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Nematode sperm
Reviews
Nematode
Sperm
A.L. Scott Parasitic nematode infections remain a major public health problem in many parts of the world. Because most of the current strategies aimed at controlling parasitic nematode infections have met with only limited success, it may be time to consider alternative approaches. An aspect of nematode biology that has drawn little attention as a target for control is the reproductiw" process. Although there are mmlerous facets of the overall reproductive biology of nematodes that hold potential as targets for intervention, Alan Scott here focuses on the male reproductive system, and outlines some of the known unique processes and characteristics of sperm formation and sperm function that could be exploited to block fertilization. The information on the behavior, developmental biology and biochemistry of nernatode sperm presented here was pieced together with the data and observations front a variety of nematode species, with a majority of the information corning from studies on the free-living Caenorhabditis eh-e~ans and the parasitic species Ascaris suum and Brugia malayi.
Anatomy of the male reproductive system For most nematode species, the male reproductive system is composed of a single testis that runs the length of the worm. Within the testis of each reproductively active male can be found cells from all of the stages of spermatogenesis (Fig. 1). The testis consists of a distal zone of germ-cell formation, a zone of maturation and a proximal storage area for the immature sperm consisting of a seminal vesicle a n d / o r a vas deferens. Most parasitic nematode species have telogonic testes, "vhvre germ-cell production is confined to the extren:e distal portion of the testis, in some nematode species, a seminal vesicle is positioned at the proximal end of the growth zone and serves as a specialized storage area for sperm until copulation. A muscular region surrounds a specialized region of the vas deferens that forms an ejaculatory duct which opens into a cloaca. Male nematodes have a number of secondary sexual organs including a cloaca, a bursa, spicules, a gubernaculum and cement glands, all of which aid in grasping the female during copulation I. The cloaca is most commonly located on the ventral surface and is surrounded by a number of sensillae. In some nematodes, the area around the cloaca forms an expanded structure called the copulatory bursa which may involve the entire ventral surface of the tail 1,2. Male nematodes possess one or two copulatory spicules which are formed from hardened cuticle. The function of the spicules is to dilate the vulva and vagina of the female during copulation. The spicules have neurons running up their center, which terrninate in sensory Alan L. Scott is at the Department of Molecular Microbiology and Immunology, School of Hygiene and Public Health, The Johns Hopkins University, 615 North Wolfe Street, Baltimore. MD 212052 ! 79, USA. Tel: + I 410 955 3442, Fax: 4, I 410 955 0105, e-maih [email protected] ! .sph.jhu.edu Parasitology Today, vol. 12, no. I I, 1996
dendrites called genital sensillae; these are thought to allow the spicule to feel its way into the female without damaging her reproductive tissues. The gubernaculum is a grooved plate which functions as a guide for the spicules.
Spermatogenesis Spermatogenesis proceeds linearly from the spermatogonial cells in the distal tip of the testis to the spermatozoa in the vas deferens (Fig. 1). During the first steps in spermatogenesis, primary spermatocytes enter meiosis and proceed through pachytene while attached via a cytoplasmic bridge to a common cytoplasmic core structure termed the rachis (Figs 1, 2a). The spermatocytes then bud from the rachial syncycia and complete meiosis to form four haploid nuclei. The four haploid nuclei migrate to the rnargins of the cell and, along with a collection of selected organelles, bud from the central region of cytoplasm to form four spberical spermatids. A spermatid is the sessile, immature, storage forrn of nematode sperm. During the process of spermatid budding, all of the biosynthetic machinery is retained in a cytoplasmic remnant termed the residual body. Microtubules, microfilaments, endoplasmic reticulum, ribosomes and the Golgi complex remain in the residual body, which is eventually degraded and absorbed -~,4. Because all of the organelles necessary for biosynthetic activity remain with the residual body, neither spermatids nor mature sperm are capable of protein synthesis. This means that all of the molecules required for sperm differentiation and sperm function are synthesized early in spermatogenesis and ~tored in various forms in the spermatid.
Spermatids Spermatids can be round, rod-like or fusiform cells, depending on the nematode species, and range in diameter from 3 lain to 10 I~m (Ref. 2). Spermatid development in the filarid B. malayi is typical of many nematode species. Brugh7 mahlyi spermatids are rounded cells as they first bud from the residual body (Fig. l). Although in some species the mature spermatids remain rounded, the fully developed spermatids in Brughl take on a rod-like appearance and are densely packed into the vas deferens of the sexually active male (Figs 1, 2b, 2c). The nuclear material in the spermatid is highly condensed and is organized into two or three discrete, non-membrane-bound bodies (Fig. 3a). Brltgia spermatids also contain the membranebound organelles typically found in the spermatids of nearly all of the nematode species studied. These organelles include mitochondria, a large refringent body and a structure that appears to be unique to nematode sperm, the membranous organelle (MO) (Fig. 3). Also present in the cytoplasm of Brugia spermatids are structures that have been designated fibrous arrays. These structures do not appear in spermatids that remain rounded. Under the electron microscope, sagittal sections of spermatids show that the fibrous
Reviews sperm activation, there is a drastic remodciil:g of the spermatid that is characterized by the extension of a Primary pseudopod away from the cell body spermatocytes and a fusion of the MOs to the plasma membrane- (Figs 4, 5). Sperm Zone of activation is completed in a matter germ cell of minutes and at this point the cells formation become motile 7. _..l Most of the MOs in mature sperm fuse with the plasma membrane and form pores on the cell body portion of the sperm 2 (Fig. 5). MOs have a spherical head portion with an elec~ 3eomdary tron dense collar and a highly invagipermatocytes hated, microvillus-like internal netMaturation work a. The MOs first form in the /.Olte Early primary spermatocyte and combine " ~ spermatid with another unique organelle, the fibrous body (FB), to form fibrous bodv-meml:;ranous organelle (FBMO) cornplexes. FB-MO complexes 0 are transient and appear to be important in the storage and delivery of sperm proteins that were synResidual body thesized early in spermatogenesis to Storage the fully differentiated spermatozoa. /Olll2 in the rnature sperm, the MOs release a fibrous glycoprotein which ~ ~ sper at'd. adheres to the outside of the spermatid cell body. This glycoprotein is thought to be important for sperm ~ N N Malt.re sperm motility". The pseudopod, which is devoid Proximal of organeiles, contains a fTlanaentous le~li', or flocculate material (Fig. 5). The surface of the pseudopod has numerFig, I, An outline of spermatogenesls in the parasitic nematode Brugia molayi, ous projections (villipodia), which form at the tip of the pseudopod and move rapidly to the base (Fig. 