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Lectin labeling of surface carbohydrates on gametes of three bivalves: Crassostrea virginica, Mytilus galloprovincialis, and Dreissena bugensis.

ABSTRACT The distribution of carbohydrates on the surface of freshwater and marine bivalve gametes was examined based on labeling by 5 lectins. The 3 species of interest were Dreissena bugensis, Mytilus galloprovincialis, and Crassostrea virginica. The lectins used to identify the surface carbohydrates are Arachis hypogaea (PNA), concanavalin A, Griffonia simplicifolia, Lens culinaris (LcH), and wheat germ agglutinin. Live eggs and sperm from each species were examined for lectin affinity. Lectin labeling differed greatly between the 3 species, with very few, if any, conserved trends. Furthermore, there was no obvious difference in labeling between marine and dreissenid species. Of particular interest were LcH labeling of M. galloprovincialis sperm that labeled the tip of the outer sperm acrosome and PNA labeling of C. virginica gametes that was specific only to the sperm inner acrosomal membrane. Fertilization series looking at the distribution of PNA labeling of the sperm acrosome showed localization to the basal ring associated with sperm-egg binding.

KEY WORDS: lectin, carbohydrates, gametes, Mytilus, Crassostrea, Dreissena, mussel, oyster

INTRODUCTION

Bivalve species have long served as models for studying fertilization (Dan & Wada 1955, Niijima & Dan 1965a, Niijima & Dan 1965b). Of particular interest are broadcast-spawning marine bivalves including oysters (Crassostrea spp.) (Coe 1931, Galtsoff 1964, Longwell & Stiles 1968), marine mussels (Mytilus spp.) (Humphreys 1962, Humphreys 1967, Longo & Anderson 1969a, Longo & Anderson 1969b), and Spisula (Allen 1953, Rebhun 1962, Longo & Anderson 1970a, Longo & Anderson 1970b). Various aspects of fertilization in these species have been studied, including ultrastructure (Rebhun 1962, Longo 1973), sperm morphology (Niijima & Dan 1965a, Niijima & Dan 1965b, Hodgson & Bernard 1986, Eckelbarger et al. 1990), egg morphology (Longo 1978), polyspermy (Dufresne-Dube et al. 1983), polarity (Dan & Inoue 1987), mitochondrial inheritance (Zouros et al. 1994), effects of ultraviolet radiation (Li et al. 2000), and the release of lysin (Riginos & McDonald 2003).

Recently, studies have focused on understanding the mechanisms involved in sperm-egg binding and fusion during fertilization (Focarelli et al. 2001, Riginos & McDonald 2003, Moy & Vacquier 2008, Springer et al. 2008). The potential role of carbohydrates in fertilization has received new interest (Togo & Morisawa 1997, Rosati et al. 2000, Di Patrizi et al. 2001, Mengerink & Vacquier 2001, Springer & Crespi 2007, Springer et al. 2008). In Mytilus, a family of lysin proteins present in the sperm acrosome are involved in degrading the egg vitelline envelope during sperm entry (Takagi et al. 1994). These Mytilus lysins are similar to C-type lectins that often exhibit carbohydrate binding activity. Another well-characterized sperm acrosomal protein is bindin in Crassostrea gigas. Originally identified as a major component of the sperm acrosome, oyster bindin is a glycoprotein that induces agglutination of eggs (Brandriff et al. 1978). These highly polymorphic proteins have structural domains similar to domains found in fucose-binding lectins (Moy et al. 2008, Springer et al. 2008). In the bivalve Unio, carbohydrates on the egg surface were shown to be essential for sperm-egg binding (Rosati et al. 2000).

To understand better the potential importance of carbohydrates on bivalve gametes, we conducted a comparative study that looks at the reactivity of 5 lectins to the gamete surfaces of 3 bivalve species. The 2 marine species, Crassostrea virginica (Gmelin) and Mytilus galloprovincialis (Lamarck), are both economically significant species that have served as models for fertilization studies. M. galloprovincialis is an invasive marine species native to the Mediterranean Sea. C. virginica is a species of oyster that is native to the east coast of North America and the Gulf of Mexico (Stanley & Sellers 1986). The third species, Dreissena bugensis (Andrusov), is an invasive freshwater bivalve originating from southwest Asia whose recent invasion into the western United States has raised significant economic and ecological concerns (Stokstad 2007, Ram & Palazzo 2008).

