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Role of the Cytoskeleton in Sperm Entry During Fertilization in the Freshwater Bivalve Dreissena polymorpha.

M. J. MISAMORE [*]

J. W. LYNN [+]

Abstract. The present study examined the role of the cytoskeleton in sperm entry and migration through the egg cytoplasm during fertilization in the zebra mussel, Dreissena polymorpha (Bivalvia: Veneroida: Dreissenidae). Fertilization in this freshwater bivalve occurs outside the mantle cavity, permitting detailed observations of fertilization. After its initial binding to the egg surface, the sperm is incorporated in two stages: (1) a gradual incorporation of the sperm nucleus into the egg cortex, followed by (2) a more rapid incorporation of the sperm axoneme, and translocation of the sperm head through the egg cytoplasm. Initial incorporation into the egg cortex was shown to be microfilament dependent. Microfilaments were found in the sperm's preformed acrosomal filament, the microvilli on the egg surface, and in an actin-filled insemination cone surrounding the incorporating sperm. Treatment of eggs with cytochalasin B inhibited sperm entry in a dose- and time-dependent manner. Microtubule polymerization was not necessary for initial sperm entry.

Following incorporation of the sperm head, the flagellar axoneme entered the egg cytoplasm and remained active for several minutes. Associated with the incorporated axoneme was a flow of cytoplasmic particles originating near the proximal end of the flagella. Inhibition of microtubule polymerization prevented entry of the sperm axoneme, and the subsequent cytoplasmic current was not observed. After sperm incorporation into the egg cortex, no appreciable microfilaments were associated with the sperm nucleus. A diminutive sperm aster was associated with the sperm nucleus during its decondensation, but no obvious extension toward the female pronucleus was observed. The sperm aster was significantly smaller than the spindle associated with the female pronucleus, suggesting a reduced role for the sperm aster in amphimixis.

Introduction

During fertilization in the freshwater zebra mussel, Dreissena polymorpha (Bivalvia: Veneroida: Dreissenidae), gametes are spawned directly into the water column, where gamete binding occurs. External fertilization is common among marine bivalves but is unusual among freshwater bivalves, in which fertilization and larval development typically occur in the mantle cavity (Burky, 1983). Broadcast spawning of gametes, the relative ease of inducing spawning (Ram et al., 1993), and the highly transparent nature of the egg cytoplasm make zebra mussels a good model system for studying early fertilization events.

Sperm entry into the egg cytoplasm during fertilization is a two-step process (Misamore et al., 1996). During the initial phase, the sperm head and midpiece are gradually (10 [micro]m/min) incorporated into the egg cortex. Following sperm binding, an insemination cone forms on the egg surface and envelopes the fertilizing sperm head as it passes through the egg plasma membrane. Once inside the egg cortex, the sperm rotates 180[degrees] and "drifts" laterally along the egg cortex. During the second phase of sperm entry, most of the sperm axoneme is incorporated into the egg cytoplasm, and the sperm head may be rapidly (1 [micro]m/s) translocated through the egg cytoplasm. The amount of translocation apparently depends on the position of the female genome in relation to the site of sperm entry (M. Misamore, unpubl. results). After axonemal incorporation and nuclear translocation, the sperm chromatin decondenses and is encompassed by a membrane forming the male pronucleus. Concurrent with sperm fusion, meiosis is reinitiated from metaphase I arrest, polar bodies form, and the female pronucleus develops. Pronuclear fusion and cleavage occur by 60-70 min postinsemination (P1).

The gradual incorporation of the sperm head in D. polymorpha displays similarities to early sperm entry in other invertebrate species (Longo and Anderson, 1968; Schatten and Mazia, 1976; Longo, 1973b). In these systems, microfilaments are believed to play a critical role in sperm incorporation into the egg cortex (Longo, 1980; Tilney and Jaffe, 1980; Cline et al., 1983; Cline and Schatten, 1986). This theory is supported by the localization of microfilaments both in egg microvilli (Burgess and Schroeder, 1977; Spudich and Amos, 1979; Tilney and Jaffe, 1980) and in sperm acrosomes (Tilney, 1975, 1978; Tilney et al., 1973); by the fertilization modifications of microfilament distribution, particularly in fertilization cones (Longo, 1978b, 1980; Tilney and Jaffe, 1980; Schatten and Schatten, 1980; Cline and Schatten, 1986); and by the blockage of fertilization by the inhibition of microfilament polymerization using cytochalasins (Gould-Somero et al., 1977; Longo, 1978a; Byrd and Perry, 1980; Schatten and Schatt en, 1980). Movement of the sperm nucleus, or subsequent pronucleus, through the egg cortex in most invertebrate and mammalian systems is attributed primarily to the formation of a microtubule-containing sperm aster. However, many bivalve species produce only a rudimentary sperm aster.

The objective of this study was to determine the role of microfilaments and microtubules in the two-step incorporation of sperm into the egg cytoplasm in Dreissena polymorpha. The initial, gradual incorporation of the sperm into the egg cortex required microfilament polymerization, whereas the rapid translocation of the sperm nucleus through the egg cytoplasm was microfilament independent but could be inhibited by preventing flagellar incorporation into the egg. Furthermore, a flow of cytoplasmic particles was observed to be associated with the sperm axoneme, which remained active following entry into the egg.