4)"11. In Ascaris, arrays are positioned along the margins of the cell Brugia and Ancvlostoma sperm, the filarncntous just beneath the cell membrane (l:;'ig. 3a). The fibrous material is distributed throughout the pseuclopod arrays extend to the tip of the cell, but do not appear and becomes organized into a cytoskeleton composed to be attached or anchored to the tip (Fig. 3b). When of fibers arranged radially into long, branched fiber the cells are viewed in cross-section, the fibrous arcomplexes, which may traverse the length of the rays are positioned around the perimeter of the cell pseudopod"'.12.ts. (Fig. 3c). The radial arrangement of the fibrous arrays Although the sperm from a number of nernatode just under the cell membrane probably contributes to species have the basic morphology and distribution the rod-shaped appearance of the Brttgia spermatids. of organelles outlined in Figs 4 and 5, there is considerable variation in the sperrn cell form. For example, Sperm in the oxyurid Aspiculuris tetraptera the sperm have a Unlike flagellated sperm of mammals, mature nemadistinct 'tail' which contains a single, large, elongate tode sperm form pseudopodia and exhibit amoeboid mitochondrion, DNA and bundles of microtubules, or crawling motility s. Maturation of spermatids to but no MOs, and a distinct 'head' which sends out spermatozoa is triggered by mating and appears to pseudopodial projectionst L Descriptions of the varied take place within the vas deferens of C. eh'gans and morphologies of nematode sperm can be found elseAscaris. Activation is thought to be analogous to capwhere 2,~,~; I7. acitation in mammalian sperm. The exact events that result in the final maturation step are not known. For C. eh'gans3 and the filarial nematodes B. malayi and C o m p o s i t i o n of the fiber c o m p l e x e s In striking contrast to all other cells that employ Dirofilaria immits (A.L. Scott, unpublished), the spercrawling motility, nematode ~perm contain neither matids can be triggered to mature ;n vitro by proteases or agents that cause a rapid increase in intraactin nor myosin m. Instead, locomotion in nematode cellular pH, such as weak bases and the ionophore sperm depends on what appears to be a simple cytomonensin. For Ascaris, proteases as well as an unskeleton, based primarily on a family of small, basic, known substance present in extracts of Ascaris vas sperm-specific proteins that have been designated the deferens can activate spermatids in vitro 4-C'. During major sperm proteins ~MSP). "[he MSPs, all of which Digital testis
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426
rogenator cells
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Porasitology Today, vol. 12, no. I I, 1996
Reviews a,
Fig. 2. Different stages of development of Bru~;a ma~uyi sperm Primary spermatocytes assembled on the rachis (a). Spermatids packed into the storage region of the testis (b). A high magnification image of B. malayi spermatids (c). Photomicrographs were taken using Nomarski interference phase contrast. Scale bars -- 30 p.m (a), 30 ~m (b), 8 p.m (c).
have a molecular mass of 14 kDa, comprise about 15';~ of the total cellular protein and over ,t0'/, of the soluble protein "',a". The MSPs have no sequence similarity to any of the described filament-forming proteins such as actin, myosin, tubulin, tektin and spasmin 2t. The MSPs appear to be unique, nematodespecific, sperm-specific cytoskeletal proteins. In Ascaris 22, Onchocerca v01vulus20 and B. malayi (A.L. Scott, unpublished) there are two isoforms of MSP which are encc, ded oil two separate genes. In Ascaris, the two MSP isoforms (MSP~ and MSP[3) differ at only four out of 129 amino acids, while in O. voh,ulus and B. malayi the two isoforms differ at five of the 129 amino acids. Between species, the amino acid sequences of MSPs are between 80{7, and 90% identical. In contrast to the small number of genes encoding MSP in the relatively large parasitic nematodes, in the smaller, free-living nematode species C. eh'dans, the three apparent isoforms of MSP are produced from a multigene array having ~bout 30 members 23.24. MSP genes in various copy nu,nber have been identified in over 25 nematode species representing 20 different genera 25,2{'(A.L. Scott, unpublished). The MSPs are synthesized in spermatocytes and assembled into cytoplasmic paracrystalline arrays or fibrous bodies (FB)4.27. In the spermatocytes, the FBs become associated with the MOs to form FB-MO complexes 4. After meiosis, the FBs segregate into the cytoplasm of the developing spermatids. Following Parositoloe/ Today, vol. 12, no. I I, 1996
Fig. 3. Transmission electron micrographs of the mature spermatids of Brugio molayi. Sagittal section of a mature spermatid (a) showing fibrous arrays (fa), mitochondrion (m), membra. nous organelle (MO) and nucleus (n). High magnification of fibrous arrays at the tip of a spermatid (b). Cross-section of a spermatid (c) showing the position of the fibrous arrays at the inside margin of the cell. Scale bars ~ 200 nm.
spermatid budding from tile resk~ual body, tile FBs disassemble and tile MSPs arc released into the cytoplasm where they are maintained in an unpolymerized state. Upon sperm activation, the MSPs become concentrated in the pseudopod where they assemble into filaments. Crystallography and electron microscopy studies of Ascaris assembled MSP have shown that the filaments are made from a hierarchy of helices 2s,2'. The MSP subunits are arranged into helical subfilament strands. Multiple subfilament strands coil around one another to produce a 10 nm wide filament. A higher-order coiling between filaments results in the formation of fiber complexes 28,2".
Sperm motility Crawling motility in nematode sperm is dependent on the formation of MSP fiber complexes. MSP fibers radiating from these complexes become anchored into a dense layer of material lining the inside of the pseudopod membrane m. At the points where the fiber complexes attach to the pseudopod, the membrane protrudes to form the villipodia. The villipodia formed at the leading edge of the cell function as the initial 427
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drops to 6.2 and the MSP depolymerizes. Activation of spermatids to mature sperm is ,~:ccompanied by an increase in :he intracellular pH to 6.4 and the formation of MSP filaments in the pseudopod. This pH-mediated assembly-disassembly also occurs in vitro and in sperm cells treated with weak acids and weak bases 2s. Crawling cells establish a pH gradient of about 0.2 units, with the leading edge being more alkaline than the area of disassembly at the base of the pseudopod. MSP filament assembly occurs preferentially at membranes and may be facilitated by pH-sensitive interactions with proteins that are instated into the pseudopod membrane after a post-transcriptional addition of fatty acids -a°. The in vitro assembly of Ascaris MSP-containing filament arrays requires components from the rnernbrane at the leading edge of the cell and at least one other cytoplasmic factor ~z. Fertilization