All 3 species are broadcast spawners. Like many marine bivalves, M. galloprovincialis and C. virginica release eggs and sperm directly into the water column. Sperm binding and fusion, and subsequent embryonic development occur in the external milieu. The various stages of fertilization have been widely studied in both species (Longwell & Stiles 1968, Hodgson & Bernard 1986. Springer & Crespi 2007, Bushek et al. 2008). The freshwater mussel D. bugensis also broadcast spawns gametes. This reproductive strategy varies from most native North American freshwater bivalves in which fertilization and early development occur in the mantle cavity of the females (Graf & O'Foighil 2000, Glaubrecht et al. 2006). Our understanding of D. bugensis fertilization is limited to 1 paper describing sperm morphology (Walker et al. 1996). A more detailed understanding of fertilization and early development is known for the closely related zebra mussel (Dreissena polymorpha) (Fong et al. 1995, Misamore et al. 1996, Luetjens & Dorresteijn 1998, Misamore & Lynn 2000, Misamore et al. 2006).

We studied the variation in lectin labeling on the surfaces of sperm and eggs. The sperm of M. galloprovincialis, C. virginica, and D. bugensis have been well characterized (Galtsoff 1964, Hodgson & Bernard 1986, Walker et al. 1996). In all 3 species, sperm are of the primitive type (Franzen 1983) with an anterior acrosome, a cell body containing a nucleus, a mid piece with 4 to 6 mitochondria, and a single flagellum (Fig. 1) (Galtsoff 1964, Hodgson & Bernard 1986, Walker et al. 1996).

Of particular interest is the sperm acrosomal region that facilitates binding to the egg. The greatest variability in sperm morphology among these species is found in the sperm head and acrosome (Fig. 1). Sperm of C. virginica have an ovoid head capped with the acrosomal vesicle (Fig. 1A). The nucleus has an anterior invagination (nuclear fossa) containing granular material and a central axial rod (Eckelbarger et al. 1990). M. galloprovincialis sperm have a round head with a large, conical acrosome (Fig. 1B) (Hodgson & Bernard 1986). A prominent axial rod extends the length of the acrosome (Hodgson & Bernard 1986). D. bugensis sperm have a slightly curved cell body with a cylindrical nucleus and conical acrosome (Fig. 1C). The axial rod extends from the base of the nucleus to the tip of the acrosome (Walker et al. 1996). In all 3 species and most other bivalves, there is an electron-dense, ring-shaped structure termed the "dense basal ring" located at the base of the inner acrosomal region (Brandriff et al. 1978). This ring is closely associated with sperm binding to the egg surface.

In eggs, the lectin labeling associated with the jelly layer and vitelline coat was of primary interest. Oocytes of both C. virginica and M. galloprovincialis contain numerous microvilli surrounded by a vitelline envelope (Alliegro & Wright 1983). Moreover, M. galloprovincialis eggs have prominent protrusions extending from the vitelline coat (termed "vitelline coat spikes") (Focarelli et al. 1991). The eggs of D. bugensis have yet to be described; however, D. polymorpha eggs have both an external jelly layer and vitelline coat with embedded microvilli (Misamore et al. 1996).

Aside from general surface carbohydrate distribution, we were interested in how carbohydrate labeling might change during fertilization. In particular, what new carbohydrates get exposed during the acrosome reaction and what happens after subsequent sperm-egg binding? Fertilization in most species involves 2 binding events (Primakoff & Myles 2002, Wassarman 2002, Hirohashi et al. 2008). An initial interaction occurs between the intact sperm acrosome and either the egg jelly layer or the vitelline envelope. This interaction initiates the acrosome reaction, resulting in the exocytosis of the acrosomal vesicle and exposing the previously hidden inner acrosomal membrane including the basal ring. In most bivalves observed to date, sperm egg binding appears to occur between the acrosomal filament and/or basal acrosomal ring of the sperm and the vitelline envelope or, more commonly, microvilli on the egg surface (Hylander & Summers 1977). In M. galloprovineialis, the highly active sperm arrest some distance from the surface of the egg as they attach to the vitelline coat spikes. Focarelli et al. (1991) suggest that it is here that sperm undergo the acrosome reaction and subsequently bind to the egg surface. In C. gigas, the exposed acrosomal ring of acrosome-reacted sperm contains bindin that binds to the fibrous tips of the egg microvilli (Brandriff et al. 1978, Springer et al. 2008).