Materials and Methods

Collection of gametes

Specimens of Dreissena polymorpha were collected from the Mississippi River near Baton Rouge, Louisiana, or from Portage Lake near Ann Arbor, Michigan, and maintained at 9[degrees]C. Animals were individually isolated overnight in artificial pondwater (PW) (Dietz et al., 1994) to avoid cross-contamination of gametes prior to use. Spawning was induced by external exposure of animals to 2 X [10.sup.-4] M serotonin (5-hydroxytryptamine) for 12 min. Animals were washed twice with PW. At the start of spawning, females were transferred to 50-ml crystalizing dishes to complete spawning. Eggs were inseminated with about 300 sperm per egg.

Three preparation and fixation protocols were used. For light microscopy, eggs were fixed in 3.2% paraformaldehyde in mussel buffer (5 mM TAPS, 0.8 mM NaCl, 0.145 mM KCl, 1.8 mM [Na.sub.2][SO.sub.4], 0.887 mM Mg[SO.sub.4] 7[H.sub.2]O, 1.32 mM Na[HCO.sub.3], 1.19 mM Ca[Cl.sub.2] * 7[H.sub.2]O, pH = 7.6) for 3 h followed by two washes in mussel buffer. Eggs for antibody labeling were permeabilized in an extraction buffer (1.0 mM TAPS, 5 mM KCl, 0.5 mM Mg[Cl.sub.2], 1.0 mM EGTA, 0.05% Nonidet P-4O, 1% glycerol, pH 7.6) for 5 min, followed by fixation in 3.2% paraformaldehyde in a low-[Ca.sup.2+] mussel buffer (3 mM EGTA, 10 mM TAPS, 3 mM NaCl, 0.01 mM KCI, 1.5 mill Mg[SO.sub.4], 0.01% sodium azide, pH = 7.6) for 3 h, followed by two washes in phosphate buffered saline (PBS) (8.1 mM sodium phosphate dibasic, 1.8 mill sodium phosphate monobasic, 25 mill NaCl 25 mM KCl, pH = 7.8) with 0.5% bovine serum albumin. Electron microscopy samples were fixed in 2.5% glutaraldehyde in mussel buffer, washed twice with 30 mil l sodium cacodylate buffer, post-fixed in 0.5% osmium tetroxide for 1 h, dehydrated through a graded acetone series, and embedded in a modified Spurr's s medium (Spurr, 1969).

Microscopy

For real-time observations, either a Nikon Optiphot with phase contrast and epifluorescence optics or a Nikon Diaphot with differential interference contrast (DIG) was used. Confocal imaging was performed on a Noran Instruments (Madison, WI) argon laser confocal microscope with Intervision Software. A JEOL 100CX transmission electron microscope was used for high-magnification observations.

Eggs were stained with 1 [micro]g/ml Hoechst 33342 for 8 mm to label DNA. To distinguish sperm bound to the egg surface from sperm incorporated into the egg cytoplasm, a dual-labeling procedure was used. Briefly, inseminated eggs were fixed and dual-labeled with Hoechst 33342 and FITC-conjugated wheat germ agglutinin (WGA) (Sigma Chemicals, St. Louis). Hoechst 33342 labeled sperm and egg DNA; WGA-FITC labeled sperm and egg surfaces (Kreimborg and Lynn, 1996). Sperm bound to the surface were labeled with both fluorochromes; incorporated sperm were labeled only with Hoechst. To label microfilaments, samples were stained with 1 [micro]g/ml FITC-conjugated phalloidin (Sigma Chemicals) for 10 min then washed twice with mussel buffer. To label egg microtubules (MTs), permeabilized samples were exposed to a monoclonal antibody to yeast [alpha]-tubulin (Accurate Antibodies MAS-077, Westbury, NY) at 1:20 dilution for 45 min, followed by a FITC-conjugated secondary antibody. A monoclonal antibody to acetylated [alpha] -tubulin (Sigma Chemicals) at 1:100 dilution for 1 h was used to selectively label sperm axonemes. This antibody selectively labeled sperm axonemal MTs but not egg MTs (Fig. 1) (L'Hernault and Rosenbaum, 1983; L'Hernault and Rosenbaum, 1985; Fechter et al., 1996).

Inhibition experiments

Prior to exposure to the inhibitors, eggs were treated with 0.005% sodium periodate for 2 mm to increase permeability through the extracellular coats of the eggs. This pretreatment does not significantly impact sperm binding or fertilization (Misamore, unpubl. results). Eggs from one spawn were aliquoted into the various treatment groups.

Cytochalasin B (GB) (Sigma Chemicals, St. Louis, MO) was used to inhibit microfilament (MF) polymerization during fertilization. Microtubule polymerization was inhibited with colchicine and colcemid, which form complexes with tubulin on MT ends, preventing further polymerization; or with nocodazole, a synthetic benzimidazole that promotes MT depolymerization (Bray, 1992).