substrate adhesion sites. The sites expand to form contact zones. On the portion of the pseudopod surface theft does not make contact with a substrate, the villipodia form blunt projections that protrude from the surface and give the pseudopod its ruffled appearance (Fig. 4). The assembly and attachment of villipodiafiber complexes at the tip of the cell is required to extend the pseudopod and initiate crawling. The discrete zones of contact at the leading edge of the pseudopod remain fixed as the rest of the cell moves over them. Cytoskeletal flow is coupled to sperm locomotion and the villipodia-fiber complexes treadmill rearward at the same rate as the cell crawls forward. As villipodia reach the base of the pseudopod, the membrane is internalized and the fibers that make up the complexes disassociate. Because the sperm cell cannot synthesize new protein, it is presumed that the MSP molectdes are recycled into new fiber complexes. This treadmill mode of motility allows Ascaris sperm to crawl over glass substrates at speeds up to 70 I~m per minute 7. Development and locomotion of nematode sperm is dependent on the precise contro! of a sequence of MSP assembly, disassembly and reassembly. Recent work ~-8 has shown that, by varying intracellular pH, the cell is able to regulate (at least in part) the state of MSP polymerization. Using fluorescence ratio imaging of cells loaded with a pH-sensitive dye, it was determined that in spermatocytes where the MSP is maintained in the paracrystal/ine fibrous bodies, the intracellular pH is maintained at 6.8 (Ref. 28). Upon transformation to spermatids, the intracellular pH 418
After entering the female, the sperm crawl up the uterus to reach the spermatheca: a specialized area of the upper uterus that is used for long-term storage of sperm. The pseudopodia of the sperm protrude into invaginations in the spermathecal walls in order to anchor themselves in a position that will allow them to fertilize eggs ~:. Oocytes are fertilized as they move through the spermatheca in their journey down the reproductive tract. Presumably, molecule(s) on the surface of nematode sperm specifically recognize the egg to initiate fertilization, as has been shown for mammalian :~ and sea-urchin sperm .~La~. The molecular basis for sperm-egg recognition and attachment, and for sperm penetration is not known. In nematode species such as C. eh,gans, almost every sperm fertilizes an egg. The number of progeny is lirnited by the number of sperm and not by the number of oocytes, which continue to mature as sperm are depleted 2. The penetrating sperm interacts with a portion of the oocyte surface coat so that the membrane of the sperrn associates with the vitelline rnembrane of the egg. The membranes of the sperm and egg fuse, and the contents of the sperm's cell body enter the cytoplasm of the oocyte. A second membrane forms beneath the vitelline membrane, and the egg shell forms between the two rnernbranes ~,~. ,';perm as a target for control
Tile rnale reproductive system offers features that make it attractive as a target for intervention. First, although the sperm from different species of nematodes are rnorphologically diverse, it appears that crucial aspects of the developmental biology and the biochemistry of sperm are remarkably conserved between nematode species. This suggests that strategies focused on disrupting the most conserved aspects Parasitology Today, vol. 12, no. I I, 1996
Reviews of sperm biology may be effective in controlling a number of different a nematode species. The conserved nature of nematode spermatogenesis is also advantageous in that it will .~. '•a•w•" allow for the use of powerful and easy-to-manipulate model systems, /::i such as C. elegans, to define the basic j biology, biochemistry and molecular biology of sperm development and ...?::!~ function, and to screen for com-; pounds that will disrupt sperm function. Another major advantage ", to targeting sperm biology and biochemistry as a means for controlling infection is that many of the highly conserved features such as the MOs -~ ..... • . P s e u d o p o d and MSPs are also unique to nematodes. This suggests that any intervention that is designed to block sperm function by targeted disruption b =====f l:~dio~ of ~ l ~ of a nematode sperm-specific mol/f o O ecule o~ complex of molecules will ~self be highly specific and have few host-associated complications. Examples of ways nematode sperm could be specifically targeted include: (1) / disruption of the early events of spermatogenesis so that mature sperm do not form. Evidence for the o "' • potential of this approach is provided by the work of Varkey et al. .~" Cell bodyL~Y~: :::-" Pseudo P od " ,/::::~.'..?:!. .. :... ' . : . . . . who have characterized a gene in C. eh'gans (spe-6) which is required for MSP localization and assembly into FBs in spermatocytes, spe-6 mutants V V V V do not form spermatids and are Fig. S. Transmission electron micrograph of a mature sperm ceil from Brugia malayi (a). sterile; (2) blocking the formation of The mature sperm cell consists c,fa ceilbody and a pseudopod. The ceil body conmature sperm through inhibition of tains the nucleus (N) mitochondria (M) and the membranous organelles (MO). Schethe process by which spermatids are matic drawing of the fiber complex movements in the pseudopod of a nematode activated to become mature sperm; sperm crawling on a substrate (b). The arrows indicate the direction of movement (3) disruption of sperm motility. The of the villipodia and the proposed movement ot the disassembled and reprocessed strategies here could take advantage fiber constituents from the base to the tip of the pseudopod. Nucleus (N), mito. of the unique features of MSP to chondria (M), membranous organelles (MO), fibrous complexes (FC) and villipodia (V). The schematic was adapted from Ref. I0. Scale bar = I p.m. inhibit filament formation and/or association with the pseudopod membrane or a disruption of the mecha2 Bird, A. and Bird, J. (1991) Th,' Structure ,,t Ncmatod,> nism by which sperm are able to maintain a pH gradient pp 117-132, Academic Press within the pseudopod. Another way to block sperm 3 Ward, S. ,'t ,11. (1981) Sperm morphogenesis in wild-type motility may be through an inhibition of pseudoand fertilization-defective m u t a n t s of Caem,rhabditis el,,,gans. pod-substrate interactions; and (4) obstruction of the ]. Cell Biol. 91,26-44 .1 Roberts, T.M. et al. (198t~) Membrane and cytoplasmic proteins molecules involved in maintaining sperm position in are t r a n s p o r t e d i n t h e same organelle complex during nemathe uterine spermatheca or the molecules that particitode s p e r m a t o g e n e s i s . ]. Cell Biol. 102, 1787-179t~ pate in sperm-egg recognition. 5 Anya, A.O. (lq7t~) Vhysiological aspects of nematode reproWith a basic understanding of spermatogenesis and duction. Adv. Parasdol. 14, 2t~7-351 ~ Four, W.E. and McMahon, J.T. (1973) Role of the glandular vas of the interactions between sperm and egg during ferdeferens in the development of Ascaris spermalozoa. I. I'a,,~ tilization, it is feasible to devise control strategies that sit,,I. 59, 753-758 will block fertilization, and thus block transmission of 7 Sepsenwol, S. and Taft, S.J. (1990) In vitro induction of crawldisease. ing in the amoeboid sperm of the nematode parasite, Ascaris "
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Acknowledgements This work was supported by a grant fi-om the MacA~thur Foundation Consortium on the Biology of Parasitic Diseases,The Johns Hopkins Program. References
1 Lee, D.L. and Atkinson, H.J. (1977) Physhflogy ot Nematodes, pp 117-132, Columbia University Press
Parasitology Toddy. vol. 12, no. I I, 1996
suum. Cell Motil. Ciitoskeh'ton 15, 99-110 8 Kimble, J. and War~t, S. (19887 Tit; Nematode Caenorhabditis elegans, (Wood, W.B., ed.), pp 190-213, Cold Spring Harbor Laboratory 9 Roberts, T.M. and Ward, S. (1982) Centripetal flow of pseudopodial surface c o m p o n e n t s c o u l d propel the amoeboid movement of C~,,enorhabditis elegans spermatozoa. ]. Col! Biol. 92, 132-138 l0 Sepsenwol, S. ct al. (198.o) A unique cytoskeleton associated with 429
Reviews .... .............
