Video Supplement

Cell Motility and the Cytoskeleton 15:99-110 (1990)

In Vitro Induction of Crawling in the Amoeboid Sperm of the Nematode Parasite, Ascaris suum Sol Sepsenwol and Stephen J. Taft Department of Biology, University of Wisconsin, Stevens Point In a highly synchronous process, the immotile spermatids of Ascaris suum extend pseudopods and become rapidly crawling sperm when treated with an extract from the glandular vas deferens of the male under strict anaerobic conditions. Within 9-12 min, a pseudopod develops. elongates rapidly. and exhibits a continuous flow of membrane specializations, the villipodia, from tip toward base. When attached to acid-washed glass, the pseudopod pulls the cell body along at speeds exceeding 70 pmimin. The pseudopod length remains constant while retrograde flow of villipodia proceeds at the same rate as the sperm’s forward movement. Cohorts of about 15 villipodia form at the leading edgc. move rearward together, and disappear at the junction of pseudopod and cell body. These are the terminations of branched, refringent fibers. which extend the length of the pseudopod. The latter are thefiber complexes that form its cytoskeleton (Sepsenwol et al.: Journal of Cell Biology 10855-66, 1989). Locomoting cells sometimes change direction when another crawls by and follow each other. When cells are exposed to air, forward movement ceases in a predictable pattern: the forward extension of the leading edge ceases, the pseudopod shortens from the base, and the cell body continues to be pulled forward. These data contribute to a model for Ascaris sperm amoeboid motility in which independent processes of continuous extension at the leading edge and continuous shortening at the base ofthe pseudopod act to propel the cell forward. Key words: ascaris, nematode, nematoda, sperm, amoeboid motility

INTRODUCTION

The purpose of this paper is to present new data on the locomotion of nematode sperm as represented by that of the pig intestinal parasite, Ascaris suurn, and to describe the morphological events of activation leading up to full motility in more detail than previously reported, using primarily time-lapse cinemicrography . The sperm of nematodes are unique. Unlike flagellated sperm, they possess Weudopods and crawl, and they are often described as “amoeboid.” As well, nematode sperm lack significant amounts of actin or myosin, proteins previously thought to be indispensable for crawling cells. In at least Ascaris and Camorhabditis, spermatids transform rapidly from stored immotile cells to crawling spermatozoa during an activation step. Previously, Foor had described the striking ultrastructural 0 1990 Wiley-Liss, Inc.

differences between the immotile spermatids stored in the male worm’s seminal vesicle and the sperm that are found in the female reproductive tract [see Foor reviews, 1970 and 19831. The latter possess a remodeled cell body and an extended pseudopod absent in the stored gametes. Among the new specializations in motile Ascaris sperm are (a) membraneous organelles that have fused with the cell membrane to form permanent convoluted pits; (b) a large refringent body apparently formed by fusion from

Received June 29, 1989: accepted October I I , 1989 Address reprint requests to S. Sepsenwol. Dept. ot Biology. University of Wisconsin. Stevens Point, WI 54481. Data corresponding to the article are planned for inclusion in a forthcoming Cell Moriliry mid t l i r CytosXe/eto,i Video Supplement ( 1990).

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numerous granules seen in inactive cells; and (c), the pseudopod. The latter is a permanent, organelle-free extension of the cell containing filamentous material and numerous membrane protrusions, the villipodia, which move rapidly from tip to base. In 1973, Foor and McMahon demonstrated that the morphological transition to the so-called “amoeboid” form occurred through the action of a secretion from the terminal portion of the male’s reproductive tract, the glandular vas deferens. They were able to induce the morphological changes associated with activation in vitro with the addition of an homogenate of this gland but were not able to induce locomotion. Abbas and Cain [ 19791 determined some of the biochemical and physical conditions necessary for in vitro activation of Ascaris sperm but also were unable to induce locomotion. Nelson and Ward 119811, on the other hand, demonstrated that mature Ascaris sperm dissected from the female reproductive tract are capable of crawling. This raised the possibility that additional maturational factors were required for full motility in Ascaris sperm, analogous to the epididymal maturation and capacitation of mammalian sperm. This is not the case, however. As shown below, sperm will crawl if spermatids are healthy, their substrate is compatible, and high C02/low O2 is maintained throughout storage and activation. In Ascaris sperm, filamentous material fills the entire Ascaris sperm pseudopod and forms a unique cytoskeleton [Sepsenwol et al., 19891. It is composed of 3-10 nm fibers arranged radially into long, branched 150-250 nmfiher complexes, which course the length of the pseudopod. Fibers from adjacent complexes interdigitate to form a solid mat over the base of the attached pseudopod. Terminations of unattached complexes protrude into the membrane to form the villipodia. The fibers label with antibodies against M S P , a 14-15kD protein abundant in and unique to nematode sperm. The cytoskeleton can be visualized in live sperm with video light microscopy and has been shown to move centripetally in concert with the villipodia. Activation and motility have also been studied in sperm of Caenorhahditis elegans, a free-living soil nematode. These cells are about 3 times smaller than Asmris’, but their morphology and motility are similar. The native sperm-activation agent in Cuerzorhahditis is not known. A number of artificial agents, however, transform immotile spermatids into crawling sperm, including the cationophore monensin, triethanolamine, and certain proteases [Nelson and Ward, 1980; Ward et al., 19831. Of these, only proteases will activate Ascaris sperm [Abbas and Cain, 1979; Sepsenwol et al., 19861. In crawling Ctrc.riorhnhditis sperm, Roberts and coworkers described a centripetal membrane flow in the pseudopod [Roberts and Ward, 1982a,b] and described