To determine the dose-dependent effects of the inhibitors on fertilization, periodate-treated eggs were incubated in the inhibitors (final concentrations: CB--6.2, 12.4 [micro]M in 0.6% DMSO; colchicine--50 to 200 [micro]M; colcemid--51 [micro]M in 0.4% DMSO; nocodazole--0.1, 50 [micro]M) or the appropriate PW control (with or without DMSO) for 10 min prior to insemination. Eggs were inseminated in the presence of the inhibitors without washing. Real-time observations were captured using video microscopy from 30 s to 40 mm PI. Samples were fixed at 10 and 30 mm PI and dual-stained with Hoechst 33342 and WGA-FITC. Stained eggs (n = 100) were scored to determine sperm entry, number of bound sperm, meiotic stage of female DNA, and presence of polar bodies. To determine whether the effects of the inhibitors are reversible, eggs were incubated in the inhibitors for 10 min, washed twice with PW, and inseminated.

Based on preliminary findings, a time-exposure series was also performed to determine the temporal effect of CB on early sperm entry. Cytochalasin B (12.4 [micro]M) was added to periodate-treated eggs at the following time points: 10 mm before insemination or 0 mm, 2 mm, or 4 mm PI. Control eggs were incubated in 0.6% DMSO 10 mm prior to insemination.

Statistical analysis

For each trial, eggs from a single female were divided into the treatment groups. From each treatment, 100 eggs were scored for incorporated sperm nuclei and polar body formation. Replicate trials (n = 5) for the dose-dependent and time-dependent experiments were performed, and means of numbers of eggs with incorporated sperm were calculated for all treatments. A two-way analysis of variance (ANOVA) blocking for variability between trials (i.e., different batches of eggs) as one factor and treatment as the second factor was performed using SigmaStat software, version 2.0 (SPSS Science, Chicago, IL). Tukey multiple comparisons were performed if significant differences ([alpha] = 0.05) were found.

Results

Morphology

As background pertinent to the results, an expanded description of the basic gamete morphology is presented here. Additional descriptions of Dreissena polymorpha gametes are given by Franzen (1983), Denson and Wang (1994), Misamore et al. (1996), and Walker et al. (1996). The acrosome of D. polymorpha sperm contains four distinct regions, including an axial rod 1.4 [micro]m in length. The filaments composing the axial rod are about 6 nm in diameter (Fig. 2) and extend from the apex of the acrosome to the anterior margin of the nucleus (Misamore et al., 1996). FITC-phalloidin labeling was localized to a spike-like structure in the acrosomal region of both bound and acrosome-reacted sperm (Fig. 2, inset).

The surface of spawned eggs is uniformly covered in microvilli at an estimated density of 50 microvilli/[micro][m.sup.2] over the entire egg surface. The microvilli are 0.8 [micro]m in length and 0.07 [micro]m in diameter (Misamore et al., 1996). The internal core of the microvilli consists of 5-nm parallel fibers perpendicular to the egg cortex. The distal tips of the microvilli have many tufts of filamentous material perpendicular to the long axis of the microvilli (Fig. 3).

Initial sperm entry

Shortly after sperm binding, the sperm acrosome opened, exposing the membrane-bound axial rod. The preformed axial rod was long enough to traverse the egg investment coat to the egg surface (Fig. 4). No additional polymerization or elongation of the axial rod was observed. Microvilli directly subjacent to the open acrosome remained perpendicular to the egg surface. Small fibrous structures positioned peripheral to bound sperm and angled toward the bound sperm were observed with scanning electron microscopy (Misamore et al., 1996). However, no reorientation of microvilli toward the sperm was discernible. The filamentous tufts of the distal microvillar tips did not appear to be associated with a specific region of the acrosome. No change in microvilli length was observed up to 10 min PI.

About 2-3 min PI, an insemination cone formed on the egg surface. As the sperm passed through the oolemma, the cone initially appeared as a conical structure about 1.6 [micro]m in diameter and 2.7 [micro]m in length. Once the sperm head reached the egg cortex, the distal end of the cone became pointed as the egg cytoplasm closed around the sperm head. Filaments 6 nm in diameter extend the length of the fertilization cone in the egg cytoplasm (Fig. 5). FITC-phalloidin labeled the fertilization cone as a well-defined region on the oolemma surrounding the sperm nucleus (Fig. 6). After the sperm had entered the egg cortex, no definitive phalloidin labeling was associated with the sperm head; however, high background fluorescence may have obscured minimal labeling.

About 5-7 min PI, the first polar body began to form. There was a clearing of cortical vesicles and a reduction ia microvilli length associated with the site of polar body extrusion. When the egg stained with phalloidin-FITC, a brightly fluorescing region was seen on the surface where the polar body first emerged (Fig. 7). After expulsion of the female dyad, a cleavage furrow formed, separating the polar body cytoplasm from the remaining egg cytoplasm. This polar body furrow corresponds with a fluorescing ring at the base of the first polar body (Fig. 7).

