Role of major sperm protein (MSP) in the protrusion and retraction of Ascaris sperm.

Nematode sperm offer a unique perspective for investigating amoeboid cell motility. These cells display the hallmark features of amoeboid movement but power their locomotion with a cytoskeleton composed of major sperm protein (MSP) filaments in place of the familiar actin cytoskeleton found in other crawling cells. Thus, properties of sperm can be compared to those of actin-rich cells to identify the shared features that are essential to motility. Sperm are simple cells in which cytoskeletal dynamics are tightly coupled to protrusion of the leading edge and retraction of the cell body. These features have facilitated reconstitution of both protrusion and retraction in cell-free extracts and enabled identification of accessory components in the motility apparatus as well as elucidation of the mechanical basis of movement. Six MSP accessory proteins have been isolated including four components of the sperm cytoskeleton and two enzymes that play key roles in regulating cytoskeletal dynamics and locomotion. Analysis of this versatile in vitro motility system has identified motor-independent mechanisms for protrusion and retraction that are based on changes in filament-packing density. These changes result in expansion and contraction of the MSP-filament network that generate the forces for movement. We discuss how the mechanisms of motility that operate in nematode sperm may contribute generally to the movement of crawling cells.

Dephosphorylation of major sperm protein (MSP) fiber protein 3 by protein phosphatase 2A during cell body retraction in the MSP-based amoeboid motility of Ascaris sperm.

The crawling movement of nematode sperm requires coordination of leading edge protrusion with cell body retraction, both of which are powered by modulation of a cytoskeleton based on major sperm protein (MSP) filaments. We used a cell-free in vitro motility system in which both protrusion and retraction can be reconstituted, to identify two proteins involved in cell body retraction. Pharmacological and depletion-add back assays showed that retraction was triggered by a putative protein phosphatase 2A (PP2A, a Ser/Thr phosphatase activated by tyrosine dephosphorylation). Immunofluorescence showed that PP2A was present in the cell body and was concentrated at the base of the lamellipod where the force for retraction is generated. PP2A targeted MSP fiber protein 3 (MFP3), a protein unique to nematode sperm that binds to the MSP filaments in the motility apparatus. Dephosphorylation of MFP3 caused its release from the cytoskeleton and generated filament disassembly. Our results suggest that interaction between PP2A and MFP3 leads to local disassembly of the MSP cytoskeleton at the base of the lamellipod in sperm that in turn pulls the trailing cell body forward.

Reconstitution in vitro of MSP-based filopodium extension in nematode sperm.

The major sperm protein (MSP) motility system in nematode sperm is best known for propelling the movement of mature sperm, where it has taken over the role usually played by actin in amoeboid cell motility. However, MSP filaments also drive the extension of filopodia, transient organelles composed of a core bundle of MSP filaments, that form in the late in sperm development but are not found on crawling cells. We have reconstituted filopodial extension in vitro whereby thin bundles of MSP filaments, each enveloped by a membrane sheath at their growing end, elongated at rates up to 17 microm/min. These bundles often exceeded 500 microm in length but were comprised of filaments only 1 microm long. The reconstituted filopodia assembled in the same cell-free sperm extracts that produced MSP fibers, robust meshworks of filaments that exhibit the same organization and dynamics as the lamellipodial filament system that propels sperm movement. The filopodia and fibers that assembled in vitro both had a membranous structure at their growing end, shared four MSP accessory proteins, and responded identically to agents that alter MSP-based motility by modulating protein phosphorylation. However, filopodia grew three- to four-fold faster than fibers. The reconstitution of filopodial extension shows that, like the actin cytoskeleton, MSP filaments can adopt two architectures, bundles and meshworks, each capable of pushing against membranes to generate protrusion. The reconstitution of both forms of motility in the same in vitro system provides a promising avenue for understanding how the forces for membrane protrusion are produced.

Rediscovering Boveri's centrosome in Ascaris (1888): its impact on human fertility and development.