how contacts are made between membrane specializations and the substrate [Roberts and Streitmatter, 19841. Using charged latex beads, labeled phospholipid micelles, lectins, and antibodies against membrane proteins, they demonstrated that both cytoplasmic proteins and membrane phospholipids are inserted at the tip of the pseudopod and move back toward the cell body at similar rates. Caenorhabditis sperm pseudopods contain filaments, granular material and MSP but no fiber complexes, and cytoskeletal elements were not considered in a model for its motility [Roberts and Ward, 1982b; see review by Roberts et al., 19891. These studies of Ascaris sperm crawling on glass provide important evidence for a model of Ascaris sperm amoeboid motility, and perhaps nematode sperm in general, in which translocation is generated by an association of pseudopod membrane and its dynamic cytoskeleton. Each of the sequences described here is contained in a videotape supplement [Sepsenwol and Taft, 19901. MATERIALS AND METHODS Collection of Worms

Male worms were collected from jejunumiileum of freshly killed swine, washed in 37-40°C tap water, transferred to 0.15 M NaCl + 0.1 M phosphate, pH 7.2-7.4 (“PBS”) at 37-40°C, and transported in insulated containers to the laboratory. Worms were maintained in frequent changes of PBS at 40°C. Preparation of Vas Deferens Supernatant (“VDX”) and Protease for Sperm Activation

Seventy vas deferens were dissected from fresh or frozen-thawed tails of male worms, homogenized on ice in 5 ml 20 mM HEPES, pH 7.0, and centrifuged at 25,0008 for 20 min. The supernatant was filtered through a 0.2 pm nitrocellulose membrane to remove particulates. The ultrafiltrate (“VDX”), ca. 1 mg/ml total protein, was stored frozen at -80°C. S. griseus protease (Sigma, type XXI), 25 pglml, was dissolved in 20 mM HEPES in 0.15 M NaCl, pH 7.4 (‘ ‘HEPES-saline buffer”). Preparation and Activation of Spermatids

The seminal vesicle was dissected from a male worm, and its contents, about 100 pl of semen (ca. lo7 spermatids), were emptied into 8 ml of HEPES-saline buffer at 40”C, pre-gassed 15 min with 15% CO,/N,. Twenty microliters of VDX or protease was added to 700 pl of the gassed sperm suspension. Time-Lapse Phase Photomicrography

Approximately 40 pl of the activating cell suspension was transferred immediately to a well of silicone

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grease on an acid-washed glass microscope slide (Gold Seal brand, Becton Dickinson Company, Oxnard, CA) and sealed with a coverslip under a 15% CO,/N, atmosphere. Cells were observed on a Zeiss Photomicroscope using a 40 X objective, phase-contrast optics and an infrared-heated stage to maintain the slide temperature at 37-39°C. Images were recorded on 16 mm Kodak PlusX reversal film using a Bolex model H16M movie camera controlled by a Nikon cine time-lapse system running at 1 frame per second. Events were timed by counting frames in time-lapse films. Enhanced Video DIC Micrography

Crawling cells activated as above were observed on a Zeiss Axiomat with DIC optics using a 63 X apochromat objective and a Dage 68M Newvicon camera. Usually, the video signal was processed directly with a Quantex model 9200EX image-processing system to suppress background, 4-frame averaged ( I / 15 sec) to enhance contrast, and stored at 1 imageisec on a Panasonic optical disk recorder. In some cases, the unprocessed video signal was stored on optical disk and later processed with the Quantex. Individual frames from the optical disk were photographed directly from the screen of a Conrac high-resolution BIW monitor onto Kodak Panatomic-X film. Perfusion of Activated Sperm With Gas Mixtures and Peroxide

Sperm were activated as above, and approximately 100 pl of suspension was transferred to an open-ended 10 X 50 mm chamber made of two coverglasses separated by #I; glass strips. When cells activated to stage 3, approximately 0.5 ml of gassed buffers with or without hydrogen peroxide was perfused through the chamber by blotting. The cells were observed with video light microscopy, and results were recorded on tape. Preparation of Sperm for Low-Voltage Scanning Electron Microscopy (LVSEM)