This initial passage of the sperm through the insemination cone into the egg cortex was blocked by inhibiting micro-filament polymerization with CB ia a dose-dependent manner (Fig. 8). In the presence of CB, sperm were able to bind to the egg surface but did not enter into the egg cytoplasm. When eggs were exposed to 12.4 [micro]M CB, sperm entry was completely inhibited, whereas exposure to 6.2 [micro]M GB significantly reduced the number of eggs with incorporated sperm relative to the control (P [less than] 0.001) (Fig. 8; Fig. 9B', C'). Polar body formation was completely inhibited by both 6.2 and 12.4 [micro]M CB. The female chromosomes were positioned at the egg periphery and persisted through anaphase (Fig. 9). Although a brief extrusion of cytoplasm was observed during the period of normal polar body formation (5 min PI), no cytoplasmic separation occurred and the extrusion was reabsorbed by 10 min PI.

Sperm entry into the egg cortex was not affected by inhibition of MT polymerization. In both normal (control) fertilizations and fertilizations in the presence of MT inhibitors, D. polymorpha sperm bound to the egg surface within 30 s of insemination, and an insemination cone formed by 3 mm PI. The insemination cones of both control and MT-inhibited trials were morphologically similar, and the sperm head and mitochondria passed through the oolemma and entered the egg cytoplasm within 4 min of binding (Fig. 9D, D'). Compared to the control, there was no significant difference (Fig. 10) in the numbers of eggs exhibiting sperm entry in the samples treated with colchicine (P = 0.935) or nocodazole (P = 0.774). Colcemid did significantly decrease sperm entry relative to controls (P = 0.032); however, greater than 68% of the eggs had sperm incorporated. While not affecting sperm entry, the MT inhibitors disrupted the first meiotic spindle, and female chromosomes appeared scattered and did not reach anaphase I (Fig . 9D, D'). Subsequent polar body formation was likewise prevented.

Sperm translocation through egg cytoplasm

Following incorporation into the egg cortex, the sperm head passed through the insemination cone, rotated 180[degrees] in the egg cortex--positioning its basal end toward the egg center, and moved laterally along the egg cortex (Fig. 11A--D; Fig. 12A--C). The direction of lateral movement was towards the quadrant of the egg containing the female genome. Following rotation, most of the axoneme entered the egg cytoplasm. During the initial axonemal incorporation when the sperm head was immediately ([less than or equal to]10 [micro]m) subjacent to the cortex, the sperm head frequently pivoted around the entry point through angles exceeding 90[degrees] (Fig. 13). As more of the axoneme entered the cytoplasm, the axoneme extended deeper into the egg cortex relative to the sperm head, which remained near the egg cortex (Fig. 14A, A'). Once the bulk of the axoneme passed through the oolemma, the sperm head frequently moved rapidly through the egg cytoplasm. This movement did not occur in the sinusoidal pattern typi cal of sperm swimming. Rather, a portion of the axoneme extended ahead of the trailing sperm head along the direction of movement.

Although they did not impact entry of the sperm head into the egg cortex, MT inhibitors suppressed the rotation and lateral movement of the sperm head along the cortex. Furthermore, MT inhibitors prevented the incorporation of the sperm axoneme into the egg cortex (Figs. 12A-C; 14B, B'; 15; 16). Under these conditions, the sperm heads exhibited rapid, sporadic oscillations immediately subjacent to the insemination cone for several minutes after passage through the cone. This sporadic oscillation seen in the presence of MT inhibitors corresponded temporally to the period of expected axoneme incorporation in control fertilizations. In most inhibitor observations, the sperm remained immediately subjacent to the cone up to 7 min PI and the axoneme ultimately fractured, allowing the sperm nucleus to move deeper into the ooplasm--although more slowly than in controls (Fig. 12). During this period the sperm flagellum remained outside the egg, extending out through the insemination cone. On occasion, the unincorpora ted portion of the flagellum detached from the egg surface and remained active for several minutes. In contrast to control observations, probing MT-inhibited eggs with the flagellar monoclonal antibody failed to label an axoneme associated with decondensing sperm nuclei (Fig. 14B, B').

Once sperm passed through the egg cortex, inhibition of MF polymerization did not affect sperm head or axoneme incorporation or subsequent nuclear decondensation. Exposure of eggs to 12.6 [micro]M CB at various times pre- and postinsemination showed a temporal effect of CB on insemination (P [less than] 0.001) (Fig. 17). Eggs incubated in CB 10 min prior to insemination showed no sperm incorporation or polar body formation (see Fig. 8). Eggs exposed immediately prior to insemination (0 min) showed almost complete blockage of sperm entry and were not significantly different from the preincubation trial (P [less than] 0.05). Addition of CB at 2 min PI significantly reduced sperm entries (P [less than] 0.05) relative to controls but did not cause complete inhibition (Fig. 17). Sperm entry was not significantly different from controls when CB was added 4 min PI. Sperm entry and rotation in the egg cytoplasm occurred in eggs treated at 4 min PI with CB. At about 3 min PI, sperm-head translocation and axoneme inco rporation were observed. The presence of the axoneme in the egg cytoplasm was verified using the monoclonal antibody to acetylated [alpha]-tubulin.