crawling in the amoeboid sperm of the nematode, Ascaris suum. 1. Cell Biol. 108, 55-66 11 Roberts,T.M. and King, K.L. (1991)Centripetal flow and directed reassembly of the major sperm protein (MSP) cytoskeleton in the amoeboid sperm of the nematode, Ascaris suum. Cell Motil Cvtoskeh'ton. 20, 228-241 12 U'gwunna, S.C. and Foor, W.E. (1982) Development and fate of the membranous organelles in spermatozoa of Ancylostoma caninum. 1. Parasitol.65, 834-844 13 Ugwunna, S.C. (1990) Extrusion of the residual body in srermatids of Ancylostoma canimtm (Nematoda, Strongyioidea). ]. Morphol. 203, 283-292 14 Lee, D.L. and Anya, O.A. (1967)The structure and development of the spermatozoon of Aspiculuris tetraptera (Nematoda). J. Cell Sci. 2, 537-544 15 Foor, W.E. (1970) Spermatozoan morphology and zygote formation in nematodes. Biol. Reprod. (Suppl. 2), 177-202 16 Chitwood, B.G. and Chitwood, M.B. (1974) Introduction to Nematoh)gy, pp 191-201, University Park Press 17 Wright, E.J. and Summerville, R.i. (1984) Postinsemination changes in amoeboid sperm of a nematode, Nippostrongylus brasiliensis. Gamete Res. 10, 397-413 18 Nelson, G.A. et al. (1982) Caenorhabditis elegans spermatozoan locomotion: amoeboid movement with almost no actin. ]. Cell Biol. ~)2,121-131 1~) Nelson, G.A. and Ward, S. (1981) Amoeboid motility and actin in Ascaris lumbricoides sperm. Exp. Cell Res. 131, 49-60 20 Scott, A.L. et al. (1989) Major sperm protein genes from Onchocerca voiwdus. Mol. Biochem. Parasitol. 36, 119-126 21 Roberts, T.M. (1987) Fine (2-5 nm) filaments: new types of cytoskeletal structures. Cell Motil. Cyh)skeleton 8, 130-142 22 King, K.L. et al. (1992) Structure and macromolecular assembly of two isoforms of the major sperm protein (MSP) from the amoeboid sperm of the nematode, Ascaris suum. ]. Cell Sci. 101,847-857
23 Burke, D.J. and Ward, S. (1983) Identification of a large multigene family encoding the major sperm protein of C. elegans. ]. Mol. Biol. 171, 1-29
24 Klass, M. et al. (1988) Conservation of the 5' flanking sequences of transcribed members of the Caenorhabditis elegans major sperm protein gene f~mily. [. Mol. Biol. 199, 15-22 25 Scott, A.L et al. (198~0M:~ic,:i.~erm protein and actin genes in free-living and parasitic nematodes. Parasitology98, 471-478 26 Schnieder, T. (1993) The diagnostic antigen encoded by gene tragment Dv3-14: a major sperm protein of Dictyocaulus viviparus. Int. I. Parasitol.23, 383-389 27 Ward, S. and Klass, M. (1982) The localization of the major sperm protein in sperm and spermatocytes. Dev. Bhd. 92, 203-208 28 King, K.U et al, (1994) Regulation of the Ascaris major sperm protein (MSP) cytoskeleton by intracellular pH. Cell Motil. C'yt:)~ke!,'ton.27, 193-205 29 StewarL M. et al. (1994) The motile major sperm protein (MSP) of Ascaris suum forms filaments constructed from two helical subfilaments. J, Mol. Biol. 243, 60-71 30 Pavalko, F.M. and Roberts, T.M. (1989) Posttranslational insertion of a membrane protein on Caenorhabditis elegans sperm occurs without de novo protein synthesis. ]. Cell. Biochem. 41, 57-70 31 Italiano, J.E. et al. (1996) Reconstitution in vitro of the motile apparatus from the amoeboid sperm of Ascaris shows that filament assembly and bundling move membranes. Co'!! 84, 105-114 32 Ward, S. and Carrel, J.S. (1979) FertUizafion and sperm competition in the nematode Caenorhabditis elegans. Dev. Bh)l.73, 304-321 33 Wassarman, P.M. (1990) Profile of a mammalian sperm receptor. Development 108, 1-17 34 Foitz, K.R. and Lennarz, W.J. (1993) The molecular basis of sea urchin gamete interactions at the egg plasma membrane. Dev. Biol. 158, 46-61 35 Kennedy, L. et al. (1989) Analysis of the membrane-interacting domain of the sea urchin sperm adhesive protein bindin. Biochemistry 28, 9153-9158 36 Varkey, J.P. et al. 0993) The Caenorhabditis elegans spe-6 gene is required for major sperm protein assembly and s h o w s second site non-complementation with an unlinked deficiency. Gen('tics 133, 79-86
Anticoagulants in Vector Arthropods K,R, Sta)'l
Kecae~h Stark and Anthony A. James are at the Department of Molecular Biology and Biochemistry, University of California, hMne, CA 92697-3900, USA. Tel: +1 714 814 5930, Fax: + I 714 824 2814, e-maih [email protected] 43O
a n t i h e m o s t a t i c activities e m p h a s i z e ~ that platelet a g g r e g a t i o n , v a s o c o n s t r i c t i o n a n d c o a g u l a t i o n represent significant obstacles to h e m a t o p h a g y I. T h e chara c t e r i z a t i o n of i n d i v i d u a l a n t i h e m o s t a t i c s u b s t a n c e s f r o m a variety of a r t h r o p o d s has revealed a t r e m e n d o u s d i v e r s i t y in their s t r u c t u r e a n d function, illust r a t i n g the e v o l u t i o n a r y c o n v e r g e n c e o n the functional s o l u t i o n s to c h a l l e n g e s of hematopi~agy. For e x a m p l e , v a s o d i l a t o r y s u b s t a n c e s h a v e b e e n identified as p r o s t a g l a n d i n - l i k e rnolecules in ticks, nitric o x i d e - b i n d i n g p r o t e i n s in t r i a t o m i n e bugs, a n d n o v e l v a s o a c t i v e p e p t i d e s in s a n d f l i e s a n d m o s q u i t o e s 2. A l t h o u g h a d i v e r s i t y of p l a t e l e t a n t i - a g g r e g a t i n g factors h a v e b e e n described, a n a p y r a s e activity that f u n c t i o n s by i n h i b i t i n g A D P - s t i m u l a t e d platelet a g g r e g a t i o n by the d e g r a d a t i o n of A D P has b e e n f o u n d in m o s t h e m a t o p h a g o u s a r t h r o p o d s 1. A p y r a s e a p p e a r s to be c o n s e r v e d as a b i o c h e m i c a l a c t i v i t y a m o n g h e m a t o p h a g o u s species, b u t the structural characterization of this e n z y m e from a n u m b e r of species m a y reveal a p o l y p h y l e t i c f a m i l y of m o l ecules. W e h a v e f o u n d f u r t h e r e v i d e n c e of the c o n v e r g e n t aspects of h e m a t o p h a g y b y e x a m i n i n g the site of a c t i o n of a n t i c o a g u l a n t s in m o s q u i t o e s a n d o t h e r hematophagous arthropods.
