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Identification and role of carbohydrates on the surface of gametes in the zebra mussel, Dreissena polymorpha.


Dreissena polymorpha, the invasive zebra mussel, is a freshwater bivalve native to the Aral Sea in central Asia. Since its accidental introduction into the Great Lakes region of the United States in the mid-1980s (Hebert and Muncaster, 1989), it has spread throughout the eastern half of the United States and as far south as Louisiana. Recently, zebra mussels and the related quagga mussels (Dreissena bugensis) have spread to the western United States (Stokstad, 2007; Benson, 2008). It has been predicted that the zebra mussel will eventually colonize the majority of permanent, inland freshwaters of the United States and southern Canada (Strayer, 1991; McMahon, 1992; Claudi and McMahon, 1993). Unlike most other freshwater bivalves in North America in which fertilization and brooding of larvae occur in the female's mantle cavity (Glaubrecht et al., 2006), zebra mussels broadcast spawn their gametes. Eggs and sperm are released directly into the external media where fertilization and larval development occur. This reproductive strategy is similar to that of many marine bivalves that also exhibit external fertilization (Longwell and Stiles, 1968; Longo and Anderson, 1969a, b; Stiles and Longwell, 1973; Ackerman et al., 1994).

Many marine bivalves that broadcast spawn are used as fertilization models. Studies have included Crassostrea spp. (Kyozuka and Osanai, 1985), Spisula solidissima (Longo, 1973, 1976; Hylander and Summers, 1977), and Mytilus edulis (Longo and Anderson, 1969a), As with most broadcast spawners, species-specific binding of gametes is a critical aspect of external fertilization (Gould and Stephano, 2003). Prominent among these mechanisms is the growing importance of carbohydrates in sperm-egg adhesion (Mengerink and Vacquier, 2001). Species and gamete specificity could be accomplished through the role of surface carbohydrates and proteins.

Carbohydrates are important in sperm-egg binding in many vertebrate models. In Xenopus laevis, glycosylated proteins on the egg are required for sperm binding to take place (Olson and Chandler, 1999). In mice, three main glycoproteins (ZP1, ZP2, and ZP3) important for fertilization have been identified. ZP3 is believed to mediate the initial binding of sperm and the acrosome reaction, while ZP2 is involved in adhesion to the sperm's inner acrosomal membrane (Dell et al., 1999). Unreacted mouse sperm bind to specific O-linked oligosaccharides on ZP3 and are believed to play a role in sperm-egg binding (Florman and Wassarman, 1985; Wassarman et al., 2004).

Many invertebrates also utilize carbohydrates during gamete activation and binding. For example, a fucose sulfate polymer on the egg jelly of sea urchins binds to a receptor on the sperm surface, inducing the acrosome reaction (Vacquier and Moy, 1997). In the freshwater bivalve Unio elongatulus, a glycoprotein, Gp273, has been isolated as the ligand molecule for sperm-egg interaction during fertilization (Di Patrizi et al., 2001). Furthermore, a fertilization molecule present on oyster sperm was shown to be a glycoprotein (Brandriff et al., 1978).

Within the last 15 years, several studies have focused on various aspects of zebra mussel fertilization and early development (Ram and Nichols, 1993; Fong et al., 1995; Luetjens and Dorresteijn, 1998; Misamore et al., 1996, 2006). The ultrastructural aspects of fertilization in zebra mussels have been well documented (Misamore et al., 1996, 2006). Sperm initially bind to the egg surface by their acrosomal region. The zebra mussel acrosome consists of a prominent acrosomal filament and an electron-dense basal region. The egg surface consists of numerous microvilli surrounded by a vitelline envelope. Upon contact with the egg, the sperm undergoes the acrosome reaction, exposing the acrosomal filament and the inner acrosomal membrane. Intimate contact is made between the acrosomal filament and inner acrosomal membrane of the sperm and the egg vitelline coat and microvilli. Although the inner acrosomal membrane is the site of sperm-egg binding, no studies have focused on the specific mechanisms of gamete binding. In the present study, we looked at the distribution and possible role of carbohydrates on zebra mussel gametes prior to and during fertilization. We also addressed the timing and potential mechanisms for detaching nonfertilizing sperm as part of the zebra mussel block to polyspermy.

Materials and Methods

Gamete collecting and handling

Zebra mussels, Dreissena polymorpha (Pallas, 1771), were collected from Ann Arbor, Michigan. Animals were kept in aquaria filled with artificial pond water (PW) (Dietz et al., 1994) at 9 [degrees]C and unfed until needed. Twenty-four hours prior to use, the animals were individually isolated and allowed to acclimate to room temperature overnight (~21[degrees]C). All procedures were carried out at room temperature (20-22 [degrees]C).