At about 8 min PI, sperm nuclei began to decondense in control (Fig. 18) and MF and MT inhibitor trials. Sperm mitochondria separated from the nucleus and moved away from the nucleus concurrent with the decondensation of the sperm chromatin. At the same time, or immediately afterward, a small sperm aster started to develop (Fig. 19). The aster formed at the base of the decondensing sperm head and was only detectable with the monoclonal yeast [alpha]-tubulin antibody, visualized with confocal microscopy. In contrast, the female meiotic spindle and astral array were clearly discernible with light microscopy. In control fertilizations, the sperm aster was detectable as a diminutive structure as late as 40 min PI. During this period the female aster and associated bundle were substantially larger (Fig. 19) (Walker, 1996). Colchicinetreated eggs exhibited no obvious sperm aster, and the female meiotic spindle was not observed. Since egg activation was determined based on the resumption of meiosis as visualized by movement of chromosomes into anaphase I, disruption of the meiotic spindle made the status of egg activation in MT inhibitor treatments difficult to ascertain.

Flow of cytoplasmic particles

Concurrent with axoneme entry, sporadic "vibrations" were observed in the egg cytoplasm between the sperm head and the sperm entry site near the expected position of the axoneme. A directed flow of cytoplasmic particles was similarly observed originating near the base of the sperm head and penetrating as deep as 10 [micro]M into the ooplasm (http://www.mbl.edu/BiologicalBulletin/VIDEO/BB.video.html). The vibrations in the egg cytoplasm began shortly after rotation and lateral displacement of the sperm head, lasted from 1-3 min, and ceased shortly before the sperm nucleus began to decondense. Although the extent varied between fertilizations, this cytoplasmic flow was evident in greater than 70% of the 20-30 similar filmed observations and on about 80 more unfilmed occasions. These observations are the norm rather than the exception in the hundreds of fertilizations observed during several reproductive seasons. Furthermore, polyspermic eggs exhibited multiple currents associated with the polynumery sperm. Whe n flagellar incorporation was inhibited, no flow of cytoplasmic particles was observed in eggs. Similarly, the flow of cytoplasmic particles was also observed in eggs exposed to MT inhibitors followed by washing prior to insemination.

Discussion

Initial sperm entry

The incorporation of the sperm components through the oolemma into the egg cortex in Dreissena polymorpha occurs in two morphologically distinct steps (Misamore et al., 1996). During the initial incorporation, the sperm head and midpiece gradually enter into the egg cortex at a rate of 1 [micro]m/min (Misamore et al., 1996). A distinct, cylindrical insemination cone encompasses the sperm as it passes through the oolemma. The insemination cone of D. polymorpha consisted of many 6-nm-thick filaments (Fig. 5) and labeled with FITC-phalloidin (Fig. 6), suggesting the presence of microfilaments. The cone assumed a more pyramidal configuration once the sperm entered the egg cortex. This is similar to morphological changes in the insemination cone of sea urchins, in which an initially rounded cone becomes a "spike-like" cone following sperm entry (Tilney and Jaffe, 1980; Cline and Schatten, 1986). When treated with the inhibitor to MF polymerization, cytochalasin B, this initial sperm entry was blocked (Fig. 9C, C' ), and no fertilization cone formed. These findings suggest that the initial incorporation of the sperm into the egg cortex was dependent on the polymerization of MFs.

Although the critical involvement of microfilaments in sperm incorporation has been reported for many marine invertebrates (Gould-Somero et al., 1977; Longo, 1978a, 1980; Byrd and Perry, 1980; Schatten and Schatten, 1980; Cline and Schatten, 1986; Schatten et al., 1986) and the present freshwater model, exactly how sperm pass through the oolemma is not well understood. Microfilaments are associated with several processes during fertilization in D. polymorpha that could potentially account for the inhibition of initial sperm entry. The major sites of polymerized MFs include the sperm acrosome, the egg microvilli, and the fertilization cone. Furthermore, MFs are critical for cytokinesis during polar body formation (Longo, 1972; Longo et al., 1993).

The sperm of several marine bivalves including Spisula and Mytilus possess preformed acrosomal processes that do not undergo a polymerization-driven elongation (Hylander and Summers, 1977; Longo, 1978a). Following activation, Mytilus sperm extend a preformed acrosomal process without the polymerization of new MFs (Dan, 1967; Longo, 1977, 1983). Like these marine bivalves, D. polymorpha has sperm that possess a preformed acrosomal process (Fig. 2) that does not elongate and is apparently insensitive to CB treatments at the dosages tested. Although it was not possible to expose only sperm to CB because washing disrupted the fragile acrosomes, polymerization of sperm MFs is not believed to be the critical component in sperm entry. This conclusion is based on several pieces of evidence. First, no elongation of the acrosomal process was observed during fertilization in D. polymorpha. Second, several studies in which washing of sperm was possible have shown that CB does not affect the fertilization capability of s perm (Sanger and Sanger, 1975; Longo, 1978a; Byrd and Perry, 1980). Third, the relative polarity of MFs in sperm acrosomes and egg microvilli is inappropriate to allow a myosin-actin ratcheting mechanism to draw the sperm into the egg in urchins (Tilney, 1978), and this is presumably the case for D. polymorpha as well. Finally, the addition of CB at 2 min PI allowed sufficient time for the sperm binding to occur prior to inhibition; however, sperm entry was still suppressed, suggesting MF involvement at a stage later than sperm binding.

