Positive Selection in the Carbohydrate Recognition Domains of Sea Urchin Sperm Receptor for Egg Jelly (suREJ) Proteins
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《分子生物学进展》
* Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, La Jolla; and Department of Genome Sciences, University of Washington, Seattle
Correspondence: E-mail: vvacquier@ucsd.edu.
Abstract
A wealth of evidence shows that protein-carbohydrate recognition mediates the steps of gamete interaction during fertilization. Carbohydrate-recognition domains (CRDs) comprise a large family of ancient protein modules of approximately 120 amino acids, having the same protein fold, that bind terminal sugar residues on glycoproteins and polysaccharides. Sea urchin sperm express three suREJ (sea urchin receptor for egg jelly) proteins on their plasma membranes. suREJ1 has two CRDs, whereas suREJ2 and suREJ3 both have one CRD. suREJ1 binds the fucose sulfate polymer (FSP) of egg jelly to induce the sperm acrosome reaction. The structure of FSP is species specific. Therefore, the suREJ1 CRDs could encode molecular recognition between sperm and egg underlying the species-specific induction of the acrosome reaction. The functions of suREJ2 and suREJ3 have not been explored, but suREJ3 is exclusively localized on the plasma membrane over the sperm acrosomal vesicle and is physically associated with sea urchin polycystin-2, a known cation channel. An evolutionary analysis of these four CRDs was performed for six sea urchin species. Phylogenetic analysis shows that these CRDs were already differentiated in the common ancestor of these six sea urchins. The CRD phylogeny agrees with previous work on these species based on one nuclear gene and several mitochondrial genes. Maximum likelihood shows that positive selection acts on these four CRDs. Threading the suREJ CRDs onto the prototypic CRD crystal structure shows that many of the sites under positive selection are on extended loops, which are involved in saccharide binding. This is the first demonstration of positive selection in CRDs and is another example of positive selection acting on the evolution of gamete-recognition proteins.
Key Words: positive selection ? fertilization ? acrosome reaction ? sperm lectins ? sperm receptors ? C-type lectins ? maximum likelihood ? sexual antagonism
Introduction
Sea urchin spermatozoa (sperm) are model cells for studying flagellar motility (Gibbons 1996; Brokaw 2002; Imai and Shingyoji 2003), the acrosome reaction (Darszon et al. 2001; Neill and Vacquier 2004), chemotaxis toward egg-released molecules (Ward et al. 1985; Garbers 1989; Kaupp et al. 2003), and species-specific binding to eggs (Glabe and Lennarz 1979; Metz et al. 1994; Vacquier, Swanson, and Hellberg 1995; Kamei and Glabe 2003). Although many of the proteins mediating these processes are known in some detail, relatively little is known about the evolution of sperm proteins involved in the underlying signal-transduction events.
The acrosome reaction (AR) is a general feature of animal sperm that renders the sperm capable of penetrating egg investments and fusing with the egg plasma membrane. We are studying the sea urchin sperm plasma membrane proteins that bind egg jelly (EJ) molecules to induce the AR. The interaction of EJ with sperm causes ion channels to activate, resulting in the influx of Ca2+ and Na+ and the efflux of K+ and H+. In addition to these ion fluxes, there is the depolarization of the sperm plasma membrane, an increase in intracellular pH of approximately 0.25 units, and increases in cAMP, cGMP, and protein kinase activity (Garbers 1989; Darszon et al. 2001; Neill and Vacquier 2004).
Monoclonal antibodies (Mabs) were produced that react with a 210-kDa glycoprotein present on the flagellar plasma membrane and also on the plasma membrane covering the sea urchin sperm acrosomal vesicle (Trimmer 1987). Mabs to this protein induce the AR and compete with EJ for AR induction (Moy et al. 1996). When this sperm protein was purified and attached to agarose beads, the fucose sulfate polymer (FSP) of EJ was the only EJ component binding the beads (Vacquier and Moy 1997). The FSP component of EJ is an indispensable inducer of the AR (Vacquier and Moy 1997; Hirohashi and Vacquier 2002a). This 210-kDa protein was named suREJ1 (sea urchin receptor for egg jelly–1), and cloning it showed that its NH2-terminal region possessed two carbohydrate recognition domains (CRDs) of the intron-containing, calcium-dependent (C-type lectin) variety (Taylor et al. 1990; Drickamer 1999; Drickamer and Fadden 2002; Taylor and Drickamer 2003).