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Parasitology Today, vol. 12, no I I, 1996
Nematode
Sperm
A.L. Scott Parasitic nematode infections remain a major public health problem in many parts of the world. Because most of the current strategies aimed at controlling parasitic nematode infections have met with only limited success, it may be time to consider alternative approaches. An aspect of nematode biology that has drawn little attention as a target for control is the reproductiw" process. Although there are mmlerous facets of the overall reproductive biology of nematodes that hold potential as targets for intervention, Alan Scott here focuses on the male reproductive system, and outlines some of the known unique processes and characteristics of sperm formation and sperm function that could be exploited to block fertilization. The information on the behavior, developmental biology and biochemistry of nernatode sperm presented here was pieced together with the data and observations front a variety of nematode species, with a majority of the information corning from studies on the free-living Caenorhabditis eh-e~ans and the parasitic species Ascaris suum and Brugia malayi.
Anatomy of the male reproductive system For most nematode species, the male reproductive system is composed of a single testis that runs the length of the worm. Within the testis of each reproductively active male can be found cells from all of the stages of spermatogenesis (Fig. 1). The testis consists of a distal zone of germ-cell formation, a zone of maturation and a proximal storage area for the immature sperm consisting of a seminal vesicle a n d / o r a vas deferens. Most parasitic nematode species have telogonic testes, "vhvre germ-cell production is confined to the extren:e distal portion of the testis, in some nematode species, a seminal vesicle is positioned at the proximal end of the growth zone and serves as a specialized storage area for sperm until copulation. A muscular region surrounds a specialized region of the vas deferens that forms an ejaculatory duct which opens into a cloaca. Male nematodes have a number of secondary sexual organs including a cloaca, a bursa, spicules, a gubernaculum and cement glands, all of which aid in grasping the female during copulation I. The cloaca is most commonly located on the ventral surface and is surrounded by a number of sensillae. In some nematodes, the area around the cloaca forms an expanded structure called the copulatory bursa which may involve the entire ventral surface of the tail 1,2. Male nematodes possess one or two copulatory spicules which are formed from hardened cuticle. The function of the spicules is to dilate the vulva and vagina of the female during copulation. The spicules have neurons running up their center, which terrninate in sensory Alan L. Scott is at the Department of Molecular Microbiology and Immunology, School of Hygiene and Public Health, The Johns Hopkins University, 615 North Wolfe Street, Baltimore. MD 212052 ! 79, USA. Tel: + I 410 955 3442, Fax: 4, I 410 955 0105, e-maih [email protected] ! .sph.jhu.edu Parasitology Today, vol. 12, no. I I, 1996
dendrites called genital sensillae; these are thought to allow the spicule to feel its way into the female without damaging her reproductive tissues. The gubernaculum is a grooved plate which functions as a guide for the spicules.
Spermatogenesis Spermatogenesis proceeds linearly from the spermatogonial cells in the distal tip of the testis to the spermatozoa in the vas deferens (Fig. 1). During the first steps in spermatogenesis, primary spermatocytes enter meiosis and proceed through pachytene while attached via a cytoplasmic bridge to a common cytoplasmic core structure termed the rachis (Figs 1, 2a). The spermatocytes then bud from the rachial syncycia and complete meiosis to form four haploid nuclei. The four haploid nuclei migrate to the rnargins of the cell and, along with a collection of selected organelles, bud from the central region of cytoplasm to form four spberical spermatids. A spermatid is the sessile, immature, storage forrn of nematode sperm. During the process of spermatid budding, all of the biosynthetic machinery is retained in a cytoplasmic remnant termed the residual body. Microtubules, microfilaments, endoplasmic reticulum, ribosomes and the Golgi complex remain in the residual body, which is eventually degraded and absorbed -~,4. Because all of the organelles necessary for biosynthetic activity remain with the residual body, neither spermatids nor mature sperm are capable of protein synthesis. This means that all of the molecules required for sperm differentiation and sperm function are synthesized early in spermatogenesis and ~tored in various forms in the spermatid.
Spermatids Spermatids can be round, rod-like or fusiform cells, depending on the nematode species, and range in diameter from 3 lain to 10 I~m (Ref. 2). Spermatid development in the filarid B. malayi is typical of many nematode species. Brugh7 mahlyi spermatids are rounded cells as they first bud from the residual body (Fig. l). Although in some species the mature spermatids remain rounded, the fully developed spermatids in Brughl take on a rod-like appearance and are densely packed into the vas deferens of the sexually active male (Figs 1, 2b, 2c). The nuclear material in the spermatid is highly condensed and is organized into two or three discrete, non-membrane-bound bodies (Fig. 3a). Brltgia spermatids also contain the membranebound organelles typically found in the spermatids of nearly all of the nematode species studied. These organelles include mitochondria, a large refringent body and a structure that appears to be unique to nematode sperm, the membranous organelle (MO) (Fig. 3). Also present in the cytoplasm of Brugia spermatids are structures that have been designated fibrous arrays. These structures do not appear in spermatids that remain rounded. Under the electron microscope, sagittal sections of spermatids show that the fibrous
Reviews sperm activation, there is a drastic remodciil:g of the spermatid that is characterized by the extension of a Primary pseudopod away from the cell body spermatocytes and a fusion of the MOs to the plasma membrane- (Figs 4, 5). Sperm Zone of activation is completed in a matter germ cell of minutes and at this point the cells formation become motile 7. _..l Most of the MOs in mature sperm fuse with the plasma membrane and form pores on the cell body portion of the sperm 2 (Fig. 5). MOs have a spherical head portion with an elec~ 3eomdary tron dense collar and a highly invagipermatocytes hated, microvillus-like internal netMaturation work a. The MOs first form in the /.Olte Early primary spermatocyte and combine " ~ spermatid with another unique organelle, the fibrous body (FB), to form fibrous bodv-meml:;ranous organelle (FBMO) cornplexes. FB-MO complexes 0 are transient and appear to be important in the storage and delivery of sperm proteins that were synResidual body thesized early in spermatogenesis to Storage the fully differentiated spermatozoa. /Olll2 in the rnature sperm, the MOs release a fibrous glycoprotein which ~ ~ sper at'd. adheres to the outside of the spermatid cell body. This glycoprotein is thought to be important for sperm ~ N N Malt.re sperm motility". The pseudopod, which is devoid Proximal of organeiles, contains a fTlanaentous le~li', or flocculate material (Fig. 5). The surface of the pseudopod has numerFig, I, An outline of spermatogenesls in the parasitic nematode Brugia molayi, ous projections (villipodia), which form at