[FIGURE 1 OMITTED]

MATERIALS AND METHODS

Gamete Collection

C. virginica were obtained during the months of May and June from Grand Isle Sea Grant Oyster Hatchery, Grand Isle, LA. They were maintained in 378-L aquaria with artificial seawater (1.1 M MgS[O.sub.4], 1.1 M KCl, 0.68 M Mg[Cl.sub.2], 0.82 M Ca[Cl.sub.2], 10 x trace elements, 0.55 M N-Tris(hydroxymethyl) methyl-3-aminopropanesulfonic acid [TAPS], 0.45 M NaCl) chilled to 16[degrees]C. C. virginica were opened and gonadal tissue was viewed under a microscope to identify sex. Eggs were stripped from females by slicing the gonadal tissue with a razor blade in a crisscross pattern and were rinsed with artificial seawater into a 250-mL beaker. The stripped eggs were filtered through a 75-[micro]m filter to remove gonadal tissue and excess debris, and were left in an artificial seawater suspension for 30 min to hydrate after extraction. After 30 min, the suspension was passed through an additional 21-[micro]m filter, with the eggs being retained on the screen and any remaining debris discarded with the filtrate. The eggs were washed off the filter into a beaker containing artificial seawater. Sperm were kept in vivo until ready to use, at which time they were stripped from the gonad and rinsed in an artificial seawater suspension.

D. bugensis were collected by the New York Museum of Natural History during May. They were maintained in 227-L aquaria filled with artificial pond water (0.1 mM KCl, 0.7 mM MgS[O.sub.4], 0.8 mM NaHC[O.sup.3], 0.6 mM Ca[Cl.sub.2]) chilled to 9[degrees]C. Animals were individually isolated overnight in 120-mL specimen cups containing cold pond water and allowed to acclimate overnight to room temperature (~21[degrees]C). Isolation was required to avoid cross-contamination of gametes prior to use. All experiments were carried out at room temperature (~20-22[degrees]C). Prior to spawning, individual D. bugensis were rinsed with deionized water and transferred to 25-mL flat-bottom tubes. Spawning was induced by external exposure to 0.2 mM 5-hydroxytryptamine (serotonin) for 12 min. Animals were washed twice with deionized water and returned to pond water. Males typically began spawning 10-20 min after serotonin exposure. Females began spawning 40-50 min after serotonin exposure. At the initiation of spawning, females were transferred to 50-mL crystallizing dishes to complete spawning.

M. galloprovincialis were obtained during the month of February from Penn Cove Shellfish, LLC, Coupeville, WA, and were kept dry overnight at 4[degrees]C. Spawning was induced the following morning by placing the mussels in a shallow dark-colored pan with sufficient room temperature (20-22[degrees]C) artificial seawater water to cover the animals. The pan was placed on an orbital shaker and rotated sufficiently to jostle the animals mildly to induce further spawning. Once an animal began to spawn, it was immediately washed with deionized water and isolated in a beaker of artificial seawater to complete spawning.

Lectin Labeling and DNA Staining

Lectins used to label the gametes of the various species were as follows: concanavalin A (Con A), wheat germ agglutinin (WGA), Griffonia simplicifolia (GSII), lens culinaris (LcH), or Arachis hypogaea (PNA). Samples were labeled with 30 [micro]g/mL Fluorescein isothiocyanate [FITC]-conjugated lectin (EY Laboratories, San Mateo, CA) for a minimum of 10 min. Lectin labeling of gametes was performed on live gametes to ensure surface labeling and to avoid lectin permeability issues during fixation. As a negative control, 30 [micro]g/mL lectin was incubated in 100 mM hapten sugar for 1 h. Hapten sugars used were N-acetyl glucosamine for GSII and WGA, [alpha]-methyl mannoside for Con A and LcH, and lactose for PNA. To visualize DNA of either eggs or sperm, samples were stained with 1 [micro]g/mL bisbenzamide (Hoechst 33342; Sigma Chemicals, St. Lewis, MO) for a minimum of 5 min.