Collection and handling of gametes followed Misamore and Lynn (2000). Briefly, spawning was induced by submerging individual animals in 0.2 mmoll [1.sup.-1] 5-hydroxytryptamine (serotonin) for 12 min, washing twice with deionized water, and resubmerging them in PW (Ram and Nichols, 1993). Males typically began spawning 5-10 min after serotonin treatment. Females generally began to spawn 30-60 min after serotonin treatment. During fertilization experiments, eggs from a first spawning were fertilized with sperm from a second spawning to ensure a high number of freshly spawned sperm. In general, spawned eggs were used within 2 h, and sperm were used within 30 min. Final concentrations of eggs were approximately 30,000-40,000 eggs per female, and sperm concentrations were roughly [10.sup.3]-[10.sup.6] sperm per male. For fertilization time series, samples were fixed with 3.2% paraformaldehyde in mussel buffer (Misamore and Lynn, 2000).

Lectin labeling and DNA staining

Seven lectins were used in this study: Wheat germ agglutinin (WGA), concanavalin A (Con A), Lens culinaris (LcH), Glycine max (SBA), Arachis hypogaea (PNA), Lotus tetragonolobus (LTA). and Griffotnia simplicifolia (GSII) (Table 1). These were selected in part on the basis of the lectin or its hapten sugar being present on gametes and possibly playing a role in fertilization in other systems (Oikawa et al., 1973; Longo, 1981; Mozingo and Hedrick, 1999; Velilla et al., 2004). Lectins were prepared from stocks of 1mg/ml in deionized water. For labeling gametes, live eggs or sperm were mixed with 30 [micro]g/ml FITC (fluorescein isothiocyanate)-conjugated lectin (EY Labs, San Mateo, CA) for a minimum of 10 min. To determine lectin localization during fertilization, eggs and sperm were mixed and labeled with 30 [micro]g/ml of FITC-conjugated lectin 1 min postinsemination (PI). All lectins were labeled using live gametes to reduce potential artifacts from labeling of fixed samples. To further emphasize specificity, lectins were pretreated with their hapten sugars for 10 min prior to treating the gametes. The following hapten sugars were used to block each lectin: N-acetyl-D-glucosamine for WGA and GSII; methyl-[alpha]-D-mannopyranoside for Con A and LcH; L-fueose for LTA; N-acetyl-D-galactosamine for SBA, lactose-PNA, (Sigma Chemicals, St. Louis, MO).

To visualize nucleoproteins, samples were labeled with either bisbenzimide (Hoechst 33342, Sigma Chemicals, St. Louis, MO) or DAPI (Invitrogen/Molecular Probes, Carlsbad, CA). Samples were stained with l[micro],g/ml final concentration of Hoechst 33342 or DAPI and allowed to incubate for a minimum of 5 min (Misamore et al., 1996).
Table 1

List of lectins and associated carbohydrates

   Lectin name     Lectin        Target
                   abbreviation  carbohydrate

Triticum vulgaris  WGA           [beta]-N-Acelylglucosamine(1.4)
(Wheat Germ                      [much greater than]

Canavalia          ConA          [alpha]-Mcthyl-mannopyraniside
ensiformis                       [greater than] [alpha]-D-Mannose
(Concanavalin A)                 [greater than][alpha]-D-Glucose
                                 [greater than]

Lens culinaris     LcH           D-Mannose and D-glueose

Glycine max        SBA           [beta]-N-Acetyl-D-galactosamine,

Arachis hypogaea   PNA           [beta]-Galactose

Lotus              LTA           L-Fucose

Griffonia          GSII          N-Acetyl-D-glucoeosamine
simplicifolia                    ([alpha],[beta])

   Lectin name        Carbohydrate

Triticum vulgaris  GlcNAc[beta](1,4)q
(Wheat Germ

Canavalia          Neu5Ac
(Concanavalin A)

Lens culinaris

Glycine max        GalNAc
Arachis hypogaea


Griffonia          GlcNAc

Affinities are based on manufacturer data for use with their
specifically isolated lectins (EY Labs, San Mateo, CA).

To label with gold-conjugated GSII lectin (EY Labs, San Mateo, CA), sperm were incubated with 0.5 [micro]g/ml lectin for 10 min, washed with 20 mmol [1.sup.1] sodium cacodylate buffer, fixed in 2.5% glutaraldehyde/1% paraformaldehyde in 20 mmol [1.sup.1] sodium cacodylate buffer for 30 min, then washed with deionized water (Misamore et al., 1996). Samples (1 [micro]l) were placed on 400-mesh copper grids coated with carbon and allowed to air dry. Samples were viewed using a JEOL 100CX transmission electron microscope.