Three milliliters of dilute sperm suspension was added to each of six 60 mm plastic petri dishes containing clean #1-1/2 coverslips, followed by 20 pl VDX. One dish was sealed and monitored by LM for stage of activation. Others were incubated in a 15% CO,/N, incubator at 40°C. Cells on coverslips were fixed at intervals of 1 to 20 min with an equal volume of fixative at room temperature for 1 h. Fixatives at final concentrations were 2% glutaraldehyde with either 0.05% saponin or 2.5% freshly prepared paraformaldehyde in 100 mM HEPES, pH 7.4. Fixed cells were washed in buffer, post-fixed 60 min in 1% OsO,, dehydrated to 100% ethanol, and critical-point dried with anhydrous CO,( 1). Specimens were platinum-coated by ion beam sputtering

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in an argon atmosphere and imaged with a Hitachi model S-900 low-voltage scanning electron microscope operating at 2 kV. To view the attachments of cells to the substrate, some critical-point-dried cells were torn free of the coverslip by pressing the coverslips gently against doublestick tape (Scotch brand, 3M Corporation, St. Paul MN; personal communication, H. Ris, University of Wisconsin, Madison). The ripped cells on coverslips and the complementary fragments on tape were coated with platinum and observed by LVSEM. Reagents

Biochemical reagents were purchased from Sigma Chemical Company (St. Louis). RESULTS Favorable Conditions for Activation and Crawling

Although Ascaris sperm partially activate under a variety of conditions, they crawled only if ( I ) spermatids were fresh (from Ascuris collected less than 24 h before activation and maintained at 37-40°C) and (2) all worm and sperm media were saturated with a 15% COJN, atmosphere. (Worms incubated in media saturated with N, alone died in 24 h.) Under these conditions the crude native activator preparation, VDX, will induce pseudopod formation and crawling in >95% of sperm cells at a concentration as low as 1 pgiml total protein. On the other hand, S . griseus protease at 25 pgiml induces the morphological changes in Ascaris spermatids associated with activation, but cells do not crawl. Both morphological transformation and crawling will proceed in the presence of 1-10 mM sodium azide. In Ascaris sperm, oxygen appears to be toxic. (See “Aerobic Aging of Activated Sperm” below .) Events of Activation

Ascaris sperm activation is marked by three directly observable processes: ( 1) so-called refringent granules in the cell body coalesce, (2) the pseudopod develops, and ( 3 ) pseudopodial membrane specializations, the villipodia, begin to flow from tip toward cell body. If the pseudopod of a fully activated cell makes proper contact with the substrate, the spermatozoon begins to crawl. Figure I . a composite of representative micrographs from the time-lapse film, shows the stages of in vitro activation from immotile cell through the onset of crawling. Figure la shows the inactive ovoid spermatid (“stage O”), its axes averaging 16 X 18 p m , filled with numerous small refringmt ,granules. Other inclusions not distinguishable at the LM level are mitochondria, a condensed nucleus, and numerous ~ n c ~ ~ ~ i l ~ r c i ~OPieoiis

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Fiy. I .

Ascaris Sperm Motility

ganelles (MO). MOs, found in all nematode sperm, migrate to and fuse with the plasma membrane during early activation, forming prominent external pores [Burghardt and Foor, 1978; see also Fig. 2a,c,d]. Formation of the Pseudopod

The first evidence of activation is the formation of small blebs on the surface within 1 min of adding VDX (Fig. 2a, “stage I ” ) . At this point the cells are attached to the glass substrate and do not detach with mild washing. (Unactivated cells do not attach to glass under the same conditions.) The ripped area of the cell in Figure 2a delimits this region of attachment and exposes the organelles beneath. The adherent patch persists until cells begin to crawl. Fibrillar material attached to the membrane, refringent granules, and MOs are in evidence at the site, but in this case MOs have not yet fused with the membrane attached to substrate. Other attached membrane patches contained 0-3 fused MOs associated with the inner face of the membrane. With time, more blebs erupt over the surface (Figs. l b , 2b). These stubby projections come together to form a short pseudopod (Fig. lc, ”stage 2”). The pseudopod rapidly extends, exhibiting complex surface folds. Ruffling and the rearward migration of the finger-like villipodia from the leading edge of the pseudopod toward the cell body are evident as early as 4.5 min. Once established, the flow of villipodia is constant throughout activation, ranging from 25 to 130 pmimin in different experiments. The pseudopod is maximally extended to about 15 pm by 8 min; total average cell length is 28 pm. Unattached, the pseudopod oscillates rapidly in a circular motion above the attached cell body (Figs. Id, 2c, “stage 3”). These dynamic aspects of the activation process are best seen in the videotape supplement. Figure 2c ,d, scanning electron micrographs of ripped stage 3 cells, shows how the internal structures at the area of cell-body attachment have changed. Several

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large refringent bodies have formed from the small granules seen in Figure 2a. Large numbers of MOs have fused with the cell body’s plasma membrane as evidenced by the prominent pores covering the outside (Fig. 2c) and by the shells of many fused MOs still adhering to plasma membrane attached to the glass (Fig. 2d). Note also the adherent filamentous material surrounding the MOs on the inner face of the plasma membrane, presumably part of the assembling pseudopod fiber complexes. The Onset of Motility