The involvement of microvilli in sperm entry varies greatly between species, but a role has been suggested in hamsters (Yanagimachi and Noda, 1970), urchins (Tilney and Jaffe, 1980), annelids (Anderson and Eckberg, 1983), and bivalves (Longo, 1987). Furthermore, Wilson and Snell (1998) propose that microvillus-like structures may be essential for most types of cell-cell fusion events. Hylander and Summers (1977) proposed a generalized model of fertilization in Mollusca. According to their model, sperm binding occurs between an inner acrosomal region of the sperm and microvilli tufts. Similar microvillar tufts were observed in D. polymorpha; however, no obvious association between these tufts and the inner acrosomal membrane was observed. Furthermore, microvilli appeared to remain perpendicular to the egg surface and did not reorient toward bound sperm as reported for Spisula (Longo and Anderson, 1970; Hylander and Summers, 1977). Misamore et al. (1996) reported extracellular fibers extending toward attached sperm; however, these fibers are substantially smaller than the egg microvilli.

Microfilament presence in insemination cones has been well documented. In urchins, insemination cones may form from the fusion of microvilli (Schatten and Schatten, 1980), and MFs in the cones are polymerized into discrete bundles (Tilney and Jaffe, 1980) from monomeric actin in the egg cortex (Spudich and Amos, 1979). Molluscan insemination cones are markedly smaller than urchin cones and MFs are not consistently reported in the cones (Longo, 1983). As in Mytilus and Spisula (Longo and Anderson, 1970; Longo, 1983), in D. polymorpha MFs in fertilization cones run the length of the ooplasmic projection, but not in discrete bundles as observed in urchins (Fig. 5). While the exact mechanisms involved remain unclear, insemination cones are implicated in sperm entry (Longo, 1980). The inhibition of sperm entry by CB also suppresses the formation of insemination cones (Longo, 1980; Schatten and Schatten, 1980, 1981).

Cytochalasin B was shown to have a reversible, dose-dependent effect on fertilization: partial inhibition occurred at 6.2 [micro]M, CB and complete inhibition at 12.4 [micro]M Byrd and Perry (1980) reported similar dose-dependent findings in two urchin species, Strongylocentrotus purpuratus and Lytechinus pictus. Sperm entry was decreased at 2.5 [micro]g/ml (5 [micro]M CB in the former species and at 5 [micro]g/ml (10 [micro]M) in the latter; inhibition was complete at 5 [micro]g/m1 (10 [micro]M) and 10 [micro]g/ml (20 [micro]M) respectively. Gould-Somero et al. (1977) found that slightly lower levels of CB partially (1 [micro]M) or completely (2 [micro]M) blocked sperm entry. That the dose-dependent responses are similar is somewhat remarkable considering the variability in the gametes, extracellular coats, and fertilization mechanisms between these diverse species.

Sperm entry was effectively blocked when CB addition preceded or was concomitant with insemination (Fig. 17). Addition of CB at 2 min PI resulted in fewer eggs exhibiting sperm penetration, but at 4 min PI sperm entry was not significantly affected. These findings suggest that CB was able to rapidly (within 1-2 min) block sperm entry, and that the period of susceptibility to CB inhibition was completed by 4 min PI. The first 4 min PI during D. polymorpha fertilization corresponds to the 1 [micro]m/min gradual-incorporation phase into the egg cortex. After 4 min, PI, CB was unable to inhibit sperm-axoneme incorporation, mitochondria detachment, or male-chromatin decondensation and pronuclear formation. CB impact on fertilization is limited to the first 6 mm PI in several urchin species (Longo, 1980; Byrd and Perry, 1980; Schatten and Schatten, 1980), and echiuroid worms (Gould-Somero et al., 1977). The restriction of MF involvement in sperm incorporation to the first 6 mm following insemination applies across a wide taxonomic range.

Unlike MF polymerization, MT polymerization was not required for the initial incorporation of the sperm nucleus into the egg cortex in D. polymorpha. Initial entry of sperm into eggs in marine invertebrates (Schatten and Schatten, 1981; Schatten et al., 1982, 1989) and algae (Swope and Kropf, 1993) also does not require MTs. Sperm were able to enter the egg cortex in MT inhibitors at a rate (1 [micro]m/min) similar to that observed in normal fertilizations. MT inhibitors were able to penetrate the egg and were effective at disrupting the meiotic spindle, thereby preventing polar body formation. Furthermore, no MT array was observed to be associated with entering sperm nuclei when [alpha]-tubulin monoclonal antibody was used to label fertilized eggs.

Sperm nuclear translocation and flagellar incorporation

After passing through the fertilization cone and entering the egg cortex, D. polymorpha sperm rotated 180[degrees], positioning the basal end of the nucleus centrad (Fig. 11). The first fluorochrome-detectable MTs associated with entering sperm were the diminutive sperm asters adjacent to decondensing sperm chromatin (Fig. 19). Small sperm asters have been reported for other bivalve species (Longo and Anderson, 1969, 1970; Longo et al., 1993). In contrast, the sperm aster is significantly larger in most invertebrate and mammalian systems and is believed to be responsible for the migration of the male and female pronuclei during syngamy. For example, in sea urchins the sperm aster extends toward the female pronucleus and is thought to effect the migration of the two pronuclei (Zimmerman and Zimmerman, 1967; Longo and Anderson, 1968; Longo, 1976; Schatten, 1981; Bestor and Schatten, 1981; Sluder et al., 1985). The role of the sperm aster in D. polymorpha is not fully understood as it does not extend toward the female pronucleus.