Quantification of sperm binding and incorporation

To evaluate sperm binding, the numbers of sperm bound within an equatorial focal plane were determined in samples fixed at various time points PI. This technique ensured that only sperm bound to the egg were counted, and not sperm in proximity (in more polar fields of focus) that were not bound to the egg surface. To quantify fertilization, the number of incorporated sperm nuclei inside each egg was counted in samples fixed at various time points PI. Two criteria were used to distinguish incorporated sperm from bound sperm (Misamore and Lynn, 2000). First, the position of the sperm relative to the egg surface (on the surface versus inside the egg cytoplasm) was used as an initial determining factor. Second, for sperm in more polar planes of focus where distinguishing the exact position of the sperm relative to the egg surface is more difficult, the state of the sperm chromatin was used. The chromatin of bound sperm remains tightly packed, with distinct lateral margins. The chromatin of incorporated sperm begins decondensing shortly after sperm entry, and the lateral margins become less defined (Misamore et al., 1996, 2006).

Both for sperm-binding and sperm-incorporation experiments, 40 eggs from each of three independent fertilizations (different males and females) were scored at 5, 10, 15, 20, and 30 min PI. The use of parametric statistical tests, such as analysis of variance with appropriate square root transformations of the data (Freeman and Tukey, 1950), or nonparametric tests based on ranks, such as Kruskal-Wallis (Zar, 1999), were compromised by the high frequencies of counts of 0 and 1 in the data. Instead, the SAS MULTTEST procedure (Richter and Higgins, 2006) was used to test for differences in the numbers of bound sperm and incorporated sperm as a function of time. This procedure incorporates random samplings without replacement of the count data into 20,000 alternative permutations of the observed counts among the five time periods, and uses the distributions of the counts in these alternative permutations to assess the probability of obtaining the observed distribution of counts across time. The MULTTEST procedure was configured to test for differences between successive time intervals.

Induction of the acrosome reaction

To induce the acrosome reaction, thereby exposing the inner acrosomal components for lectin labeling, sperm were incubated with ionomycin calcium (Sigma Laboratories) to elevate intracellular calcium levels. Ionomycin has been used as a tool for inducing the acrosome reaction in the sperm of other animals, including human sperm (Thomas and Meizel, 1988). An initial stock of 1 mg/ml ionomycin in DMSO was diluted to a final concentration of 4 [micro]g/ml. To determine the effectiveness of ionomycin in inducing the acrosome reaction in zebra mussels, sperm from three independent spawns were treated with either 4 [micro]g/ml ionomycin or 0.4% DMSO. To aid in visualizing intact acrosomes, sperm were labeled with FITC-conjugated WGA as described above. Fifty sperm from each treatment were scored for the presence or absence of intact acrosomes. In the ionomycin-treated sample, 78% of the sperm were acrosome-reacted, whereas only 9% of the DMSO control sperm had undergone spontaneous acrosome reactions.

Sugar inhibition of fertilization

The ability of several sugars to block sperm-egg binding was investigated to identify possible carbohydrate moities involved in gamete binding. Eggs were pretreated with 5 mmol [1.sup.1] solutions of GlcNAc, fucose, galactose, or PW for 10 min prior to insemination. Due to the extremely low osmolarity of PW (Dietz et al., 1994), sugar concentrations were as high as possible without osmotically compromising the eggs or sperm. Inseminated eggs were fixed 3 min PI, and sperm binding was assessed. Numbers of equatorial bound sperm from three independent trials of 50 eggs each were scored. A Kruskal-Wallis nonparametric ANOVA was performed, followed by Dunn's multiple comparisons where significance differences were identified.

Effects of alteration of egg surface on sperm binding and detachment

To determine how modifying egg surface proteins might affect sperm binding, eggs were pretreated with trypsin prior to fertilization. Eggs were treated with 0.01% trypsin, 0.01% bovine serum albumin (BSA), or PW for 1 min, washed once with PW, and inseminated with a slightly higher concentrations of sperm. Samples were fixed at 5 min, and numbers of bound sperm were determined as described above for three independent trials of 50 eggs each. A Kruskal-Wallis nonparametric ANOVA was performed, followed by Dunn's multiple comparisons where significance differences were identified.

Detachment of nonfertilizing sperm

To understand the timing and possible mechanism of the detachment of nonfertilizing sperm during zebra mussel fertilization, numerous eggs were observed in real-time and with video microscopy for several independent fertilizations. Additionally, to determine the possible role of trypsin-like enzymes on sperm detachment, eggs were treated with (soybean) trypsin inhibitor (SBTI). Eggs were inseminated and sperm binding was allowed to progress. One minute after insemination, SBTI was added to a final concentration of 0.01%. Both PW and BSA (0.01% final concentration) controls were performed. Samples were fixed at 5 and 20 min PI. To compare sperm binding and detachment, the proportions of the 50 eggs with bound sperm were determined after 5 and 20 min for each of the three trials, hereafter termed A, B, and C. The proportions with bound sperm for the three treatments were compared using G-statistic analyses of a 2 X 3 contingency table (Zar, 1999) at both 5 and 20 min. Where significant differences among proportions were observed at 20 min, G-tests were also performed on two subsequent 2X2 contingency tables that compare (1) the proportion of eggs with bound sperm between PW and BSA and (2) between SBTI and the proportions for the PW and BSA data combined. For these 2 X 2 analyses, Yates correction for continuity was applied to the data (Zar, 1999), and the significance level was adjusted to 0.025 to account for two non-independent tests at P = 0.05.