the tip of the pseudopod and move rapidly to the base (Fig. 4)"11. In Ascaris, arrays are positioned along the margins of the cell Brugia and Ancvlostoma sperm, the filarncntous just beneath the cell membrane (l:;'ig. 3a). The fibrous material is distributed throughout the pseuclopod arrays extend to the tip of the cell, but do not appear and becomes organized into a cytoskeleton composed to be attached or anchored to the tip (Fig. 3b). When of fibers arranged radially into long, branched fiber the cells are viewed in cross-section, the fibrous arcomplexes, which may traverse the length of the rays are positioned around the perimeter of the cell pseudopod"'.12.ts. (Fig. 3c). The radial arrangement of the fibrous arrays Although the sperm from a number of nernatode just under the cell membrane probably contributes to species have the basic morphology and distribution the rod-shaped appearance of the Brttgia spermatids. of organelles outlined in Figs 4 and 5, there is considerable variation in the sperrn cell form. For example, Sperm in the oxyurid Aspiculuris tetraptera the sperm have a Unlike flagellated sperm of mammals, mature nemadistinct 'tail' which contains a single, large, elongate tode sperm form pseudopodia and exhibit amoeboid mitochondrion, DNA and bundles of microtubules, or crawling motility s. Maturation of spermatids to but no MOs, and a distinct 'head' which sends out spermatozoa is triggered by mating and appears to pseudopodial projectionst L Descriptions of the varied take place within the vas deferens of C. eh'gans and morphologies of nematode sperm can be found elseAscaris. Activation is thought to be analogous to capwhere 2,~,~; I7. acitation in mammalian sperm. The exact events that result in the final maturation step are not known. For C. eh'gans3 and the filarial nematodes B. malayi and C o m p o s i t i o n of the fiber c o m p l e x e s In striking contrast to all other cells that employ Dirofilaria immits (A.L. Scott, unpublished), the spercrawling motility, nematode ~perm contain neither matids can be triggered to mature ;n vitro by proteases or agents that cause a rapid increase in intraactin nor myosin m. Instead, locomotion in nematode cellular pH, such as weak bases and the ionophore sperm depends on what appears to be a simple cytomonensin. For Ascaris, proteases as well as an unskeleton, based primarily on a family of small, basic, known substance present in extracts of Ascaris vas sperm-specific proteins that have been designated the deferens can activate spermatids in vitro 4-C'. During major sperm proteins ~MSP). "[he MSPs, all of which Digital testis
t
426
rogenator cells
/
Porasitology Today, vol. 12, no. I I, 1996
Reviews a,
Fig. 2. Different stages of development of Bru~;a ma~uyi sperm Primary spermatocytes assembled on the rachis (a). Spermatids packed into the storage region of the testis (b). A high magnification image of B. malayi spermatids (c). Photomicrographs were taken using Nomarski interference phase contrast. Scale bars -- 30 p.m (a), 30 ~m (b), 8 p.m (c).
have a molecular mass of 14 kDa, comprise about 15';~ of the total cellular protein and over ,t0'/, of the soluble protein "',a". The MSPs have no sequence similarity to any of the described filament-forming proteins such as actin, myosin, tubulin, tektin and spasmin 2t. The MSPs appear to be unique, nematodespecific, sperm-specific cytoskeletal proteins. In Ascaris 22, Onchocerca v01vulus20 and B. malayi (A.L. Scott, unpublished) there are two isoforms of MSP which are encc, ded oil two separate genes. In Ascaris, the two MSP isoforms (MSP~ and MSP[3) differ at only four out of 129 amino acids, while in O. voh,ulus and B. malayi the two isoforms differ at five of the 129 amino acids. Between species, the amino acid sequences of MSPs are between 80{7, and 90% identical. In contrast to the small number of genes encoding MSP in the relatively large parasitic nematodes, in the smaller, free-living nematode species C. eh'dans, the three apparent isoforms of MSP are produced from a multigene array having ~bout 30 members 23.24. MSP genes in various copy nu,nber have been identified in over 25 nematode species representing 20 different genera 25,2{'(A.L. Scott, unpublished). The MSPs are synthesized in spermatocytes and assembled into cytoplasmic paracrystalline arrays or fibrous bodies (FB)4.27. In the spermatocytes, the FBs become associated with the MOs to form FB-MO complexes 4. After meiosis, the FBs segregate into the cytoplasm of the developing spermatids. Following Parositoloe/ Today, vol. 12, no. I I, 1996
Fig. 3. Transmission electron micrographs of the mature spermatids of Brugio molayi. Sagittal section of a mature spermatid (a) showing fibrous arrays (fa), mitochondrion (m), membra. nous organelle (MO) and nucleus (n). High magnification of fibrous arrays at the tip of a spermatid (b). Cross-section of a spermatid (c) showing the position of the fibrous arrays at the inside margin of the cell. Scale bars ~ 200 nm.
spermatid budding from tile resk~ual body, tile FBs disassemble and tile MSPs arc released into the cytoplasm where they are maintained in an unpolymerized state. Upon sperm activation, the MSPs become concentrated in the pseudopod where they assemble into filaments. Crystallography and electron microscopy studies of Ascaris assembled MSP have shown that the filaments are made from a hierarchy of helices 2s,2'. The MSP subunits are arranged into helical subfilament strands. Multiple subfilament strands coil around one another to produce a 10 nm wide filament. A higher-order coiling between filaments results in the formation of fiber complexes 28,2".
Sperm motility Crawling motility in nematode sperm is dependent on the formation of MSP fiber complexes. MSP fibers radiating from these complexes become anchored into a dense layer of material lining the inside of the pseudopod membrane m. At the points where the fiber complexes attach to the pseudopod, the membrane protrudes to form the villipodia. The villipodia formed at the leading edge of the cell function as the initial 427
F
drops to 6.2 and the MSP depolymerizes. Activation of spermatids to mature sperm is ,~:ccompanied by an increase in :he intracellular pH to 6.4 and the formation of MSP filaments in the pseudopod. This pH-mediated assembly-disassembly also occurs in vitro and in sperm cells treated with weak acids and weak bases 2s. Crawling cells establish a pH gradient of about 0.2 units, with the leading edge being more alkaline than the area of disassembly at the base of the pseudopod. MSP filament assembly occurs preferentially at membranes and may be facilitated by pH-sensitive interactions with proteins that are instated into the pseudopod membrane after a post-transcriptional addition of fatty acids -a°. The in vitro assembly of Ascaris MSP-containing filament arrays requires components from the rnernbrane at the leading edge of the cell and at least one other cytoplasmic factor ~z. Fertilization