A markedly larger MT array is associated with the female chromatin in D. polymorpha (Walker, 1996). A dense bundle of MTs is observed immediately subjacent to the polar bodies. Emanating from the MT bundle toward and surrounding the female pronucleus is a prominent cone-shaped array of MTs. This MT bundle is believed to anchor the female pronucleus and guide its centrad movement into the egg (Walker, 1996). The large female aster is also believed to play an important role in movement of the male pronucleus. An analogous structure may also be present in both Spisula and Mytilus (Longo, 1973a). Furthermore, as has been found in urchins (Zimmerman and Zimmerman, 1967), colcemid prevents pronuclear migration in D. polymorpha (Walker, 1996).

Following sperm rotation, most of the sperm axoneme was incorporated into the egg cytoplasm and the sperm head often was rapidly (1 [micro]m/s translocated through the egg cytoplasm (Misamore et al., 1996). Microfilament polymerization appeared to play little or no role in the second stage of sperm entry. Addition of CB after initial sperm entry (4 min PI trials) failed to prevent the rapid translocation of the sperm nucleus or the incorporation of the flagella. Furthermore, no obvious association of MFs and incorporated sperm was observed using epifluorescence or electron microscopy.

Conversely, MT polymerization played a prominent, yet somewhat unconventional, role in sperm nuclear translocation and flagellar incorporation. Following entry of the sperm head into the cortex, the bulk of the sperm axoneme was incorporated into the egg cytoplasm in D. polymorpha. During this incorporation, dramatic movements of the sperm head were observed, as well as a lateral migration of the sperm head along the egg cortex. These movements may be analogous to "jerking" movements exhibited by urchin sperm during axoneme incorporation (Schatten, 1981). Schatten (1981) suggests that the continued movement of the sperm tail may be involved in its movement through the fertilization cone and into the cytoplasm proper. In D. polymorpha, there is an obvious correlation between movements of the flagellum as it enters the egg cytoplasm and movements exhibited by the sperm head in the egg cortex. The exact mechanisms involved in flagellar incorporation are not known. Video microscopic observations of both urchins ( Schatten, 1981) and zebra mussels (this study) suggest that flagellar movement may be involved. Furthermore, flagellar incorporation in D. polymorpha was blocked by MT inhibitors. Microtubule polymerization appears to be essential for flagellar incorporation in D. polymorpha. This finding is in contrast to the results of studies with urchins, in which nocodazole did not inhibit axoneme incorporation (Schatten and Schatten, 1981; Fechter et al., 1996). Furthermore, the exaggerated movement of the sperm head immediately subjacent to the insemination cone during the inhibited axoneme incorporation further supports the concept that flagellar movement takes part in axoneme incorporation.

In contrast, Epel et al. (1977) reported that deflagellated sperm heads were able to bind and enter sea urchin eggs. Attempts to duplicate those experiments with D. polymorpha in the present investigations were unsuccessful. Finally, Schatten and Schatten (1981) reported that MT inhibitors increased the lateral displacement of the sperm head along the cortex and that the formation of the sperm aster may signal the end of this lateral movement (Schatten, 1982). In D. polymorpha, lateral displacement of the sperm head was restrained by the attached yet unincorporated flagellum. Once the flagellum was severed, however, lateral displacement and decondensation were observed.

Following axonemal incorporation and quiescence, the sperm mitochondria separated from the nucleus as it began to decondense. As in other invertebrate species (Schatten and Schatten, 1981), sperm decondensation in D. polymorpha was not affected by either MF or MT inhibitors. In zebra mussels, nuclear decondensation and mitochondrial separation are apparently unaffected by MT inhibitors, suggesting that flagellar detachment is also unaffected. In sea urchins, microtubules appear to be essential for detachment of the sperm tail, its migration toward the female pronucleus, and its disassembly (Schatten and Schatten, 1981; Fechter etal., 1996). Similarly, activation of D. polymorpha eggs by sperm was unaffected by the presence of either MF or MT inhibitors. Several studies have shown that early egg activation occurs during fertilization even in the presence of CB (Byrd and Perry, 1980; Schatten and Schatten, 1980; Dale and DeSantis, 1981; Lynn, 1989). In these studies, initiation of the cortical granule release or an electrophysiological response were used as indicators of egg activation. Like those of most molluscs (Humphreys, 1967; Longo, 1983), the eggs of D. polymorpha do not release cortical granules immediately following egg activation. Since D. polymorpha eggs are inseminated at metaphase I arrest, the resumption of meiosis can serve as an indicator of egg activation (Longo, 1972, 1978a; Longo et al., 1993). In this study, D. polymorpha sperm readily bound to the egg surface in the presence of MF or MT inhibitors and apparently induced the resumption of meiosis, since eggs devoid of bound sperm remained in metaphase arrest.