Light and fluorescent microscopy were performed on a Zeiss Axiovert 200 and a Nikon Optiphot equipped with phase-contrast and epifluorescence. Digital micrographs were captured using a Zeiss AxioCam MRm and Axiovision software. Confocal microscopy was performed on a Leica SP1 confocal microscope. Adobe Photoshop was used for final image processing. Scanning and transmission electron microscopy were performed using a JOEL model JSM-6100 scanning and JEOL 100-CX transmission electron microscopes respectively. Sample preparation for electron microscopy followed Misamore et al, 1996.


Lectin labeling of gametes

For both eggs and sperm, a group of commonly used FITC-conjugaled lectins was tested for affinity to either male or female gametes (Table 2). WGA uniformly labeled the egg (Fig. 1 A). Similarly, Con A (Fig. 1B) and LeH (Fig. 1C) also uniformly labeled the egg. Conversely, LTA (Fig. 1D), SBA (Fig. 1E), PNA (Fig. 1F), and GSII (Fig. 1G) showed no detectable labeling of the egg even when labeling times were extended to 1 h. The labeling of the eggs was associated with either the egg plasma membrane or the vitelline coat. There was no lectin labeling of the egg jelly layer. Labeling of all lectins was blocked with 100 mmol [1.sup.-1] solutions of their hapten sugar (Fig. 1A"-C").
Table 2
Summary of lectin labeling of gametes

Lectin(*)       Eggs             Sperm

WGA        Entire surface  Entire surface
Con A      Entire surface  Activated acrosome
LcH        Entire surface  Activated acrosome
LTA        No label        Activated acrosome
SBA        No label        Activated acrosome
PNA        No label        Activated acrosome
GSII       No label        Activated acrosome

* Con A, Canavalia ensiformis; GSII, Griffonia simplicifolia;
LcH, Lens culinaris; SBA, Glycine max; LTA, Lotus tetragonolobus;
PNA, Arachis hypogaea; WGA, Triticum vulgare (wheat germ

WGA labeled the entire surface of the sperm (Fig. 2A). There was uniform distribution of the lectin along the length of the flagella, midpiece, and sperm head. Unlike WGA, the other lectins (Con A. LcH, LTA, SBA. PNA, and GSII) failed to label unreacted sperm (Fig. 2C, E, G, I, and K respectively).

While six of the seven lectins failed to label the plasma membrane of sperm with intact acrosomes, all lectins intensely labeled the inner acrosomal membrane once it became exposed and accessible to the lectin. In unreacted sperm, WGA labeled the outer surface of the intact acrosome, but no labeling was observed on the inner acrosomal region (Fig. 2A). In sperm induced to undergo the acrosome reaction by ionomycin, WGA intensely labeled the inner acrosomal region (Fig. 2B). GSH was unable to label unreacted sperm (Fig. 2K) but intensely labeled the inner acrosomal region and acrosomal filament in acrosome-reacted sperm (Fig. 2L). This labeling was specific to both the acrosomal filament and inner acrosomal membrane (Fig. 3). Similar results of inner acrosomal labeling in reacted sperm were observed with Con A (Fig. 2D). LcH (Fig. 2F), LTA (Fig. 2H), SBA (Fig. 2J). and PNA (Fig. 2N).

Lectin binding to acrosome-reacted sperm was reduced by pretreating the sperm in the lectin's hapten sugar but was not completely eliminated (Fig. 4). In WGA-treated sperm, the non-acrosomal, plasma membrane labeling was completely blocked by 100 mmol [1.sup.-1] GlcNAc (Fig. 4A. B). However, labeling of the inner acrosomal membrane was reduced but not completely eliminated by hapten sugar blocking even when sugar concentrations were doubled (200 mmol [1.sup.-1]). lectin concentrations reduced from 30 to 0.4 [micro]g/m] lectin (Fig. 4C), and blocking time extended to 1h. Likewise, inner acrosome-specific labeling of Con A was reduced but not completely eliminated with the reduced lectin and elevated sugar concentrations (Fig. 4D). Similar reduction but not complete inhibition of acrosomal labeling was observed for LcH, LTA, SBA,PNA, and GSH with their hapten sugars (data not shown).