We rediscover and review the brilliant work of Theodore Boveri, over a 100 years ago, on the centrosome of the round worm Ascaris and show how it impacts on our understanding of human fertilization and embryogenesis. Boveri was able to make fundamental predictions on the mechanics of fertilization and the dominant role of the sperm centrosome (Boveri's rule), which is now applicable to most animals. Using advanced digital imaging by light and electron microscopy, we explore centrosomal dynamics during Ascaris fertilization and the first cell cycle during cleavage. Twenty figures are presented in this visual publication. Humans follow Boveri's rule, as do most mammals excluding some rodents, and there is a remarkable similarity of the events of fertilization and cleavage in Ascaris and humans, the latter of which has been documented since 1991. The role of the sperm centrosome (centriole) in egg activation, polarity, embryogenesis, infertility and cancer is discussed. An attempt is made to portray the images Boveri may have visualized, in his painstaking drawings presented in his thesis in 1888. We now know the origins of the centrosome in human somatic cells--predominantly from the sperm cell. The impact of Boveri's work on human development is highlighted in this age of assisted reproductive technology.

Structure and macromolecular assembly of two isoforms of the major sperm protein (MSP) from the amoeboid sperm of the nematode, Ascaris suum.

Ascaris sperm are amoeboid cells that crawl by extending pseudopods. Although amoeboid motility is generally mediated through an actin-based cytoskeleton, Ascaris sperm lack this system. Instead, their major sperm protein (MSP) forms an extensive filament system that appears to fulfil this function. Because their motility appears to be essentially the same as that of their actin-rich counterparts, Ascaris sperm offer a simple alternative system for investigation of the molecular mechanism of amoeboid movement. To examine the structure and composition of the cytoskeleton, we stabilized the extremely labile native MSP filaments by detergent lysis of sperm in the presence of either glutaraldehyde or polyethylene glycol (PEG). Biochemical analysis showed that the cytoskeleton contained two isoforms of MSP, designated alpha- and beta-, that we purified and sequenced. Both contain 126 amino acids and have an acetylated N-terminal alanine, but differ at four residues so that alpha-MSP is 142 Da larger and 0.6 pH unit more basic than beta-MSP. Neither isoform shares sequence homology with other cytoskeletal proteins. In ethanol, 2-methyl-2,4-pentanediol (MPD), and other water-miscible alcohols each isoform assembled into filaments 10 nm wide with a characteristic substructure repeating axially at 9 nm. These filaments were indistinguishable from native fibers isolated from detergent-lysed sperm. Pelleting assays indicated a critical concentration for assembly of 0.2 mM for both isoforms in 30% ethanol, but alpha-MSP formed filaments at lower solvent concentration than beta-MSP. When incubated in polyethylene glycol, both isoforms formed thin, needle-shaped crystals that appeared to be constructed from helical fibers, with a 9 nm axial repeat that matched that seen in isolated filaments. These crystals probably contained a parallel array of helical filaments, and may enable both the structure of MSP molecules and their mode of assembly into higher aggregates to be investigated to high resolution.

Distribution of a fluorescent ivermectin probe, bodipy ivermectin, in tissues of the nematode parasite Ascaris suum.

A fluorescent derivative of the anthelmintic ivermectin (4''-5,7-dimethyl bodipy proprionylivermectin, referred to hereafter as bodipy ivermectin) was synthesized for an investigation of the distribution of avermectins. Injected into adult Ascaris suum at doses greater than 0.1 micron per worm, it produced a dose-dependent immobilization. Fluorescent microscopy of frozen sections revealed the distribution of the probe in the whole nematode. Staining of collagenase-isolated muscle cells was studied using bath application of bodipy ivermectin. The trypan-blue quenching technique showed that the ivermectin probe was located in the outer monolayer of the muscle membrane. The cytoplasm was not stained. The interpretation of these observations is discussed in view of the known lipophilic nature of avermectins. Staining of the muscle membrane and nerve cord is consistent with the view that avermectins act at these sites. The significance of the hypodermal and lateral line staining is also discussed.