Table I shows the wide variation in the time at which the events leading to crawling begin (stages 4 and 5). After a period ranging from 8 to 33 min, the pseudopod makes repeated contacts with the substrate, as evidenced by the birefringence of the pseudopod as it touches the glass, and exhibits active ruffling of the edges (Fig. le, “stage 4”). Approximately 2 min later, the pseudopod makes broad contact with the substrate and exhibits obvious tractile force: the cell body, still tethered to the glass, is stretched, taffy-like, by the pseudopod (Fig. 3a). The spermatozoon pulls itself free or is unstuck by other cells and begins to crawl forward, the pseudopod pulling the cell body behind it (Figs. If, 6a, “stage 5”). The early cell-body attachment is not a prerequisite to crawling: spermatids may be activated 15 or more min in a polypropylene tube, to which they will not adhere, then transferred to glass. Under these conditions, the pseudopods, but not the cell body, will attach to the glass, and some of the cells will begin to crawl. When cells were activated at 42”C, pseudopod formation proceeded more rapidly, and villipodial flow exceeded 80 pmimin (maximum observed = 130 pm/ min). Crawling began as early as 9-1 1 min, even though coalescence of refringent vesicles was incomplete. such spermatozoa, however, crawled only 30-60 s, then rounded up. Characteristics of Sperm Motility

Fig. 1. Time-lapse cine sequence of Ascuris sperm in vitro activation through full forward motility. The same 3 cells are shown in a-e. a: Inactive spermatid from seminal vesicle (“stage 0”). b: Blebs erupt over surface of spermatid: cell body attaches (”stage I ”): activation time: 5 min. c: Formation of early. stubby pseudopod; beginning coalescence of vesicles (“stage 2”); activation time: 6 min. d: Elongation of pseudopod. advanced coalescence o f vesicles into “donut” configuration (“stage 3”): activation time: 10 min. e: Pseudopod makes intermittent contact with substrate. actively ruffles: coalescence of vesicles complete (“stage 4”); activation time: 32 min. f: Onset o f crawling (”stage 5“); branched fiber complexes are visible in pseudopod (arrow); activation time: 44 niin. Cell at bottom left o f frame (asterisk) is reversing direction and will follow the cell to its right. Phase-contrast optics.

Crawling sperm exhibit the following features: ( I ) The length of the pseudopod remains constant while the cell is crawling (as in Fig. If); (2) the villipodia formed at the leading edge of the pseudopod become larger ruffled structures as they move over the top or form large, birefringent areas of contact as they move beneath the pseudopod; (3) the membrane ruffles terminate abruptly at the boundary between cell body and pseudopod (Fig. 6a); (4) prominent fibers of high refractive index roughly parallel to the axis of the pseudopod can be seen running its length and branching out toward the leading edge (Fig. I f ) . These are the assembled fiber complexes comprising the pseudopod cytoskeleton [Sepsenwol et al., 19891.

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Fig. 2. Low-voltage scanning electron micrographs of pseudopod formation, cell body attachment, and MO fusion. a: Underside of “ripped” early stage I cell that had been attached to glass; time of activation. I min. Exposed patch shows numerous cortical membraneous organelles (MO) and refringent granules (g) in the region of cell attachment. These MOs have not yet fused with the plasma membrane, nor have the refringent vesicles begun to coalesce. The fibrillar material between organelles is thought t o be precursor o f the pseudopod cytoskeleton. The blebs ( b ) erupting from the surface are the first sign of pseudopod formation. b: Stubby villi form after early blebs and precede the formation of a stubby pseudopod (see Fig. I b). c: Undcrside o f cell body o f stage 3 “ripped” spermatozoon; time of activation. 17 min. Numerous MOs have fused with the cell-body membrane

during activation forming characteristic ringed transmembrane pores (arrowheads). The pseudopod and villipodia (v) have not yet attached. Fused cytoplasmic granules (fg) are forming the large refringent body seen in fully activated sperm. d: Cell-body attachment site (coverslip side) o f stage 3. “ripped” cell from the same preparation as c; time of activation. 17 rnin. Numerous MOs cluster against the plasma membrane; some have been ripped open and show the external pore structure attached to the empty MO shells (asterisks). Small bare patches represent MOs that have been ripped free of the membrane, as seen in b. Filamentous material attached to the cytoplasmic side of the membrane (arrows) is common. Glutaraldehyde-paraf;,rmaldehyde fixation; V,,,,,. 2 kV: magnification bars = I pm.

Some of the first cells to crawl collide with others still tethered to the substrate by their cell bodies. At the moment the pseudopod of another cell touches the base

of a tethered cell body, the rapid membrane flow of both suddenly ceases. The villipodia of the tethered cell are stretched out, claw-like, without detaching (Fig. 3b).