When suREJ1 was cloned, two additional homologous cDNAs were obtained and named suREJ2 and suREJ3. suREJ2 has one N-terminal CRD and appears to be an intracellular plasma membrane protein located over the entire cell but concentrated over the giant sperm mitochondrion (Galindo, Moy, and Vacquier 2003a). Functional studies have not been done on suREJ2. suREJ3 has one N-terminal CRD and is localized exclusively on the sperm plasma membrane covering the acrosomal vesicle, suggesting it may be involved in AR signal transduction (Mengerink, Moy, and Vacquier 2002; Neill and Vacquier 2004). suREJ3 is physically associated with the sea urchin homolog of human polycystin-2 (Neill, Moy, and Vacquier 2004), an important human disease protein known to form nonselective cation channels in mammalian cells (Cantiello 2004; Delmas et al. 2004).
Collectively, the three suREJ plasma membrane proteins possess a total of four CRDs, which suggests that they function by binding specific terminal saccharide residues (Taylor and Drickamer 2003). CRDs are ancient protein domains (King, Hittinger, and Carroll 2003) of approximately 120 amino acids, which form a major portion of the C-type lectin superfamily (Ebner, Sharon, and Ben-Tal 2003) and are important in cell-cell adhesion, innate immune recognition, and cell signaling. Crystallographic studies show that all CRDs have the same protein fold (Taylor and Drickamer 2003).
Many eukaryotic gamete recognition proteins exhibit rapid evolution, which can be driven by positive Darwinian selection (Swanson and Vacquier 2002). Positive selection results from fixation of mutations that increase the fitness of the organism. To determine whether positive selection has acted on the evolution of the four CRDs of the three suREJ proteins, we analyzed the four CRD sequences from six sea urchin species.
Materials and Methods
RNA Isolation
Fresh testes (0.8 g) from Strongylocentrotus purpuratus, S. franciscanus, and Allocentrotus fragilis (all three are Northeast Pacific species) and testes fixed in 80% ethanol and stored at –20°C of S. pallidus, S. droebachiensis (both circumpolar Arctic species), and Hemicentrotus pulcherrimus (Northwest Pacific), were extracted with 8 ml TRIZOL reagent (Invitrogen) following the manufacturer's high-salt procedure. For polymorphism studies of one species, RNA was isolated from testes of five male S. purpuratus from the same population.
cDNA Synthesis
First-strand cDNA synthesis was performed using the Invitrogen Superscript protocol. Reactions contained 2.5 mg total RNA, 200 μg random primers, 4 μl 10 mM dNTP, and DEPC-treated water to a total volume of 12 μl. Reactions were heated 3 min at 70°C and frozen in dry ice–ethanol. Tubes were thawed and 4 μl of 5x first-strand buffer, 2 μl 100 μM DTT and 1 μl RNase OUT added and the tubes incubated 10 min at 23°C, and then 2 min at 42°C. One ml of Superscript II RT was added and the tubes incubated 42°C for 1 h, followed by 70°C for 15 min. One microliter of RNase H (2U/μl) was added and the tubes incubated 37°C for 20 min before storage at –20°C.
Amplification and Sequencing of suREJ CRDs
Forty-five exact-match primers to the four S. purpuratus CRD sequences were used to amplify the CRDs from the three suREJ proteins of the other five sea urchin species. PCR reactions contained 50 ng template cDNA or genomic DNA, 1.5 μl 50 mM MgCl2, 5 μl 10X Taq buffer, 2 μl 10 mM dNTPs (2.5 mM each nucleotide), 0.25 μl Taq polymerase (Bioline), and water to 50 μl total volume. Temperature cycling used a hot start of 3 min at 95°C, followed by 35 cycles of 94°C for 1 min, 42°C to 60°C for 1 to 4 min, and 72°C for 1 to 4 min and a final extension of 72°C for 5 min. PCR products were TA cloned (Invitrogen) or gel extracted (Qiagen) for sequencing. Big dye sequencing (ABI) reactions were performed, precipitated with ethanol, and PCR products were dried and sequenced by the UCSD AIDS Center or Sequegene Inc. (www.sequegene.com). GenBank accession numbers for the three suREJ proteins of S. purpuratus are U40832 (suREJ1), AY346376 (suREJ2), and AF422153 (suREJ3). The 20 CRDs of the three suREJ proteins of the other five species are GenBank numbers AY620378 to AY620397.