Sample Fixation

After lectin labeling, samples were washed and fixed 1:1 with paraformaldehyde fixative. M. galloprovincialis and C. virginica samples were fixed in 2% paraformaldehyde and 0.55 M TAPS in artificial seawater. D. bugensis samples were fixed with 4% paraformaldehyde in 5.0 mM TAPS, 0.2 mM KCl, 2 mM NaCl, 1.8m M [Na.sub.2]S[O.sub.4], 1.25 mM MgS[O.sub.4], 2.0 mM NaHC[O.sup.3], 2.0 mM Ca[Cl.sub.2], 20% MeOH. Samples were fixed overnight and then washed twice with artificial seawater for marine species or mussel buffer (5 mM TAPS, 0.8 mM NaCl, 0.145 mM KCl, 1.8 mM [Na.sub.2]S[O.sub.4], 0.887 mM MgS[O.sub.4] x 7[H.sub.2]O, 1.32 mM NaHC[O.sup.3], 1.9 mM Ca[Cl.sub.2] x 7[H.sub.2]O; pH, 7.6) for D. bugensis. All samples were kept at 4[degrees]C until later use.

Samples for scanning electron microscopy were prepared as follows: Samples were fixed overnight with glutaraldehyde fixative (5% glutaraldehyde in artificial seawater), washed 3 times in 30 mM sodium cacodylate, fixed 1:1 in 2% osmium tetroxide/0.5 M sodium cacodylate for 1 h, and washed 3 times in 0.3 M sodium cacodylate. Samples were dehydrated in an ethanol series (30%, 50%, 75%, 2 x 95%, 3 x 100%) for 5 min at each step, critical point dried, and sputter coated with 15 nm gold.

Fertilization Time Series for C. virginica

Between 50 [micro]L and 250 [micro]L C. virginica sperm was collected, depending on relative sperm concentration after strip spawning. The sperm was added to 5 mL of eggs. Inseminated egg samples were allowed to sit for 30 sec to allow the sperm to bind to the eggs before adding the various lectins. The lectins used for fertilization trials were PNA, Con A, or GSII at a final concentration of 30 [micro]g/mL. Samples were fixed at 2, 5, 10, 15, 20, and 30 min postinsemination.

Microscopy

Light and fluorescent microscopy were performed on a Zeiss Axiovert 200 (Carl Zeiss, Inc., Thornwood, NY) and a Nikon Optiphot (Nikon Inc., Melville, NY). Digital micrographs were captured using a Zeiss AxioCam MRm (Carl Zeiss, Inc., Thornwood, NY) and Axiovision software (Carl Zeiss, Inc., Thornwood, NY). Confocal microscopy was done using a Leica SP2 LSCM (Microsystems Inc., Bannockburn, IL). Scanning electron microscopy was done with a JEOL model JSM-6100 (JEOL, Ltd., Peabody, MA) scanning electron microscope. Adobe Photoshop was used for final image processing.

RESULTS

Lectin Labeling

To determine the distribution of carbohydrates on the surfaces of both eggs and sperm, several commonly used FITC-conjugated lectins (Table 1) were tested for affinity to the gametes of the 3 bivalve species. Several species-specific differences were observed.

[FIGURE 2 OMITTED]

In the quagga mussel, D. bugensis, the lectin WGA labeled the entire surface of spawned eggs (Fig. 2A, Table 2). The distribution of lectin was uniform across the entire egg surface and was restricted to the surface coats immediately surrounding the egg plasma membrane. No punctate labeling was observed. Similar results were seen in eggs labeled with Con A (Fig. 2B), LcH (Fig. 2C), and GSII (Fig. 2D). Like the previous lectins, PNA uniformly labeled the entire egg surface (Fig. 2E). Furthermore, a previously undescribed external jelly layer labeled with PNA in a mottled fashion (Fig. 2E). Labeling of the egg jelly was not observed with WGA, LcH, Con A, or GSII.

Sperm of D. bugensis showed differential distribution of lectin labeling among the 5 lectins. LcH labeled the entire surface of the sperm, including the cell body, the mitochondria, the acrosome, and the flagellum (Fig. 2H, H'). Conversely, WGA, Con A, GSII, and PNA did not label the sperm cell body, the flagellum, or mitochondria. The labeling of these 4 lectins was restricted solely to the acrosomal region of D. bugensis sperm (Fig. 2F, G, I, J, respectively).

