

Microtubule organization by the budding yeast spindle pole body.

In budding yeast microtubule organizing functions are provided by the spindle pole body (SPB), a multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle. The SPB organizes the nuclear and cytoplasmic microtubules which are spatially and functionally distinct. Microtubule formation in yeast requires the Tub4p-complex, containing the gamma-tubulin Tub4p, and two additional proteins, the SPB components Spc97p and Spc98p. The Tub4p complex assembles in the cytoplasm and is then anchored to the sides of the SPB which organize microtubules. This is achieved by the binding of Spc97p and Spc98p to so-called gamma-tubulin complex binding proteins (GTBPs) at the SPB. Spc72p is the yeast GTBP at the cytoplasmic side of the SPB, while Spc110p is the nuclear GTBP. Both GTBPs control the number of Tub4p complexes associated with the SPB and thereby the number of microtubules formed. In addition, the GTBPs may regulate the activity of the Tub4p complex. Homologues of Spc97p and Spc98p have been identified from yeast to mammalian cells and these are also part of gamma-tubulin complexes, suggesting that these related proteins may also interact with GTBPs at the centrosome. Candidates for GTBPs have been identified in mammalian and insect cells.     

Petasiger oschmarini n. sp. (Digenea: Echinostomatidae) in the grebes Podiceps grisegena (Bod.) and P. auritus (L.) (Aves: Podicipedidae) from Kamchatka,Russia

A re-examination of three sets of trematodes collected in two species of grebes, Podiceps grisegena and P. auritus, from the Kamchatka region of eastern Russia, previously identified as Petasiger neocomense, revealed that they actually represent a new species, which is named Petasiger oschmarini. Characteristic morphological features of the new species include: a head collar possessing a constant number of 19 spines; a large cirrus-sac comparable in size to the ventral sucker, containing a bipartite seminal vesicle and a well-developed pars prostatica; a large bulb-like cirrus similar in size to the testes; a long forebody; a long oesophagus; testes situated obliquely or symmetrically; and lateral vitelline fields which are not confluent in the forebody. The new species is distinguished from all Palaearctic and Nearctic members of Petasiger which possess 19 collar spines. The material described by Oschmarin (1950) as P. neocomense is considered to be conspecific.