substrate adhesion sites. The sites expand to form contact zones. On the portion of the pseudopod surface theft does not make contact with a substrate, the villipodia form blunt projections that protrude from the surface and give the pseudopod its ruffled appearance (Fig. 4). The assembly and attachment of villipodiafiber complexes at the tip of the cell is required to extend the pseudopod and initiate crawling. The discrete zones of contact at the leading edge of the pseudopod remain fixed as the rest of the cell moves over them. Cytoskeletal flow is coupled to sperm locomotion and the villipodia-fiber complexes treadmill rearward at the same rate as the cell crawls forward. As villipodia reach the base of the pseudopod, the membrane is internalized and the fibers that make up the complexes disassociate. Because the sperm cell cannot synthesize new protein, it is presumed that the MSP molectdes are recycled into new fiber complexes. This treadmill mode of motility allows Ascaris sperm to crawl over glass substrates at speeds up to 70 I~m per minute 7. Development and locomotion of nematode sperm is dependent on the precise contro! of a sequence of MSP assembly, disassembly and reassembly. Recent work ~-8 has shown that, by varying intracellular pH, the cell is able to regulate (at least in part) the state of MSP polymerization. Using fluorescence ratio imaging of cells loaded with a pH-sensitive dye, it was determined that in spermatocytes where the MSP is maintained in the paracrystal/ine fibrous bodies, the intracellular pH is maintained at 6.8 (Ref. 28). Upon transformation to spermatids, the intracellular pH 418
After entering the female, the sperm crawl up the uterus to reach the spermatheca: a specialized area of the upper uterus that is used for long-term storage of sperm. The pseudopodia of the sperm protrude into invaginations in the spermathecal walls in order to anchor themselves in a position that will allow them to fertilize eggs ~:. Oocytes are fertilized as they move through the spermatheca in their journey down the reproductive tract. Presumably, molecule(s) on the surface of nematode sperm specifically recognize the egg to initiate fertilization, as has been shown for mammalian :~ and sea-urchin sperm .~La~. The molecular basis for sperm-egg recognition and attachment, and for sperm penetration is not known. In nematode species such as C. eh,gans, almost every sperm fertilizes an egg. The number of progeny is lirnited by the number of sperm and not by the number of oocytes, which continue to mature as sperm are depleted 2. The penetrating sperm interacts with a portion of the oocyte surface coat so that the membrane of the sperrn associates with the vitelline rnembrane of the egg. The membranes of the sperm and egg fuse, and the contents of the sperm's cell body enter the cytoplasm of the oocyte. A second membrane forms beneath the vitelline membrane, and the egg shell forms between the two rnernbranes ~,~. ,';perm as a target for control
Tile rnale reproductive system offers features that make it attractive as a target for intervention. First, although the sperm from different species of nematodes are rnorphologically diverse, it appears that crucial aspects of the developmental biology and the biochemistry of sperm are remarkably conserved between nematode species. This suggests that strategies focused on disrupting the most conserved aspects Parasitology Today, vol. 12, no. I I, 1996
Reviews of sperm biology may be effective in controlling a number of different a nematode species. The conserved nature of nematode spermatogenesis is also advantageous in that it will .~. '•a•w•" allow for the use of powerful and easy-to-manipulate model systems, /::i such as C. elegans, to define the basic j biology, biochemistry and molecular biology of sperm development and ...?::!~ function, and to screen for com-; pounds that will disrupt sperm function. Another major advantage ", to targeting sperm biology and biochemistry as a means for controlling infection is that many of the highly conserved features such as the MOs -~ ..... • . P s e u d o p o d and MSPs are also unique to nematodes. This suggests that any intervention that is designed to block sperm function by targeted disruption b =====f l:~dio~ of ~ l ~ of a nematode sperm-specific mol/f o O ecule o~ complex of molecules will ~self be highly specific and have few host-associated complications. Examples of ways nematode sperm could be specifically targeted include: (1) / disruption of the early events of spermatogenesis so that mature sperm do not form. Evidence for the o "' • potential of this approach is provided by the work of Varkey et al. .~" Cell bodyL~Y~: :::-" Pseudo P od " ,/::::~.'..?:!. .. :... ' . : . . . . who have characterized a gene in C. eh'gans (spe-6) which is required for MSP localization and assembly into FBs in spermatocytes, spe-6 mutants V V V V do not form spermatids and are Fig. S. Transmission electron micrograph of a mature sperm ceil from Brugia malayi (a). sterile; (2) blocking the formation of The mature sperm cell consists c,fa ceilbody and a pseudopod. The ceil body conmature sperm through inhibition of tains the nucleus (N) mitochondria (M) and the membranous organelles (MO). Schethe process by which spermatids are matic drawing of the fiber complex movements in the pseudopod of a nematode activated to become mature sperm; sperm crawling on a substrate (b). The arrows indicate the direction of movement (3) disruption of sperm motility. The of the villipodia and the proposed movement ot the disassembled and reprocessed strategies here could take advantage fiber constituents from the base to the tip of the pseudopod. Nucleus (N), mito. of the unique features of MSP to chondria (M), membranous organelles (MO), fibrous complexes (FC) and villipodia (V). The schematic was adapted from Ref. I0. Scale bar = I p.m. inhibit filament formation and/or association with the pseudopod membrane or a disruption of the mecha2 Bird, A. and Bird, J. (1991) Th,' Structure ,,t Ncmatod,> nism by which sperm are able to maintain a pH gradient pp 117-132, Academic Press within the pseudopod. Another way to block sperm 3 Ward, S. ,'t ,11. (1981) Sperm morphogenesis in wild-type motility may be through an inhibition of pseudoand fertilization-defective m u t a n t s of Caem,rhabditis el,,,gans. pod-substrate interactions; and (4) obstruction of the ]. Cell Biol. 91,26-44 .1 Roberts, T.M. et al. (198t~) Membrane and cytoplasmic proteins molecules involved in maintaining sperm position in are t r a n s p o r t e d i n t h e same organelle complex during nemathe uterine spermatheca or the molecules that particitode s p e r m a t o g e n e s i s . ]. Cell Biol. 102, 1787-179t~ pate in sperm-egg recognition. 5 Anya, A.O. (lq7t~) Vhysiological aspects of nematode reproWith a basic understanding of spermatogenesis and duction. Adv. Parasdol. 14, 2t~7-351 ~ Four, W.E. and McMahon, J.T. (1973) Role of the glandular vas of the interactions between sperm and egg during ferdeferens in the development of Ascaris spermalozoa. I. I'a,,~ tilization, it is feasible to devise control strategies that sit,,I. 59, 753-758 will block fertilization, and thus block transmission of 7 Sepsenwol, S. and Taft, S.J. (1990) In vitro induction of crawldisease. ing in the amoeboid sperm of the nematode parasite, Ascaris "
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Acknowledgements This work was supported by a grant fi-om the MacA~thur Foundation Consortium on the Biology of Parasitic Diseases,The Johns Hopkins Program. References
1 Lee, D.L. and Atkinson, H.J. (1977) Physhflogy ot Nematodes, pp 117-132, Columbia University Press
Parasitology Toddy. vol. 12, no. I I, 1996
suum. Cell Motil. Ciitoskeh'ton 15, 99-110 8 Kimble, J. and War~t, S. (19887 Tit; Nematode Caenorhabditis elegans, (Wood, W.B., ed.), pp 190-213, Cold Spring Harbor Laboratory 9 Roberts, T.M. and Ward, S. (1982) Centripetal flow of pseudopodial surface c o m p o n e n t s c o u l d propel the amoeboid movement of C~,,enorhabditis elegans spermatozoa. ]. Col! Biol. 92, 132-138 l0 Sepsenwol, S. ct al. (198.o) A unique cytoskeleton associated with 429
Reviews .... .............