In M. galloprovincialis, Con A and GSII labeled the entire egg surface (Fig. 3A, B; Table 2). These lectins uniformly labeled the entire egg with no discernible pattern to their distributions. Conversely, PNA showed no labeling of the egg (Fig. 3C). LcH labeled the egg surface, but in a distinctly punctate fashion (Fig. 3D).

To visualize better LcH labeling of eggs, confocal microscopy showed that LcH labeled the vitelline coat spikes of the M. galloprovincialis eggs (Fig. 4A, B). Scanning electron microscopy revealed that the spikes were found regularly dispersed across the egg's surface (Fig. 4C, D). These results are similar to previous findings by Focarelli et al. (1991) that first described the presence of the vitelline coat spikes using the lectin Dolichos biflorus (DBA). WGA labeled the entire egg surface including the vitelline coat spikes (Fig. 3E).

Using fluorescent and phase-contrast microscopy, we found that lectin labeling of M. galloprovincialis sperm varied depending on the state of acrosomal reaction (Fig. 5). WGA, PNA GSII, and Con A failed to label sperm with intact acrosomes (Fig. 5A, B, D, E, respectively). LcH showed a very distinct pattern, labeling the distal tip of intact acrosomes (Fig. 5C, C'). None of the lectins labeled the cell bodies or flagella. As acrosomes underwent spontaneous acrosome reactions exposing the inner acrosomal contents to the lectin, labeling varied between lectins. PNA specifically labeled the acrosomal filament (Fig. 5B"). WGA labeled the basal region of partially and fully reacted acrosomes (Fig. 5A', A"), but not the acrosomal filament. LcH labeled both the acrosomal filament and the basal acrosomal region (Fig. 5C"). GSII and Con A failed to label M. galloprovincialis sperm (Fig. 5D, E).

[FIGURE 3 OMITTED]

In the eastern oyster, C. virginica, both WGA (Fig. 6A) and LcH (Fig. 6B) labeled the entire surface of the eggs (Table 2). This labeling was uniform and did not exhibit any distinct patterning. Conversely, Con A, GSII, and PNA did not label the egg surface (Fig. 6 B, D, E, respectively). In C. virginica sperm, WGA labeled the entire sperm surface including the cell body and flagellum (Fig. 6F). Con A, LcH, GSII, and PNA labeling was restricted specifically to the acrosomal region. Not all sperm exhibited acrosomal labeling (Fig. 6G, I); however, the acrosomal state of C. virginica is more difficult to determine using standard light microscopy.

Leetin Fertilization Series

As a result of the labeling of PNA specifically to the acrosomal region of sperm and not the eggs in C. virginica, fertilization series were performed to determine the distribution of PNA during fertilization. At 2 min postinsemination, sperm were observed bound to the egg surface (Fig. 7A, B). PNA labeling was detected as a distinct region between the sperm nucleus and the egg surface. By 10 min postinsemination, the sperm nucleus was incorporated into the egg cytoplasm. When viewed from a perpendicular angle to the egg surface, PNA labeling appeared as a distinct ringlike structure (Fig. 7C). Morphologically, similar ringlike structures, termed "basal rings," have been reported for several marine bivalves (Dan & Wada 1955; Niijima & Dan 1965a, Hylander & Summers 1977, Brandriff et al. 1978, Franzen 1983, Hodgson & Bernard 1986) as well as D. polymorpha (Fallis et al. 2010). Thus, we termed this structure a "PNA basal ring." At about 15 min postinsemination, the PNA basal ring began to break apart and become less evident (Fig. 7D). These rings were associated with the anterior region of both fertilizing and nonfertilizing sperm bound to the egg surface. In time series labeled with Con A and GSII, no discernible labeling was associated with bound sperm.

[FIGURE 4 OMITTED]

DISCUSSION

Lectin Labeling of Eggs

This study compared the distribution of carbohydrates based on the lectin affinity on the surfaces of eggs in 3 broadcast-spawning bivalve species (Table 3 [Fallis et al. 2010]). In D. bugensis, all 5 lectins labeled the egg surface. C. virginica eggs were the least reactivate, labeling only with LcH and WGA. In M. galloprovincialis, only PNA failed to label eggs. As expected, we obtained similar results with Con A, as previously reported by Focarelli et al. (1991). In addition, both WGA and LcH labeled the vitelline coat spikes in M. galloprovincialis. Focarelli et al. (1991) initially identified the vitelline coat spikes as a possible primary binding site of unreacted sperm in M. galloprovincialis using the lectin DBA, which labels N-acetyl-D-galactosamine. Here we show similar labeling of the vitelline spikes with LcH. These results suggest the presence of LcH-associated carbohydrates (D-mannose, D-glucose) on the vitelline coat spikes. Like DBA and LcH, WGA labeled the vitelline coat spikes, but differed in its labeling of the egg surface as well.