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TABLE I. Timetables for In Vitro Activations (Time-Lapse Film and Video) Stage 1

2 3 4 5 Speed of crawling

Timc-lapse film [time o f onset] 2 . 8 min 5.5 min 6.5 min 32 rnin 42 rnin 70 pmimin

Video I [time of onset] 2 min 3-4 min 5 min 8-9 min -

Video 2 [time of onset] -

4-5 min 6 min 8 min 14 25 pm/min

Video 3 [time of onset] 3 min“ 5 min

7-8 min I 1 min 90 pm/minh

“Chamber temperature = 42-43°C. hCells crawled 30-60 s before rounding up.

Fig. 3. Behavior of motile stage 5 sperm tethered to glass. a: Motile cells with cell bodies still attached. Tractile force exerted by attached pseudopod is shown by stretched cell body (*). Some cells have protruded pseudopods beneath others’. b: Tethered cell lifted from sub-

strate by another tethered cell crawling behind. Note the elongated villipodia, still attached, at the leading edge of the pseudopod (arrows). Phase-contrast optics; magnification bar, 5 pin,

When the free cell withdraws, membrane flow returns to the tethered cell. All in vitro activated spermatozoa in a given preparation initially crawl at the same speed. The speed varies directly with temperature within a range of 32°C (speed = 0) to 42°C (>SO pmimin). In the 6 experiments shown in Table 1 , crawling rates ranged from 25 to 90 pm/min at 38-42°C. The range of speeds of Ascaris sperm is rapid compared to actin-rich amoeboid cells (Table 11). As the cell moves, the membrane flow appears frozen; that is, the rearward movement of membrane ruffles formed by the villipodia exactly matches the forward movement of the spermatozoan. Such cells will crawl continuously for up to 30 min and then round up (see “Aerobic Aging of Activated Sperm” below).

sperm. The narrow range of values for stages 1 through 3 indicates that the morphological transformations are predictable from preparation to preparation under the same incubation conditions. It is not unusual for hundreds of cells within a single low-power field to enter the same stage of activation within 30 s of each other. The greatest variation among preparations is the onset of crawling (stage 5 , 9-41 rnin); yet in a normal activation, all cells in a given preparation begin to crawl within a 2-min interval.

Synchrony of Events

Table I summarizes the times at which cells enter stages 1 through 5 , in four experiments using different

Turning

While the pseudopod’s shape is roughly rectangular and stable when crawling in a straight line, it is capable of rapid change when its path is obstructed. Figure 4 is a composite of video frames of a turning cell. The change in direction begins at the leading edge of the pseudopod. Villipodia and fiber complexes suddenly appear off to a side away from the obstruction. The bulk of

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Sepsenwol and Taft TABLE 11. Translocation Rates for Ascaris Sperm and Other Amoeboid Cell Types Cell type In vitro activated A.sc.uri.s sperm In vitro activated Caeiiorhabcliti.~.sperm Streaming Dicrwstelium amoebae Activated human neutrophils Chick embryo fibroblasts

Translocation rate

Reference

25-70 + ymimin 20 ymimin 1-40 ymimin 17-25 ymimin 0.16-0.85 Fminiin

Roberts and Ward [ 1982aI McNarnara et al. [ 19871 Burton et a]. [ 19861 lshihara et al. [ 19881

~

the pseudopod and the cytoskeletal fibers within extend in that direction, and the cell body is turned as it follows the new leading edge. Sperm-Sperm Interaction

In any field of crawling sperm there were numerous examples of sperm changing and even reversing direction as another passed nearby (without touching); these “paired” sperm followed behind or crawled alongside each other for some distance. Figure I f shows one such example; this and four others are included in the videotape supplement. Effective Substrates for Crawling Cells

Ascaris spermatozoa attached and moved most rapidly on acid-washed glass. Their pseudopods attached poorly, and the cells did not crawl on normal or aminemodified polystyrene plastics, or on glass coated with 0.5% gelatin, 0.5% bovine serum albumin (fraction V), or Ascaris uterine extract. Poly-d-lysine-coated glass (MW 30,000, 0.1 mg/ml) was very effective in binding the sperm, as evidenced by the spreading and flattening of the pseudopod; however, villipodial flow ceased, and the cells did not move. Similarly, activated cells attached irreversibly to nitrocellulose paper (0.45 pm pore size, Schleicher & Schuell, Keene, NH) which was immersed in buffer, but failed to crawl. Aerobic Aging of Activated Sperm

Normal activation and motility occur in the presence of high CO, and low oxygen. If motile spermatozoa are exposed to air they cease crawling, and the pseudopod rapidly changes its morphology in a predictable manner. Figure 5 , a composite of video-DIC frames, illustrates this aging phenomenon. In Figure 5a,b, the forward third of the pseudopod stops advancing as no new villipodia are generated along the leading edge. In the posterior two-thirds of the pseudopod, however, villipodia continue to flow rearwards (Fig. 5c-e). The motile portion shortens and pulls the cell body into the immotile front, with the result that the cell body becomes surrounded by pseudopod. Thus, two processes, pseudopod extension at the tip and pseudopod shortening at its junction with the cell body, become separated after 20-