Morphology of the uterus of Ascaris lumbricoides in the region where fertilization and formation of egg-shell occur.

Morphology of the female reproductive system of Ascaris lumbricoides L. was studied in the region starting with the junction between the oviduct and the uterus (O-U) up to the junction of both uterine branches into the vagina with regard to the process of fertilization and formation of egg-shells. In the O-U junction morphology differed in two following sections: a continuous simple squamous up to simple cuboidal epithelium, and simple cuboidal up to columnar epithelium with broad intercellular spaces leading into the lumen of the tubular reproductive organ filled with sperm. The area in the O-U junction zone was found where the wall of the organ was formed by elongated club-shaped cells attached to the common basal lamina by a narrow pedicle. Intercellular spaces thus formed "crypts" which was covered with dilated parts of cells towards the tubular lumen. Crypts were found to be filled with sperm. This area resembles the structure known as the receptaculum seminis where the stored sperm survive. Epithelial cells of the uterus are of cuboidal up to columnar shape with signs of merocrine secretion. In the distal part of the uterus the secretory active cells probably produce viscous secreta allowing the transfer of the eggs towards the vagina. The cells of the uterus wall are elongated and because of their longer axis, they are orientated longitudinally. In centripetal parts, the cell walls do not have contact with each other and form elongated, deep furrows ("canyons") through which the sperm can run against the flow of uterus content up to the junction of the O-U, where they are stored in the spermatheca-like structure. At any time they are disposal for fertilization.

New telomere formation after developmentally regulated chromosomal breakage during the process of chromatin diminution in Ascaris lumbricoides.

During the process of chromatin diminution, which takes place in all presomatic cells of the early Ascaris embryo, the heterochromatic termini of the chromosomes are lost. Here we show that the newly formed ends of the reduced somatic chromosomes carry tandem repeats of the telomeric sequence TTAGGC. Comparison of a cloned somatic telomere with the corresponding germline chromosomal region revealed that these telomeric repeats are not present at or near the chromosomal breakage site. They are most likely added by a telomerase-mediated event. Chromosomal breakage, which precedes the telomere addition process, takes place within a short, specific chromosomal region (CBR); however, it does not occur at a single locus, but rather at many different sites. Altogether, our data show that chromatin diminution in Ascaris is a complex molecular process that includes site-specific chromosomal breakage, new telomere formation, and DNA degradation.

Colonization of Ascaris lumbricoides eggs by the fungus Verticillium chlamydosporium Goddard.

The process of colonization of Ascaris lumbricoides eggs by the fungus Verticillium chlamydosporium was studied by scanning electron microscopy. The preparations were made by fractionation of egg suspension exposed to the fungus for four days and frozen in liquid nitrogen according to Sterba and Milácek (1986). Ovicidal fungus forms an abundant ramifying mycelial network in the area between the eggs. However, egg-shells are penetrated only by some hyphae without any penetration organs produced (simple hyphal penetration). In a liquid medium, after penetration, hyphae inside the eggs rapidly grow among inner structures of egg-shells and on the surface of developing larvae. In the next phase, hyphae colonize the developing larva. The eggs attacked by this fungus remain morphologically unchanged for a long time except the sites of penetration. Verticillium chlamydosporium is a fungus with unique ovicidal properties. It colonizes the eggs of Ascaris lumbricoides at all stages of embryo development and also attacks larvae inside the eggs.

Antibodies against the cuticlin of Ascaris suum cross-react with epicuticular structures of filarial parasites.

The insoluble cuticlin from the cortical zone of the cuticle of adult Ascaris suum was purified and used to raise antibodies in C57/bl mice. The specificity of the antibodies for the external cortical layer was shown in the indirect immunofluorescence antibody test and by immunoelectronmicroscopy. A very high specificity for the external cortical layer was found. Some cross-reactions with cuticular collagens occurred, and increased after booster immunizations. The anti-cuticlin antibody cross-reacted with the electron-dense layers of the cortical zones of adult Acanthocheilonema viteae and Brugia pahangi. A very weak reaction was found in the cortical zone of adult Onchocerca volvulus. In the cuticles of third stage larvae of all three species mainly epitopes in the cortical zones were labelled. In no case did the anti-cuticlin antibody interact with the outermost surface of the cuticle.