[FIGURE 5 OMITTED]

When these comparisons are expanded to include previous work on zebra mussel (D. polymorpha) eggs (Fallis et al. 2010), several interesting trends are observed (Table 3). WGA and LcH labeled the eggs in the 4 species. Thus, the carbohydrates associated with these 2 lectins are present on eggs in all 4 bivalve species. Differential labeling was not restricted to dreissenid versus marine species. GSII labeled M. galloprovincialis and D. bugensis eggs, but not C. virginica or D. polymorpha. Con A labeled M. galloprovincialis and D. bugensis eggs as well as D. polymorpha, but failed to label C. virginica eggs. Finally, only D. bugensis eggs were labeled with PNA.

[FIGURE 6 OMITTED]

It is worth noting that we present some of the first observations on D. bugensis egg morphology. D. bugensis eggs are surrounded by a larger, more durable outer jelly layer when compared with D. polymorpha eggs (Misamore et al. 1996). The potential role of the jelly layer in sperm chemotaxis or induction of the acrosome reaction is unknown in dreissenids, although quantitative observations show both D. polymorpha and D. bugensis sperm are more concentrated in the egg jelly during fertilization (pers. obs.). As clearly demonstrated in sea urchins, egg jelly layers are often involved in both chemotaxis and induction of the acrosome reaction (reviewed by Hirohashi et al. 2008). The importance of glycosylation in the egg jelly and the sperm receptor has also been well documented (Mengerink & Vacquier 2001). The jelly layer of D. bugensis labeled with the PNA lectin, suggesting that either lactose or galactose may be associated with this jelly layer. It is noteworthy that we have yet to find a lectin that similarly labels the jelly layer of D. polymorpha (Fallis et al. 2010). The immediate surface of D. bugensis eggs was reactive to all 5 lectins. In contrast, D. polymorpha does not label with either PNA or GSII (Table 3).

[FIGURE 7 OMITTED]

Sperm Lectin Labeling

Sperm of C. virginica, D. bugensis, and M. galloprovincialis were highly reactive to lectin labeling. All 5 lectins labeled the acrosomes of D. bugensis, with LcH also labeling the entire surface of the sperm. Alternately, in C. virginica all 5 lectins labeled the acrosomes whereas WGA also labeled the entire sperm surface. M. galloprovincialis showed the greatest variability in lectin labeling, with 2 lectins (GSII, Con A) failing to label sperm, 2 lectins labeling the inner acrosomal region (LcH, WGA), and 2 lectins (PNA, LcH) labeling the acrosomal filament.

Because of their larger acrosome, different levels of acrosome-reacted sperm were easily detected in M. galloprovincialis. The "partial reaction" of Mytilus acrosomes was first reported in Mytilus edulis by Dan and Wada (1955). In unreacted sperm, LcH had a very distinct labeling of the tip of the intact acrosome. Focarelli et al. (1991) showed that the acrosomal tip of M. galloprovincialis sperm initially interacted with the vitelline coat spikes. The distal tip labeling of intact acrosomes in M. galloprovincialis suggests this region of the acrosome membrane may be distinct from the rest of the acrosome, at least with regard to LcH labeling. A potential role for this specific region of the acrosome requires further study.

In M. galloprovincialis, PNA only labeled the extended acrosomal filament. Labeling by LcH and PNA differed slightly in partially reacted sperm relative to fully opened acrosomes. In M. edulis, the plasma membrane of the acrosomal vesicle folds back onto itself during the acrosome reaction, exposing the inner acrosomal membrane (Dan 1970). This partial exposure could be the cause for the labeling observed in partially reacted sperm in M. galloprovincialis; however, electron microscopy is needed to confirm this inference. WGA labeled the partially reacting sperm as well as the inner acrosomal region of acrosome-reacted sperm, suggesting that the carbohydrate associated with WGA may be present on the basal region of the inner acrosomal membrane but not the acrosomal filament.