30 min in vitro. Sometimes, pseudopod membrane recedes from the edges, revealing long spikes (Fig. 6b). These spikes may wave back and forth, but no new villipodia form and there is no sign of centripetal flow. About 10-20 min following the puddling of the pseudopod, the spikes resorb, and the cell resembles a “fried egg” (Fig. 6c). The causative factor for this aging phenomenon is not known, but there is evidence that it may be due to the presence of oxygen, rather than a lack of CO, (See Table 111). Perfusing motile cells with media saturated with air instead of 15% CO,/N, or containing hydrogen peroxide will duplicate the spiking and rounding up, but medium lacking CO, does not. The destruction of pseudopod morphology in peroxide-containing media is much more rapid than air, being complete in 40 s to 2 min. Aging was not prevented when 10 mM azide was added to air-saturated HEPES-saline.

DISCUSSION

Many different kinds of sperm are stored outside the testis in a relatively inactive state and experience an activation step before reaching the site of fertilization. Among the flagellated sperm, gametes contain the complete motile apparatus. In the case of Ascaris sperm, and probably nematode sperm generally, an entire locomotory system is assembled during the short activation process. This allows male Ascaris gametes to conserve energy during their storage as spermatids and to begin their ascent of the long female reproductive tract shortly after insemination. The Ascaris Sperm Activator

This study demonstrates that the native factor in the male Ascaris’ vas deferens is sufficient to transform in vitro spermatids from the seminal vesicle into fully developed, crawling spermatozoa. Preliminary evidence indicates that the vas deferens factor responsible for sperm activation is a pepsin-sensitive glycoprotein in the molecular weight range of 60 kD (S. Sepsenwol, unpublished.) The mechanism by which it initiates activation is unknown. That some proteases activate both Ascaris and Caenorhubditis sperm suggests that nematode sperm ac-

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tivators might be proteases. Indeed, the action of VDX seems to be inhibited by the serine-protease inhibitor PMSF (unpublished). Nonetheless, its target is specific: Ascuris VDX will not activate Cuenorhubditis sperm (T.M. Roberts, Florida State University, Tallahassee, personal communication; Ascuris sperm used as positive controls). The failure to achieve crawling in Ascuris sperm activated with serine proteases, even under optimal conditions of C02/N2atmosphere, temperature, and substrate, may reflect non-specific hydrolysis of membrane proteins necessary for pseudopod attachment. Anaerobiosis and Substrate Requirements for Motility

For spermatids to activate properly with VDX and become crawling spermatozoa requires fresh spermatids, a proper substrate, anaerobiosis, and high pC0,. By comparison, Ascaris muscle mitochondria generate ATP in the absence of free oxygen and lack significant cytochrome oxidase activity [Saz, 1971; Kohler and Bachmann, 19801. Muscle cytoplasmic enzymes also fix CO, as part of this anaerobic fermentation pathway [Saz, 1981; Rioux and Komuniecki, 19841. Little is known about the metabolism of Ascaris spermatids or activated sperm; however, the dependence of activation and motility on CO, and their independence from azide is consistent with what is known of Ascaris muscle metabolism. On the other hand, both air and hydrogen peroxide effectively arrest motility and lead to the permanent destruction of pseudopod morphology. This sensitivity to oxygen may reflect an inability of sperm to handle damaging peroxides. Anaerobiosis is exceptional among nematode sperm. Those of Cuenorhabditis [Ward et al., 19831, Nipposfrongyfus[Wright and Sommerville, 19841, and Nemutospiroides [Wright and Sommerville, 19851 activate and/or crawl under aerobic conditions, and at least Cuenorhubdifis sperm are inhibited by 1-10 mM azide [Pavalko and Roberts, 19871. Cell Adhesion During Activation and Crawling

Fig 4. Video-DIC sequence of turning cell. An extension forms in the new direction from the preexisting pseudopod. A line is drawn through the longitudinal axis o f the cell body to show that it is pulled around in the new direction during the maneuver. Fiber complexes are visible in a. Time interval between frames. 10 s; magnification bar. S Pm.