crawling in the amoeboid sperm of the nematode, Ascaris suum. 1. Cell Biol. 108, 55-66 11 Roberts,T.M. and King, K.L. (1991)Centripetal flow and directed reassembly of the major sperm protein (MSP) cytoskeleton in the amoeboid sperm of the nematode, Ascaris suum. Cell Motil Cvtoskeh'ton. 20, 228-241 12 U'gwunna, S.C. and Foor, W.E. (1982) Development and fate of the membranous organelles in spermatozoa of Ancylostoma caninum. 1. Parasitol.65, 834-844 13 Ugwunna, S.C. (1990) Extrusion of the residual body in srermatids of Ancylostoma canimtm (Nematoda, Strongyioidea). ]. Morphol. 203, 283-292 14 Lee, D.L. and Anya, O.A. (1967)The structure and development of the spermatozoon of Aspiculuris tetraptera (Nematoda). J. Cell Sci. 2, 537-544 15 Foor, W.E. (1970) Spermatozoan morphology and zygote formation in nematodes. Biol. Reprod. (Suppl. 2), 177-202 16 Chitwood, B.G. and Chitwood, M.B. (1974) Introduction to Nematoh)gy, pp 191-201, University Park Press 17 Wright, E.J. and Summerville, R.i. (1984) Postinsemination changes in amoeboid sperm of a nematode, Nippostrongylus brasiliensis. Gamete Res. 10, 397-413 18 Nelson, G.A. et al. (1982) Caenorhabditis elegans spermatozoan locomotion: amoeboid movement with almost no actin. ]. Cell Biol. ~)2,121-131 1~) Nelson, G.A. and Ward, S. (1981) Amoeboid motility and actin in Ascaris lumbricoides sperm. Exp. Cell Res. 131, 49-60 20 Scott, A.L. et al. (1989) Major sperm protein genes from Onchocerca voiwdus. Mol. Biochem. Parasitol. 36, 119-126 21 Roberts, T.M. (1987) Fine (2-5 nm) filaments: new types of cytoskeletal structures. Cell Motil. Cyh)skeleton 8, 130-142 22 King, K.L. et al. (1992) Structure and macromolecular assembly of two isoforms of the major sperm protein (MSP) from the amoeboid sperm of the nematode, Ascaris suum. ]. Cell Sci. 101,847-857
23 Burke, D.J. and Ward, S. (1983) Identification of a large multigene family encoding the major sperm protein of C. elegans. ]. Mol. Biol. 171, 1-29
24 Klass, M. et al. (1988) Conservation of the 5' flanking sequences of transcribed members of the Caenorhabditis elegans major sperm protein gene f~mily. [. Mol. Biol. 199, 15-22 25 Scott, A.L et al. (198~0M:~ic,:i.~erm protein and actin genes in free-living and parasitic nematodes. Parasitology98, 471-478 26 Schnieder, T. (1993) The diagnostic antigen encoded by gene tragment Dv3-14: a major sperm protein of Dictyocaulus viviparus. Int. I. Parasitol.23, 383-389 27 Ward, S. and Klass, M. (1982) The localization of the major sperm protein in sperm and spermatocytes. Dev. Bhd. 92, 203-208 28 King, K.U et al, (1994) Regulation of the Ascaris major sperm protein (MSP) cytoskeleton by intracellular pH. Cell Motil. C'yt:)~ke!,'ton.27, 193-205 29 StewarL M. et al. (1994) The motile major sperm protein (MSP) of Ascaris suum forms filaments constructed from two helical subfilaments. J, Mol. Biol. 243, 60-71 30 Pavalko, F.M. and Roberts, T.M. (1989) Posttranslational insertion of a membrane protein on Caenorhabditis elegans sperm occurs without de novo protein synthesis. ]. Cell. Biochem. 41, 57-70 31 Italiano, J.E. et al. (1996) Reconstitution in vitro of the motile apparatus from the amoeboid sperm of Ascaris shows that filament assembly and bundling move membranes. Co'!! 84, 105-114 32 Ward, S. and Carrel, J.S. (1979) FertUizafion and sperm competition in the nematode Caenorhabditis elegans. Dev. Bh)l.73, 304-321 33 Wassarman, P.M. (1990) Profile of a mammalian sperm receptor. Development 108, 1-17 34 Foitz, K.R. and Lennarz, W.J. (1993) The molecular basis of sea urchin gamete interactions at the egg plasma membrane. Dev. Biol. 158, 46-61 35 Kennedy, L. et al. (1989) Analysis of the membrane-interacting domain of the sea urchin sperm adhesive protein bindin. Biochemistry 28, 9153-9158 36 Varkey, J.P. et al. 0993) The Caenorhabditis elegans spe-6 gene is required for major sperm protein assembly and s h o w s second site non-complementation with an unlinked deficiency. Gen('tics 133, 79-86
Anticoagulants in Vector Arthropods K,R, Sta)'l
Kecae~h Stark and Anthony A. James are at the Department of Molecular Biology and Biochemistry, University of California, hMne, CA 92697-3900, USA. Tel: +1 714 814 5930, Fax: + I 714 824 2814, e-maih [email protected] 43O
a n t i h e m o s t a t i c activities e m p h a s i z e ~ that platelet a g g r e g a t i o n , v a s o c o n s t r i c t i o n a n d c o a g u l a t i o n represent significant obstacles to h e m a t o p h a g y I. T h e chara c t e r i z a t i o n of i n d i v i d u a l a n t i h e m o s t a t i c s u b s t a n c e s f r o m a variety of a r t h r o p o d s has revealed a t r e m e n d o u s d i v e r s i t y in their s t r u c t u r e a n d function, illust r a t i n g the e v o l u t i o n a r y c o n v e r g e n c e o n the functional s o l u t i o n s to c h a l l e n g e s of hematopi~agy. For e x a m p l e , v a s o d i l a t o r y s u b s t a n c e s h a v e b e e n identified as p r o s t a g l a n d i n - l i k e rnolecules in ticks, nitric o x i d e - b i n d i n g p r o t e i n s in t r i a t o m i n e bugs, a n d n o v e l v a s o a c t i v e p e p t i d e s in s a n d f l i e s a n d m o s q u i t o e s 2. A l t h o u g h a d i v e r s i t y of p l a t e l e t a n t i - a g g r e g a t i n g factors h a v e b e e n described, a n a p y r a s e activity that f u n c t i o n s by i n h i b i t i n g A D P - s t i m u l a t e d platelet a g g r e g a t i o n by the d e g r a d a t i o n of A D P has b e e n f o u n d in m o s t h e m a t o p h a g o u s a r t h r o p o d s 1. A p y r a s e a p p e a r s to be c o n s e r v e d as a b i o c h e m i c a l a c t i v i t y a m o n g h e m a t o p h a g o u s species, b u t the structural characterization of this e n z y m e from a n u m b e r of species m a y reveal a p o l y p h y l e t i c f a m i l y of m o l ecules. W e h a v e f o u n d f u r t h e r e v i d e n c e of the c o n v e r g e n t aspects of h e m a t o p h a g y b y e x a m i n i n g the site of a c t i o n of a n t i c o a g u l a n t s in m o s q u i t o e s a n d o t h e r hematophagous arthropods.
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