When comparisons of sperm lectin labeling include D. polymorpha, no obvious trends between dreissenid and marine bivalves were observed. D. polymorpha shows identical labeling to C. virginica. Within dreissenids, LcH labeled the entire sperm surface in D. bugensis whereas WGA labeled the sperm surface in D. polymorpha.

Lectin Labeling Daring Fertilization

Using the hypothesis that sugars important for sperm-egg binding would be unique to one gamete, we were particularly interested in identifying lectins that labeled the sperm acrosomal region but not the egg. This unique carbohydrate labeling might suggest a potential importance in fertilization. In D. polymorpha, GSII specifically labels the sperm inner acrosomal membrane and filament, and the hapten sugar reduced sperm binding (Fallis et al. 2010). Similarly, Focarelli et al. (1991) showed that N-acetylgalactosamine, the hapten sugar to DBA, reduced sperm binding and fertilization. Two lectin-labeling patterns in this study stood out as potential candidates for future study. First, PNA labeled the acrosomal filament in M. galloprovincialis but failed to label the egg surface. Second, in C. virginica, PNA labeling was also restricted to just the acrosomal region and was absent from the egg surface.

To understand the relationship between PNA labeling and fertilization, we performed lectin-labeled fertilization studies in C. virginica. Fertilization series in C. virginica labeled with GSII and Con A, showed no discernible labeling pattern in bound or fertilized sperm (data not shown). However, fertilizations labeled with PNA showed a distinctive ringlike structure associated with bound sperm. This PNA basal ring was clearly evident between the bound sperm and the egg surface. The PNA basal ring remained on the egg surface whereas the sperm nucleus and mitochondria entered the egg cytoplasm. At approximately 15 min postinsemination, the PNA basal ring detached from the egg surface. Similar results were seen in the zebra mussel when sperm were labeled with GSII (Fallis et al. 2010). In zebra mussels, nonfertilizing sperm and their associated GSII basal ring detach from the egg surface in a trypsinlike enzymatic process as part of the block to polyspermy. Similar processes for PNA basal ring detachment may be occurring in C. virginica.

Togo and Morisawa (1999) found that C. gigas have a slow block to polyspermy, which occurs approximately 15 min after fertilization. The nonfertilizing sperm bound to the egg surface and underwent an acrosome reaction, but were unable to fuse with the plasma membrane. Similarly, Alliegro and Wright (1983) found a decrease in bound sperm followed by sperm detachment. Here we qualitatively observed that PNA basal rings of fertilizing sperm left of the egg surface were lost approximately 15 min after fertilization. More quantitative, detailed studies looking at possible sperm detachment need to be performed before a relationship between the loss of the PNA basal rings and a slow block to polyspermy can be made. Modifications of the egg surface typically by cortical granule exocytosis to detach nonfertilizing sperm are used in the slow block to polyspermy in invertebrate systems (Gould & Stephano 2003). However, Togo and Morisawa (1999) reported no release of proteases by the egg after fertilization. Other marine invertebrate species exhibit no obvious cortical exocytosis including the bivalves Crassostrea, Mytilus, and Spisula (Gould & Stephano 2003), and D. polymorpha (Misamore et al. 1996).

Our understanding of potential roles carbohydrates and carbohydrate-binding proteins play in bivalve fertilization continues to increase. The recent sequencing of acrosomal lysin in oyster (Springer & Crespi 2007) and oyster bindin (Moy et al. 2008) suggests that both molecules have domains that resemble carbohydrate-binding lectins (Brandriff et al. 1978). Here we found variability of carbohydrates on the surfaces of both eggs and sperm in 3 bivalve species. Several lectins specifically recognize the inner acrosomal binding region of sperm. The specific role of these carbohydrates in sperm-egg binding requires identification and sequencing of the associated glycoproteins.

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KEVYN MARIE K. MCANLIS, (1) JOHN W. LYNN (2) AND MICHAEL J. MISAMORE (1) *

(1) Department of Biology, Texas Christian University, TCU Box 298930, Fort Worth, TX 76129;

(2) Department of Biological Sciences, 202 Life Sciences Building, Louisiana State University, Baton Rouge, LA 70803

* Corresponding author: E-mail: m.misamore@tcu.edu

Note: Lectins used to label the gametes of the various species were as follows: (Con A), (WGA), (GSII), (LcH), or Arachis hypogaea (PNA).
TABLE 1.
List of lectins used and their associated carbohydrates.