The earliest observable event of in vitro activation was the adherence of spermatids to glass. While its physiological significance is uncertain, this property reflects the insertion of adhesion molecules into the cell membrane soon after exposure to VDX, tethering the cell body during pseudopod formation. The adhesion molecules in the cell body may be the same as those in the psuedopod, but the dynamics are different: once broken, the attachment of the cell body is not reformed; on the other hand, there seems to be a continuous cycle of attachment-detachment between pseudopod membrane and substrate. Pseudopod attachment occurs at the tips of villipodia that form at the leading edge, as clearly shown

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TABLE 111. Effect of Gas and Peroxide on Crawling Cells Re-perfusion medium“

Cessation of motility

Resumption of mot i I i t y

Formation of spikes + rounding up

C02/NI N2 only C0,/N2 C02/N,

Immediateh Immediateh Immediate‘ Immediate‘

40 s 40 s

>40 min >30 min 20-25 s 2.5-3.0 min

+ 9 m M HzOl + 0.9 mM H 2 0 2

-

“Sperm activated to stage 3 in coverslip chamber under normal conditions, then perfused with 500 pl of experimental buffer. ’Spermatozoa typically stopped moving (immediate = 40°C, sperm activate faster and begin to crawl sooner. Sperm-Sperm Interaction

What factors-physical or chemical-cause some crawling cells to change direction and follow others are not known. Nor is it known whether this “pack” behavior occurs in vivo. The Phenomenology of Ascaris Sperm Motility: An Extension-Shortening Model

A model has been presented for Ascaris sperm motility based on the movement and behavior of cytoskeleta1 structures, the fiber complexes, found within the

Fig. 5 . Video-DIC sequence showing “aging” o f crawling cells exposed to air. Crawling cell (a) stops extending pseudopod and (b) villipodial flow ceases in the anterior third o f the pseudopod. Villipodia continue to move centripitally i n the posterior two-thirds of the pseudopod. c-e: The rear of the pseudopod shortens, the cell body continues to be pulled forward. Magnification bar, S p m Fig. 6. Scanning electron micrographs o f normal stage 5 and aging spermatozoa. a: Stage 5 spermatozoon fixed during crawling. Note sharp boundary between pseudopod and cell body, the presence o f MO fusion pores over the cell body, and the absence of villipodia o n the cell body membrane. b: A spiked. aging spermatozoon about 10 niin after cessation of crawling in aerobic medium. Cell body has been pulled into pseudopod similar t o Figure 5e. c: “Fried egg” spcrmatozoon 20 min after cessation of crawling in aerobic medium. Glutaraldehyde-paraforinaldehyde fixation; V,,,,,. 2 kV: magnification bars. 5 pm.

pseudopod [Sepsenwol et al., 19891. In this model, fiber complexes and the villipodia formed by their extension into the pseudopod membrane are assembled at the tip, and the complexes are disassembled at the cell bodypseudopod junction. This treadmilling model implies that the pseudopod shortens as it extends to move the cell along the substrate. The “aging” of cells (Fig. 5 ) provides another confirmation of this model by demonstrating shortening in the “tethered” pseudopod. Thus. two independent processes, normally operating in unison in crawling cells, have become asynchronous: ( 1 ) c‘ontinuous extension of the pseudopod by forming new villipodia (fiber complexes and membrane) at the leading edge, and (2) continuous shortening of the pseudopod (complexes and membrane) at the CPJ. The sudden cessation of villipodial flow seen when cells crawl beneath the CPJ of tethered sperm (Fig. 2b) may reflect temporary interference with membrane internalization. Amoeboid Motility: Nematode Sperm Vs. Actin-Based Systems

For many years nematode sperm have been described as “amoeboid” because they crawl over substrate via a pseudopod. There are, however, significant differences between the pseudopodial locomotion of Ascuris sperm and the actin-based movement described for Amoeba proreus and other crawling cells. ( I ) The pseudopod is a permanent differentiation of the cell, which acts as the locomotor organelle. When turning, only the pseudopod reforms in a new direction rather than being resorbed into the cell and reformed from the cell body. (2) The movement of the pseudopodial membrane specializations in nematode sperm appears continuous, rather than pulsatile. (3) A structurally homogeneous cytoskeleton shapes the pseudopod. The formation and movement of villipodia are related to the assembly and movement of the pseudopod’s MSP cytoskeleton, the fiber complexes [Sepsenwol et al., 19891. On the other hand, what appear to be unique features o f the nematode sperm motility system may provide

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a different perspective for viewing actin-based ameoboid motility. Specifically, treadmilling of the cytoskeleton (e.g., fiber complexes or f-actin) and recycling of pseudopod membrane might suffice to generate amoeboid locomotion. A contractile interaction with another cytoskeletal element ( e . g . , myosins) may not be required. ACKNOWLEDGMENTS