Distribution of GSH labeling for fertilizing sperm

GSH labeling was further evaluated due in part to its specificity to the inner acrosomal membrane of sperm and its failure to label eggs. To determine the fate of the GSH-labeled inner acrosomal region of the sperm during fertilization, we mixed eggs and sperm together and added FITC-conjugated GSH lectin 1 min postinsemination (PI). Immediately after sperm binding to the egg surface, the GSH labeling was detected between the sperm head and the egg surface (Fig. 5A, A"). This labeling was localized to a distinct region at the tip of the sperm where it contacts the egg surface (Fig. 3, Fig. 5A, A"). Under scanning electron microscopy, this region displayed a ring-like structure associated with the sperm acrosome bound to the egg surface (Fig. 5H).

After incorporation into the egg cytoplasm, the GSII labeling remained on the surface of the egg (Fig. 5B) at 5 min PI. With the sperm nucleus, mitochondria, and flagellar axoneme inside the egg cytoplasm, the GSH labeling remained as a ring-like structure on the surface of the egg (Fig. 5C, D). Morphologically similar ring-like structures (basal rings) have been reported in several marine bivalves (Dan and Wada, 1955; Niijima and Dan, 1965; Hylander and Summers, 1977; Brandriff et al., 1978; Franzen, 1983; Hodgson and Bernard, 1986); thus we termed this characteristic labeling a "GSH basal ring." This GSH basal ring remained on the egg surface throughout the early fertilization events (Fig. 5C, D); it eventually detached from the egg surface 15-20 min PI (Fig. 5F) and was typically lost by 30-40 min. At this point in the fertilization process, the female and male pronuclei are moving toward each other and their fusion is imminent (Fig. 6A). When even higher sperm concentrations were used to increase polyspermic eggs, all incorporated sperm left distinct GSII basal rings on the surface of the egg.

Inhibition of sperm binding

When eggs were pretreated with various sugars, GlcNAc significantly inhibited sperm binding relative to the pondwater control, as indicated by a Kruskal-Wallis nonparametric ANOVA and Dunn's multiple comparisons (Fig. 7A; P [less than] 0.05). Two other sugars, fucose and galactose, showed no significant effect on sperm binding relative to the control (P [less than] 0.05). The decrease in sperm binding was specific to GlcNAc and not attributed to osmotic changes due to sugars added to the media. Disruption of surface proteins by pretreatment of eggs with trypsin had a significant effect on sperm binding relative to controls (P [less than] 0.05; Fig. 7B).


Detachment of nonfertilizing sperm

For the many sperm that bind to the egg but do not fertilize it, the sperm eventually detach from the egg surface (Fig. 6). From visual observations and video microscopy, we determined that nonfertilizing sperm detached from the egg surface at about 10-15 min PI. No obvious elevation of egg surface coats was observed. The timing of nonfertilizing sperm detachment roughly corresponds to the release of the first polar body and the initial decondensation of fertilizing sperm.

We also observed that when nonfertilizing sperm detached from the egg surface, they took their associated GSII basal ring (Fig. 6) with them. Thus, when labeled sperm are released off the egg surface, the lectin GSII is released with the detaching sperm and does not remain on the egg surface as it does with fertilizing sperm. Similarly, GSII basal rings left on the egg surface also detached at about 20 min PI (Fig. 5F).

To quantify the relationship between sperm binding, incorporation, and detachment of nonfertilizing sperm, fertilized eggs were fixed at various times PI (Fig. 8). The number of bound sperm decreased significantly over time between 5 and 10 min PI (P [less than] 0.001) and between 10 and 15 min PI (P [less than] 0.05). Sperm entry into the eggs showed significant increases in number of incorporated sperm between 5 and 10 min PI (P [less than] 0.001) and between 10 and 15 min PI (P < 0.01). Number of incorporated sperm did not significantly increase after 15 min PI (Fig. 8B) (P < 0.3).


To understand the possible role of trypsin-like proteases in sperm detachment, the effect of trypsin inhibitors on sperm detachment, the effect of trypsin inhibitors on sperm detachment was investigated. There were no significant differences in proportions of eggs having bound sperm at 5 min among the PW, BSA, and STBI treatments for trials A (G = 3.44; df = 2; P > (0.05) or C (G = 0.00; df = 2; P > 0.05) (Fig. 9). However, at 20 min PI there were significant differences for both trial A (G = 12.8; df = 2; P < 0.01) and trial C(G = 49.6; df = 2; P < 0.01) between the three treatments (Fig. 9). More eggs had bound sperm in the SBTI treatment than in the control treatments for both trial A (G = 12.2; df = 1; P < 0.005) and trial C (G = 37.6; df = 1; P < 0.005). There was no significant difference in the proportions with bound sperm between PW and BSA controls for trial A (G = 0.071; df=1; p.> 0.0125), but there was a greater proportion of bound sperm for the PW treatment in trial C (G = 8.48; df = 1; P < 0.005).