                       Lectin                    Target
   Lectin Name      Abbreviation              Carbohydrate

Triticum vulgaris       WGA        [beta]-N-acetylglucosamine(1,4)
                                     [much greater than]
  (wheat germ                      N-acetylneuraminic acid
                                     [much greater than]
  agglutinin)                      N-acetylgalactosamine
Canavalia               ConA       [alpha]-Methyl-mannopyranside >
  ensiformis                       [alpha]-D-mannose > a-D-glucose >
  (concanavalin                    [alpha]-N-acetyl-D-glucosamine
   A)
Lens culinaris          LcH        D-mannose and D-glucose
                                   [alpha]-N-acetyl-D-glucosamine
Griffonia               GSII       [alpha]-N-acetyl-D-glucosamine
  simplicifolia                    [beta]-N-acetyl-D-glucosamine
Arachis hypogaea        PNA        Lactose > terminal
                                   [beta]-D-galactose

                       Carbohydrate
   Lectin Name         Abbreviation

Triticum vulgaris   GlcNA[beta](3(1,4)
  (wheat germ       Neu5Ac
  agglutinin)       [alpha]-GalNAc
Canavalia
  ensiformis
  (concanavalin     [alpha]-GlcNAc
   A)
Lens culinaris      GlcNAc

Griffonia           [alpha]-GlcNAc
  simplicifolia     [beta]-GlcNAc
Arachis hypogaea

Affinities based on primary literature reported by the
manufacturer for use with their specifically isolated
lectins (EY Laboratories, San Mateo, CA).

TABLE 2.
Summary of lectin labeling on gametes of D. bugensis,
M. galloprovincialis, and C. virginica.

                      D. bugensis

Lectin         Egg                 Sperm

PNA      Entire surface,    Activated acrosome
           jelly layer
GSII     Entire surface     Activated acrosome
LcH      Entire surface     Entire surface
WGA      Entire surface     Activated acrosome
ConA     Entire surface     Activated acrosome

                  M. galloprovincialis

Lectin         Egg                 Sperm

PNA      No labeling        Acrosomal filament
GSII     Entire surface     No labeling
LcH      Vitelline coat     Acrosomal filament
           spikes
WGA      Entire surface,    Activated acrosome
           vitelline coat
           spikes
ConA     Entire surface     No labeling

                    C. virginica

Lectin         Egg                 Sperm

PNA      No labeling        Activated acrosome
GSII     No labeling        Activated acrosome
LcH      Entire surface     Activated acrosome
WGA      Entire surface     Entire surface
ConA     No labeling        Activated acrosome

TABLE 3.
Comparison of lectin distribution on the eggs and sperm
of the marine bivalves M. galloprovincialis and C. virginica,
and the freshwater mussels D. bugensis and D. polymorpha.

                   Marine Species

Gamete    Lectin   C. virginica    M. galloprovincialis

Eggs      PNA      No labeling     No labeling
          GSII     No labeling     Egg surface
          LcH      Egg surface     Vitelline coat
                                     spikes
          WGA      Egg surface     Egg surface
                                   Vitelline coat
                                     spikes

Sperm     ConA     No labeling     Egg surface
          PNA      AR              Acrosome filament
          GSII     AR              No labeling
          LcH      AR              AR + filament
          WGA      Sperm surface   AR
          ConA     AR              No labeling

                   Freshwater Species

Gamete    Lectin   D. bugensis     D. polymorpha *

Eggs      PNA      Egg surface     No labeling
          GSII     Egg surface     No labeling
          LcH      Egg surface     Egg surface

          WGA      Egg surface     Egg surface

Sperm     ConA     Egg surface     Egg surface
          PNA      AR              AR
          GSII     AR              AR
          LcH      Sperm surface   AR
          WGA      AR              Sperm surface
          ConA     AR              AR

* Fallis et al. (2010).

AR, inner acrosomal membrane; filament, acrosomal filament.
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Author:McAnlis, Kevyn Marie K.; Lynn, John W.; Misamore, Michael J.
Publication:Journal of Shellfish Research
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Geographic Code:1USA
Date:Apr 1, 2010
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