The authors would like to thank Mai Nguyen Thieu, Harold Nguyen, and Thanh Duong for their early contributions to this project, the staff at the Integrated Microscopy Resource, Madison (G. Schatten, Director) for their valuable technical assistance with the video DIC, and Hans Ris for his interpretive insights and editorial comments. This work was supported by grants from the UWSP-UPDC, the National Science Foundation (DCB 8610475), and the National Institutes of Health (R15-GM37435-01). The Madison Integrated Microscopy Resource is supported by NIH Biomedical Research Technology Program Grant P4 1-RR00570. REFERENCES Abbas, M., and Cain, G.D. (1979): In vitro activation and behavior of the ameboid sperm of Ascaris suum (Nematoda). Cell Tissue Res. 200:273-284. Burghardt. R.C., and Foor, W.E. (1978): Membrane fusion during spermiogenesis in Ascaris. J . Ultrastruct. Res. 62: 190-202. Burton, J.L.. Law, P., and Bank, H.L. (1986): Video analysis of chemotactic locomotion of stored human polymorphonuclear leukocytes. Cell Motil. 6:485-491. Foor, W.E. (1970): Spermatozoan morphology and zygote formation in nematodes. Biol. Reprod. [Suppl.] 2:177-202. Foor, W.E. (1983): Nematoda. In Adiyodi. K.G., and Adiyodi, R.G. (eds.): Reproductive Biology of Invertebrates, Vol. 2., “Spermatogenesis and Sperm Function.” New York: John Wiley and Sons, pp. 221-256. Foor, W.E., and McMahon, J.T. (1973): Role of the glandular vas deferens in the development of Ascaris spermatozoa. J . Parasitol . 59:753 -758. Ishihara, A., Holifield, B., and Jacobson, K. (1988): Analysis of lateral redistribution of a monoclonal antibody complex plasma membrane glycoprotein which occurs during cell locomotion. J. Cell Biol. 106:329-343. Kohler. P., and Bachmann, R . (1980): Mechanisms of respiration and

phosphorylation in Ascaris muscle mitochondria. Mol. Biochem. Parasitol. 1:75-90. McNarnara, G.T., Barclay. S.L., and Futrelle, R.P. (1987): Morphogenetic movements during cell aggregation in Dicposrrlium discoidrum wild-type and streamer F mutants defective in cGMP metabolism. J . Cell Biol. 105:260a. Nelson, G.A., and Ward, S . (1980): Vesicle fusion. pseudopod extension and amoeboid motility are induced in nematode spermatids by the ionophore monensin. Cell 19:457-464. Nelson, G.A., and Ward, S . (1981): Amoeboid motility and actin in Ascaris lumbricoidc2s sperm. Exp. Cell Res. 131:149-160. Pavalko, F.M., and Roberts. T.M. (1987): Caenorhabditis elegans spermatozoa assemble membrane proteins onto the cell surface at the tips of pseudopodial projections. Cell Motil. 7:169-177. Rioux. A., and Komuniecki. R . (1984): 2-Methylvalerate formation in mitochondria of Ascaris suum and its relationship to anaerobic energy generation. J . Comp. Physiol. [B] 154:349-354. Roberts, T.M., Sepsenwol, S . , and Ris, H. (1989): Sperm motility in nematodes: Crawling movement without actin. In Schatten, H., and Schatten. G. (eds.): “The Cell Biology of Fertilization.” Orlando: Academic Press, pp. 41-60. Roberts, T.M., and Streitmatter, G. (1984): Membrane-substrate contact under the spermatozoon of Caenorhabditis elegans, a crawling cell that lacks filamentous actin. J. Cell Sci. 69: 117126. Roberts, T.M., and Ward, S . (1982a): Membrane flow during nematode spermiogenesis J Cell R i d 92:l 13-120. Roberts, T.M.. and Ward, S. (1982b): Centripetal flow of pseudopodial surface components could propel the amoeboid movement of Caenorhabditis rlegnns spermatozoa. J . Cell Biol. 92: 132138. Saz, H.J. ( 197 I ) : Anaerobic phosphorylation in Ascaris mitochondria and the effects of antihelminthics. Comp. Biochem. Physiol. [B] 391627-637. Saz, H.J. (1981): Energy metabolism of parasitic helminths. Annu. Rev. Physiol. 43:323-341. Sepsenwol. S . . Braun. T.. and Nguyen, M. (1986): Adenylate cyclase activity is absent in inactive and motile sperm in the nematode parasite, Ascaris suum. J. Parasitol. 72:962-964. Sepsenwol, S . , Ris, H., and Roberts, T.M. (1989): A unique cytoskeleton associated with crawling in the nematode, Ascaris mum. J. Cell Biol. 1 0 8 5 - 6 6 , cover. Sepsenwol, S . . and Taft, S.J. (1990): Ascaris sperm motility [videotape supplement]. Submitted for publication. Ward. S . , Hogan. E., and Nelson, G . A . (1983): The initiation of Caenorhabditis elegans spermiogenesis in vivo and in vitro. Dev. Biol. 98:70-79. Wright, E.J., and Sommerville, R.1. (1984): Postinsemination changes in the amoeboid sperm of a nematode, Nippostrongy/us brasrliensis. Gamete Res. 10:397-413. Wright, E.J., and Sommerville, R.I. (1985): Structure and development of the spermatozoon of the parasitic nematode, Nemutospiroides dubius. Parasitology 90: 179-192.

In vitro induction of crawling in the amoeboid sperm of the nematode parasite, Ascaris suum.

In a highly synchronous process, the immotile spermatids of Ascaris suum extend pseudopods and become rapidly crawling sperm when treated with an extr...
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