Biotin as a probe of the surface of Ascaris suum developmental stages.

Sulfo-NHS-biotin (aqueous soluble) and NHS-biotin (organic soluble) labeled similar SDS-2ME (sodium dodecyl sulfate/beta-mercaptoethanol) soluble cuticular proteins of second stage larvae (L2) and third stage Ascaris suum larvae (L3). Comparable analysis of biotin-labeled fourth stage larvae (L4), young adults, and mature adult Ascaris suum revealed strong labeling of several SDS-2ME soluble cuticular proteins with NHS-biotin, while sulfo-NHS-biotin appeared to strongly label a single SDS-2ME soluble cuticular protein. Both biotin probes labeled only cuticular proteins, since no evidence of internal labeling was observed in any developmental stage examined by either electroblot analysis or by electron microscopy. Our data suggest a greater cuticular permeability to the organic soluble biotin reagent in the later developmental stages (greater than L3) of A. suum than to the aqueous soluble biotin reagent, and may indicate the presence of a hydrophobic barrier in the cuticle of the later stages of the parasite.

Surface properties of membrane vesicles prepared from muscle cells of Ascaris suum.

To facilitate biochemical, pharmacological, and biophysical studies on the membrane of the body muscle of Ascaris suum, a method for preparing intact vesicles was developed. Vesicles were prepared by incubating a muscle flap preparation with 1 mg/ml collagenase in a saline solution and then washing in saline without enzyme. The vesicles then formed gradually over the next hour as outgrowths of the original surface membrane from the bag region of the muscle. The vesicles were harvested readily by suction using a Pasteur pipette. The structure of the vesicles was examined with the transmission electron microscope. The whole-cell patch-clamp technique showed that the vesicles had a high input resistance and that the membrane was complete. The vesicle membrane was shown to contain Ca-activated Cl channels and gamma-aminobutyric acid-activated Cl channels. The vesicles also were shown to be suitable for fluorescence recovery after photobleaching studies designed to examine lateral and vertical movement of a lipid probe (5-N [octadecanoyl]-aminofluorescein) in the membrane. This probe had a mean lateral diffusion coefficient (DL) of 8.1 x 10(-9) cm2/sec, but only a proportion (68.4%) of the probe was mobile. The latter observation illustrated the nonuniform nature of the membrane. Ivermectin (10(-7) M) had no effect on DL or percent recovery. Trypan blue quenching experiments showed that the lipid probe remained in the outer monolayer of the membrane. These observations illustrate the experimental value of the vesicles; they are potentially useful in discerning anthelmintic mode of action and in drug screening.

[Morphologic characteristics of the eggs of Ascaris lumbricoides in coprological findings in humans]. / Morfologické zvlástnosti vajícok Ascaris lumbricoides v koprologických nálezoch u ludí.

On microphotographs the morphological peculiarities of the eggs of Ascaris lumbricoides are demonstrated. Diagnostic problems caused by these anomalies are discussed. Study of the variations of this parasite can contribute to a better understanding of the morphology, ecology and biology of intestinal parasites.

A unique cytoskeleton associated with crawling in the amoeboid sperm of the nematode, Ascaris suum.