In trial B there were significant differences in the proportions of eggs with bound sperm at 5 min (G = 9.99; df = 2; P [less than] 0.01), with the SBTI treatment having the smallest proportion. However, at 20 min PI the pattern was reversed, with a significantly greater proportion of eggs with bound sperm in the STBI treatment than for the combined data from the PW and the BSA treatments (G = 13.3; df = 1; P [less than] 0.005) and no significant difference between the PW and the BSA treatments (G = 0.065; df = 1; P [greater than] 0.025). Thus in all three treatments, there were significantly greater proportions of eggs with bound sperm in the SBTI treatment relative to both controls.


Carbohydrates have been shown to be an integral part of many molecules that mediate fertilization (Dell et al., 1999; Mengerink and Vacquier, 2001). Considerable effort over the past decades has focused on specific proteins involved in sperm-egg binding, such as bindin in sea urchins (Vacquier and Moy, 1977), fertilin (Cho et al., 1998) and izumo (Inoue et al., 2005) in mammals, Spe9 in Caenorhabditis elegans (Singson et al., 1998), and Fusl and Hap2 in Chlamydomonas (Misamore et al., 2003; Liu et al., 2008). More recently, efforts have focused on the role of carbohydrates in fertilization (Dell et al., 1999); Di Patrizi et al, 2001; Mengerink and Vacquier, 2001; Biermann et al., 2004; Tanghe et al., 2004; Wassarman et al., 2004). In mammals, modification of the carbohydrates located on ZP proteins has been shown to greatly alter sperm binding (Florman et al., 1984). Moreover, several reproductive proteins have been shown to contain lectin-like domains that proteins have been shown to contain lectin-like domains that specifically recognize carbohydrates, including lysin in Mytilus (Springer and Crespi, 2007), SuREJ in sea urchins (Mah et al., 2005), and bindin in oysters (Moy et al., 2008).

In the freshwater bivalve Unio elongatulus, several detailed studies have characterized specific carbohydrates associated with the surface of the eggs required for fertilization (Focarelli and Rosati, 1995; Focarelli et al., 1995, 1988, 2003; Di Patrizi et al., 2001). Two main glycoproteins have been identified in the vitelline coat (Focarelli et al., 2001). One, gp273, is the target of sperm binding: O-linked, but not N-linked, oligosaccharides derived from gp273 were shown to specifically inhibit sperm-egg binding, with fucose playing an essential role (Focarelli and Rosati, 1995).

In the present study we have begun to characterize the carbohydrates associated with the gametes of zebra mussels. We found several carbohydrate moieties on both eggs and sperm. Their distribution across the egg was highly uniform. In sperm, several carbohydrates were localized specifically in the inner acrosomal region. Furthermore, one particular carbohydrate (GlcNAc) was shown to localize only to the inner acrosomal region in the sperm, and pretreating eggs with this carbohydrate significantly reduced sperm binding.

Carbohydrates on zebra mussel eggs

Zebra mussel eggs possess a relatively small, transparent outer jelly layer (Misamore et al., 1996). This layer is not overtly distinct, but it appears to play a role in sperm chemotaxis since sperm clearly aggregate around and within it (pers. obs.). None of the lectins utilized in this study labeled the zebra mussel jelly layer, suggesting that none of the carbohydrates associated with these lectins are found in the egg jelly. In contrast, the lectin from Arachis hypogea (PNA) labeled the jelly layer of the related quagga mussel Dreissena bugensis (pers. obs.). Similarly, WGA bound to the outermost layer of the egg jelly of Xenopus laevis (Mozingo and Hedrick, 1999), and Con A, WGA, and GSII labeled various layers of newt egg jelly (Okimura et al., 2001).

A more distinct vitelline envelope immediately surrounds the egg plasma membrane (Misamore et al, 1996). Embedded in the vitelline layer are numerous microvilli that evenly surround the egg surface. Three of the lectins uniformly labeled zebra mussel eggs. This suggests that the sugars associated with WGA, Con A, and LcH are located on either the egg vitelline coat or plasma membrane, Attempts to specifically localize lectin labeling to either the vitelline envelope or egg plasma membrane using gold-conjugated lectins and electron microscopy have been unsuccessful to date due in part to the fragile nature of the vitelline envelope. However, we believe the vitelline envelope is the most likely location for lectin labeling given its significance in sperm-egg binding (Misamore et al., 1996), the uniform, non-punctate labeling under confocal microscopy, and the failure of gold-labeled lectin to bind to envelope-denuded egg plasma membranes (data not shown).

With all the lectins, the distribution appeared uniform across the egg. This supports the theory that zebra mussel eggs exhibit little or no polarity across the egg surface prior to fertilization since sperm binding can occur uniformly across the egg surface (Misamore et al., 1996). This contrasts with the highly polar nature of Unio eggs, which exhibit a distinct polarity using LTA and Con A (Focarelli et al., 1988). Additionally, zebra mussels also do not exhibit the prominent vitelline spikes found in the marine bivalve Mytilus (Focarelli et al., 1991).