Flow of cytoplasmic particles

During axoneme incorporation, a significant cytoplasmic movement was noted in the region of the axoneme. Flows of cytoplasmic particles were observed in numerous regions near the site of sperm entry and conspicuously originating at the basal region of the sperm head. The impetus or functional significance of this flow remains in question. Two possible mechanisms for generating these currents include beating by a functional axoneme displacing the particles or plus-end-directed transport along axonemal MTs by motor proteins associated with either the cytoplasmic particles or the sperm axoneme.

There appear to be few, if any, reported instances where flagella retain dynamic function once incorporated into the egg cytoplasm (Schatten, 1981; 1982). The last movement typically associated with flagellar bending occurs shortly after sperm binding, and movement of the sperm nucleus once inside the egg is typically associated with cytoskeletal elements, specifically the sperm aster (Schatten, 1982; Longo, 1987). Technical limitations make it difficult to isolate movements attributed to incorporated axonemes from egg-derived events; nevertheless, several pieces of evidence support the concept of an active axoneme inside the egg.

First, the sperm axoneme retains the ability to generate movement following demembranation during incorporation. Active movement by isolated, demembraned sperm axonemes has been demonstrated in other species (Bray, 1992). Second, video microscopy of mechanically-ruptured, fertilized D. polymorpha eggs revealed incorporated sperm vigorously moving within the collapsing egg membrane (Misamore, pers. obs.). Third, there is an apparent alteration of the flagellar beat pattern in incorporated sperm. During the rapid translocation of the sperm through the egg cytoplasm (Misamore et al., 1996), the sperm head trails the proximal portion of the axoneme. The proximal third of the axoneme becomes the leading portion of the moving sperm cell. Similar types of flagella-driven movement can be seen in other systems. For example, the single-celled flagellate Euglena moves via a singular flagellum that extends slightly more anterior than the cell body before bending posteriorly (Bray, 1992). Helical waves running the length of the flagellum propel the cell forward, resulting in a rotational movement to the cell body. Hamster sperm exhibit a pronounced change in beat pattern upon entry into the dense cumulus oophorous surrounding the egg. Penetrating sperm frequently progress with the proximal portion of the flagellum extending slightly forward, with the head ratcheting through the dense cumulus oophorous (Yacnagimachi, 1994). The change in flagellar beating seen in D. polymorpha may be attributable to the greater viscosity of the egg cytoplasm relative to the external milieu.

A second potential source of the observed cytoplasmic flow could result from active movement of particles down the exposed axoneme MTs by molecular motors. The plusend-directed flow of particles suggests the presence of a kinesin or kinesin-like motor. Initial attempts to label incorporated axonemes with kinesin antibodies have been unsuccessful (data not shown); however, support for this hypothesis is as follows. Microtubule motors are relatively abundant in the egg cytoplasm. Gilksman and Salmon (1993) reported substantial MT gliding along surfaces coated with an ooplasm extract. Scholey et al. (1985) have isolated a kinesin from urchin eggs. Porter et al. (1987) showed that this egg kinesin exhibited plus-end movement along isolated axonemes, and kinesin-coated beads translocate along centrosome MTs. Furthermore, Kozminski et al. (1995) found that a flagellar kinesin, FLA10, facilitated movement of intraflagellar particles, or rafts, along the length of the axoneme of Chlamydomonas flagella. Clearly, more detailed testing of this hypothesis is needed.

Finally, no flow of cytoplasmic particles was observed in D. polymorpha zygotes when axoneme incorporation was blocked with MT inhibitors. The observed flow is either directly, or at least indirectly, related to the incorporated flagellum. The significance of this particle flow down the axoneme remains an intriguing question.

In summary, initial sperm entry into the egg cortex is a gradual, MF-dependent process, while subsequent flagellar incorporation is MT dependent. Dynamic movement of the incorporated sperm head and flagella is observed inside the egg cytoplasm, and a flow of cytoplasmic particles associated with the incorporated axoneme was observed. D. polymorpha serves as a good model for studying fertilization and exhibits many similarities to other fertilization models. However, as demonstrated in this study, its remarkably transparent cytoplasm allows extremely detailed observations of the intracellular interactions between eggs and their incorporated sperm, revealing previously undescribed phenomena that warrant further study in both this and other systems.

Acknowledgments

We gratefully acknowledge the following individuals for their invaluable contributions to this research: Susan J. Nichols, J. Rachel Walker, Katie Kreimborg, Steven Smith, Thomas Dietz, and the staff of the M.D. Socolofsky Microscopy Center. This research was funded in part by a grant from Sea Grant of Louisiana NOAA grant #46RG00960, project R/ZMM-2.

(*.) Present address: Dept. Cell Biology, Univ. of Texas-Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9039. E-mail: michael.misamore@email.swmed.edu

(+.) To whom correspondence should be addressed. E-mail: zolynn@lsu.edu

Abbreviations: CB, cytochalasin B; MF, microfilament; MT, microtubule; PI, post insemination; PW, pondwater.

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