Nematode sperm extend pseudopods and pull themselves over substrates. They lack an axoneme or the actin and myosins of other types of motile cells, but their pseudopods contain abundant major sperm protein (MSP), a family of 14-kD polypeptides found exclusively in male gametes. Using high voltage electron microscopy, a unique cytoskeleton was discovered in the pseudopod of in vitro-activated, crawling sperm of the pig intestinal nematode Ascaris suum. It consists of 5-10-nm fuzzy fibers organized into 150-250-nm-thick fiber complexes, which connect to each of the moving pseudopodial membrane projections, villipodia, which in turn make contact with the substrate. Individual fibers in a complex splay out radially from its axis in all directions. The centripetal ends intercalate with fibers from other complexes or terminate in a thickened layer just beneath the pseudopod membrane. Monoclonal antibodies directed against MSP heavily label the fiber complexes as well as individual pseudopodial filaments throughout their length. This represents the first evidence that MSP may be the major filament protein in the Ascaris sperm cytoskeleton. The large fiber complexes can be seen clearly in the pseudopods of live, crawling sperm by computer-enhanced video, differential-interference contrast microscopy, forming with the villipodia at the leading edge of the sperm pseudopod. Even before the pseudopod attaches, the entire cytoskeleton and villipodia move continuously rearwards in unison toward the cell body. During crawling, complexes and villipodia in the pseudopod recede at the same speed as the spermatozoon moves forward, both disappearing at the pseudopod-cell body junction. Sections at this region of high membrane turnover reveal a band of densely packed smooth vesicles with round and tubular profiles, some of which are associated with the pseudopod plasma membrane. The exceptional anatomy, biochemistry, and phenomenology of Ascaris sperm locomotion permit direct study of the involvement of the cytoskeleton in amoeboid motility.

ESR studies on iron-sulfur clusters of complex II in Ascaris suum mitochondria which exhibits strong fumarate reductase activity.

Complex II of Ascaris suum mitochondria, which functions as fumarate reductase in physiological conditions, contains three types of iron-sulfur clusters. These correspond to clusters S-1, S-2 and S-3 and are distinguishable by low-temperature ESR studies. Cluster S-1 is reduced by succinate, giving ESR signals with gz, gy and gx values at 2.033, 1.939 and 1.920. The existence of cluster S-2 is suggested by an enhancement of the S-1 spin relaxation induced upon reduction of S-2 by dithionite. Cluster S-3 is ESR detectable under air-oxidized conditions and gives a strong signal at g = 2.025. Cluster S-3 was only partially reduced even with an excess amount of sodium succinate, which is a common characteristic of fumarate reductase but this is not seen in the mitochondrial complex II.

Electron-transfer complexes of Ascaris suum muscle mitochondria. III. Composition and fumarate reductase activity of complex II.

Complex II of the anaerobic respiratory chain in Ascaris muscle mitochondria showed a high fumarate reductase activity when reduced methyl viologen was used as the electron donor. The maximum activity was 49 mumol/min per mg protein, which is much higher than that of the mammalian counterpart. The mitochondria of Ascaris-fertilized eggs, which require oxygen for its development, also showed fumarate reductase activity with a specific activity intermediate between those of adult Ascaris and mammals. Antibody against the Ascaris flavoprotein subunit reacted with the mammalian counterparts, whereas those against the Ascaris iron-sulfur protein subunit did not crossreact, although the amino acid compositions of the subunits in Ascaris and bovine heart were quite similar. Cytochrome b-558 of Ascaris complex II was separated from flavoprotein and iron-sulphur protein subunits by high performance liquid chromatography with a gel permeation system in the presence of Sarkosyl. Isolated cytochrome b-558 is composed of two hydrophobic polypeptides with molecular masses of 17.2 and 12.5 kDa determined by gradient gel, which correspond to the two small subunits of complex II. Amino acid compositions of these small subunits showed little similarity with those of cytochrome b-560 of bovine heart complex II. NADH-fumarate reductase, which is the final enzyme complex in the anaerobic respiratory chain in Ascaris, was reconstituted with bovine heart complex I, Ascaris complex II and phospholipids. The maximum activity was 430 nmol/min per mg protein of complex II. Rhodoquinone was essential for this reconstitution, whereas ubiquinone showed no effect. The results clearly indicate the unique role of Ascaris complex II as fumarate reductase and the indispensability of rhodoquinone as the low-potential electron carrier in the NADH-fumarate reductase system.