Our visual observations show that treatment of eggs with trypsin prior to insemination substantially disrupts the egg coat and blocks nearly all sperm binding. This differs from Crassostrea, in which trypsin treatment of the eggs did not inhibit sperm binding (Togo and Morisawa, 1999).

Carbohydrates on zebra mussel sperm

WGA was the only lectin to label the outer acrosomal membrane. In sea urchins, WGA was shown to specifically bind to the sperm surface to block the acrosome reaction (Podell and Vacquier, 1984). However, Misamore et al., (2006) found that pretreatment of both eggs and sperm with 10 mg/m1 WGA did not impact fertilization.

The inner acrosomal membrane labeled strongly with all seven lectins (Fig. 2), suggesting the presence of the sugars associated with these lectins. Since Con A, LcH, and WGA labeled sugars on both the inner acrosomal membrane of sperm (Fig. 2) and the egg (Fig. 1), the specific role of these carbohydrates in either sperm binding to an egg receptor or egg carbohydrate binding to a sperm receptor is more difficult to confirm. Hapten sugars easily blocked egg (Fig. 1) and sperm surface (Fig. 4) labeling, but were less successful in blocking the specific inner acrosomal labeling. One possible explanation is that the carbohydrates on the inner acrosomal membrane are highly concentrated and have greater affinities for the lectins than for the blocking sugars or the gamete surface carbohydrates. This is supported by the intensity of the acrosomal labeling by all the lectins. The strong labeling of all lectins suggests that the zebra mussel inner acrosomal membrane is highly glycosylated.


GSII specifically localized to the sperm acrosome (Fig. 2). Moreover, immunogold labeling showed that GSII localizes specifically to the inner acrosomal membrane and the acrosomal filament (Figs. 2, 3). These two regions are the location of sperm-egg binding during fertilization (Misamore et al., 1996). This labeling suggests the presence of GlcNAc on the inner acrosomal membrane and acrosomal filament. Furthermore, eggs pretreated with GlcNAc prior to insemination showed reduced sperm binding. Conversely, fucose and galactose did not reduce sperm binding, but lectins specific for these carbohydrates (PNA and LTA) did label the inner acrosomal membrane.





Throughout fertilization, the GSII labeling retained a ring-like distribution (Figs. 5, 6). A consistent feature in most marine bivalve sperm is an electron-dense region at the base of the acrosomal vesicle. Termed the basal ring in Mytilus (Niijima and Dan, 1965), it was later isolated by Brandriff et al., (1978) in oyster and shown to agglutinate eggs. Zebra mussels exhibit a prominent, complex acrosome consisting of a central actin-filled acrosomal filament and a complex electron-dense basal region (Fig. 3C; Misamore et al., 1996). In activated or bound sperm, the inner acrosomal membrane forms a ring-shaped structure that likely corresponds to the GSII basal ring reported here and similar structures in marine bivalves (Brandriff et al., 1978). The specific localization of GlcNAc to the region of sperm binding plus the ability of GlcNAc to block binding suggests a possible role of GlcNAc in sperm-egg binding in zebra mussels. To date, most studies examining the role of carbohydrates in fertilization have focused on carbohydrates associated with the egg (Mengerink and Vacquier, 2001). Fertilization molecules on sperm often contain lectin-like domains recognizing a carbohydrate on the egg (Mengerink and Vacquier, 2001; Springer and Crespi, 2007; Moy et al., 2008). The labeling of the sperm acrosome but not the egg and the blocking of binding by the hapten sugar suggest that carbohydrates on the sperm may play an important role in sperm binding in zebra mussels. Identification of the specific molecule associated with the GSII labeling and demonstration of binding of that molecule to the egg surface are required to conclusively verify that a GlcNAc residue on the sperm acrosome is essential for zebra mussel fertilization.

While the sperm nucleus, mitochondria, and flagella enter into the egg (Misamore et al., 2006). the GSII basal ring remains on the egg surface (Fig. 5). suggesting that the inner acrosomal membrane remains on the egg surface. This is further supported by the observations of Misamore et al., (2006) showing, on the basis of WGA lectin labeling, that the rest of the sperm plasma membrane remained on the egg surface after sperm incorporation.

Sperm detachment and the block to polyspermy

The block to polyspermy in zebra mussels remains unclear. Previously we have shown that at high levels of insemination laboratory-spawned animals are susceptible to polyspermy (Misamore et al., 1996). The mechanisms for preventing polyspermy have been studied in numerous invertebrates (see review by Gould and Stephano, 2003). Evidence for a fast block to polyspermy has been suggested in several marine bivalve species including Spisula (Finkel and Wolf, 1980). Mytilus (Dufresne-Dube et al., 1983; Togo et al., 1995), and oysters (Alliegro and Wright, 1983; Togo and Morisawa, 1999). As first identified in sea urchins (Jaffe, 1976), a membrane depolarization based on external sodium is believed to generate a fast polyspermic block in Crassostrea gigas (Togo and Morisawa, 1999) and Mytilus edulis (Togo et al., 1995). Presence of an altered membrane potential block in zebra mussels remains unknown; however, the low external ionic concentrations required by zebra mussels (Dietz et al., 1994) adds an additional complicating element to this issue.

In Mytilus edulis, polyspermy is inhibited in part by suppression of the acrosome reaction in supernumerary sperm binding after the first fertilizing sperm (Togo et al., 1995). Conversely, supernumerary Crassostrea gigas sperm did undergo the acrosome reaction (Togo and Morisawa, 1999). The.GSII labeling of bound sperm showed that supernumerary zebra mussel sperm also undergo the acrosome reaction.

At about 10--15 min PI, nonfertilizing sperm detached from the outer surface of fertilized eggs. Zebra mussels exhibit no obvious mechanical block to polyspermy such as elevation of a fertilization envelope or obvious cortical granule exocytosis (Misamore et al., 1996). As demonstrated here, nonfertilizing sperm detach from the egg surface roughly at the time of first polar body release and the initial decondensation of fertilizing sperm (Misamore et al., 2006).

Live and epifluorescence observations indicate that the GSII basal ring is released from the egg surface with the detaching sperm (Fig. 6). During detachment of nonfertilizing sperm or rings left by incorporated sperm (Fig. 5F), the GSII ring appears to remain intact after detachment, with no portion remaining on the egg surface. This suggests that the events responsible for detaching bound sperm are not directly destroying the GlcNAc moieties recognized by the GSII lectin.

A trypsin-like enzyme appears to play a role in sperm detachment in zebra mussels. Treating eggs with trypsin prior to insemination disrupts the egg surface, preventing subsequent sperm binding. We routinely use 0.01% trypsin treatment to remove bound sperm during studies focusing on sperm incorporation. Furthermore, SBTI treatment inhibited sperm detachment from the egg surface (Fig. 9). Similar results have been shown in sea urchins, where a trypsin-like enzyme alters sperm-binding sites (Longo et al., 1974). Whether this trypsin-like enzyme is targeting a GlcNAc receptor on the egg surface or a completely different system requires further investigation.

When sperm are followed through time PI, several trends are observed (Fig. 8). As expected, the number of bound sperm per egg decreased significantly over time (Fig. 8A). Similarly, the number of incorporated sperm increased over time and then leveled off after 15 min PI (Fig. 8B), suggesting that no additional sperm incorporation can occur due to the detachment of nonfertilizing sperm. A similar complete block to polyspermy at 15 min PI was reported for oysters (Togo and Morisawa, 1999); however, Crassostrea are not believed to release proteases.

In summary, the distribution of several carbohydrates on the surfaces of zebra mussel gametes was described in detail. Several of the lectins labeled both eggs and sperm, while others were specific just to the inner acrosomal membrane and filament of sperm. A GSII basal ring similar in structure to basal rings in marine bivalves was observed in zebra mussels. Like oyster basal rings, this zebra mussel GSII basal ring appears to be glycosylated. The labeling of GlcNAc by GSII to the site of sperm binding and the inhibition of sperm binding by GlcNAc suggest a possible role in sperm-egg binding in zebra mussels. Detachment of sperm during the zebra mussel block to polyspermy does not appear to directly target GlcNAc, but it does require protease activity. Further work, including the adaptation of isolation techniques used in marine systems to the freshwater zebra mussel, is needed to fully identify the specific glycoprotein associated with the GSII basal ring and its precise role in sperm-egg binding.


The authors acknowledge Drs. Ernest Couch and John Pinder of Texas Christian University; Dr. S. Jerrine Nichols and the US Geological Survey Great Lakes Science Center; and the Socolofsky Microscopy Center, Department of Biological Sciences, LSU. Funding in part was provided by the Texas Christian University Research and Creative Activities Fund.

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(1) Department of Biology, Texas Christian University, Fort Worth, Texas; (2) National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health, Bethesda, Maryland; and (3) Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana

Received 30 January 2009; accepted 22 October 2009.

* To whom correspondence should be addressed. E-mail:

Abbreviations: AR. acrosome reaction; BSA, bovine serum albumin; Con A, Canavalia ensiformis (concanavalin A); GlcNAc, N-acetyl-D-glucosamine; GSII, Griffonia simplicifolia; LcH, Lens culinaris LTA, Lotus tetragonolobus; PI, postinsemination; PNA.Arachis hypogaea; PW, pond water; SBA, Glycine max; SBTI, soy bean trypsin inhibitor; WGA, Triticum vulgare (wheat germ agglutinin).
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Author:Fallis, Lindsey C.; Stein, Kathryn K.; Lynn, John W.; Misamore, Michael J.
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Date:Feb 1, 2010
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