[Three recent cases of ascariasis in northern Kyushu].

Ascariasis is considered to be one of the rare infectious diseases in Japan, but recently it has been slightly increasing. This paper reports three ascariasis cases who seemed to be infected recently in the Kitakyushu area, Japan. Case 1: A 59-year-old woman in Kitakyushu City passed a round worm after continuous abdominal pain. The patient was discharged from the hospital because of no further abnormal intestinal symptoms and findings. Case 2: An 85-year-old woman in Nakama City, who suffered from cerebral infarction, vomited a round worm before hospitalization. Many ascarid eggs were detected after admission, and after treatment with pyrantel pamoate (Combantrin) two round worms were passed and egg detection became negative. Case 3: A 77-year-old man in Saikawa Town vomited 3 round worms after gastrectomy due to early gastric cancer. Many unfertilized eggs were also detected from the stool together with hook worm eggs, but no eggs were found after administration of pyrantel pamoate. Morphological examination was made by a scanning electron microscope on the denticles on the dentigerous lip ridges of the worms to differentiate from possible infection with a pig parasite, Ascaris suum. The three cases were diagnosed as ascariasis due to human Ascaris lumbricoides based on the following evidences that the expelled worms had 1) less pointed tips of the denticles and shallower or wider interdenticle notches, and 2) far more denticles of smaller size along the dentigerous ridges, compared with Ascaris suum. The necessity of differentiating pig- from human-ascarids, when considering human infection with Ascaris suum, is discussed.

Intermediate filaments in the giant muscle cells of the nematode Ascaris lumbricoides; abundance and three-dimensional complexity of arrangements.

The body muscle cells of the nematode Ascaris lumbricoides are characterized by massive amounts of intermediate filaments (IF). These occur in all three regions of this giant cell type. They traverse the cytoplasm of the balloon-like belly, which houses the nucleus, and occur as bundles in the arm-like extensions to the nerve. The organization of IF in the third region, the contractile fiber, was analyzed further by serial sections and three-dimensional reconstruction. IF bundles traverse the glycogen-rich lumina of the fiber and reach as baskets around the sarcomeres. Together with numerous dense bodies they form the Z-band-like arrangements. IF bundles reach the plasma membrane at hemidesmosome-like specializations often situated at deep membrane invaginations filled with a fibrillar component of the extracellular matrix. The ultrastructural appearance of IF bundles is connected to the contractional state of the sarcomeres. They appear straight in extended muscle but coil up upon contraction. In the pharynx massive IF bundles are oriented longitudinally. A second type of IF bundles follows the radially oriented sarcomeres. These reveal pronounced Z-band type structures with massive disks. IF surround the sarcomeres and seem to terminate at these disks. We discuss possible functions of the complex IF organization in body muscle and pharynx.

Lagochilascariasis humana en Venezuela: descripción de un caso fatal / Human lagochilascariasis in Venezuela: report of a lethal case

Levamisol (fenilimidotiazol), considerado um potente imunoestimulante, quando administrado a camundongos suíços näo causou aumento significante nos pesos do timo, figado ou baço, apesar de a droga ter sido usada em diferentes tempos antes da remoçäo desses órgäos. Doses elevadas da droga usadas no esquema profilático de 4 dias näo tiveram efeito anti-malárico. Entretanto quando dada a camundongos com malária, 24 horas antes, ao mesmo tempo ou 24 horas após inoculaçäo de uma cepa de Plasmodium berghei cloroquina-sensível ou uma cepa cloroquina-resistente o levamisol reduziu, ainda que discretamente, a parasitemia nos grupos tratados, sendo a dose de 1 mg/kg o melhor esquema. Foi observado também atraso na mortalidade por malária nos grupos tratados com o levamisol. No entanto, todos os animais morreram. Os dados sugerem que o levamisol tem efeito imunoestimulante, ainda que discreto, na resposta imune de animais, deprimida pela malária