Sequence Analysis
Sequencher and MacVector were used to align DNA and translated sequences. ClustalW was used for multiple alignments. The PHYLIP program dnamL was used for phylogenetic trees (Felsenstein 2004). A transition/transversion ratio (= 1.9) and other parameters were estimated from the data. Blast (Altschul et al. 1997) was used to search the Prosite, Pfam, InterPro, CCD, and SMART databases. Nonsynonymous (dN) and synonymous (dS) nucleotide substitutions were calculated using CODEML from the PAML software package (Yang 1997). For the analysis of the variation in the dN/dS ratio between sites, we calculated the likelihood of a neutral model where no codons could have a dN/dS ratio greater than 1 (L0) and compared it with the likelihood of a model in which a subset of sites could have a dN/dS ratio greater than 1 (L1). The negative of twice the difference in the log-likelihood obtained from these two models (–2[log(L0) – log(L1)]) was compared with the 2 distribution with degrees of freedom equal to the difference in number of estimated parameters. The variation in the dN/dS ratio between sites was modeled using both a discrete (PAML models M0 and M3) and beta (PAML models M7 and M8) distributions. The M0 versus M3 results were consistent with the M7 versus M8 results, but only the latter are presented because it is a more robust test of adaptive evolution. We checked for convergence by performing the analyses from different initial dN/dS ratios (Bielawski and Yang 2003).
Results
Phylogeny of the 24 suREJ CRDs
Alignment of the 24 CRDs of the four suREJ proteins of the six sea urchin species shows that 18 out of 120 amino acid positions (15%) are identical (fig. 1). The five Cys residues are completely conserved, as are many positions occupied by aromatic residues. Sequencing genomic DNA shows the presence of two introns in all CRDs at positions 49 and 84. The consensus residues of the 28 positions that define intron-containing, calcium-dependent CRDs (Taylor and Drickamer 2003) are shown in bold letters above the alignment. Fifteen of these positions agree with the consensus in at least 16 of the CRDs, supporting the classification of these CRDs in this large protein domain family. The largest single block of conserved residues is between positions 34 and 47.
FIG. 1.— Alignment of all 24 CRDs of the three suREJ proteins of the six sea urchin species. The consensus sequence for the intron-positive, calcium-dependent variety of CRDs is shown in bold above the sequence. Arrow heads mark the conserved positions of the two introns. Dashes are inserted for optimal alignment. Dark gray boxed residues indicate identity in at least 12 sequences. Spu, S. purpuratus; Afr, A. fragilis; Spa, S. pallidus; Sdr, S. droebachiensis; Hpu, H. pulcherrimus; Sfr, S. franciscanus; R1C1, suREJ1CRD1; R1C2, suREJ1CRD2; R2C, suREJ2CRD; R3C, suREJ3CRD. These designations are used for all figures.
A neighbor-joining tree of the 24 CRD sequences (fig. 2) shows that with the exception of the S. franciscanus suREJ2CRD, 23 of the CRDs cluster together as to the type of CRD and not as to species. The tree topology shows that the four suREJ CRDs were already differentiated from each other in the common ancestor of these six species. This is also supported by the fact that in the six pairwise comparisons of the four CRDs of S. purpuratus the percent identity ranges from 40% to 46%, whereas pairwise comparisons of the same CRD between any two species range from 69% to 97% in suREJ1CRD1, 65% to 97% in suREJ1CRD2, 45% to 95% in suREJ2CRD, and 67% to 94% in suREJ3CRD (table 1).
FIG. 2.— Neighbor-joining tree of the 24 suREJ CRDs (500 replicas). Bootstrap values are shown on nodes. The CRDs were already differentiated from each other before the speciation of these six sea urchins.
Table 1 Percent Identities and dN/dS in Pairwise Comparisons of CRDs
Positive Selection in suREJ CRDs
Two methods were used to test for positive selection (adaptive evolution). First, the average dN/dS ratios across the entire CRD sequences in all 15 pairwise comparisons of the six species were calculated (table 1). Plots of dN versus dS show that for suREJ1CRD2, all 15 comparisons fall above the line of neutral expectation (fig. 3). Although none of the pairwise comparisons are significantly different from 1 after correction for multiple testing (Bonferroni 1936), these results do indicate the likely action of positive Darwinian selection driving the divergence of this domain. For the majority of the other CRD comparisons, the dN/dS ratios are high (0.5) compared with average dN/dS ratios of most genes (0.2). Many genes with a ratio of 0.5 or higher are in fact subjected to positive selection (Swanson et al. 2004).
FIG. 3.— Plots of dN (vertical axis) versus dS (horizontal axis) for the 15 possible pairwise comparisons of the four CRDs. The method is an average across the CRD. In suREJ1CRD2, all comparisons fall above the line of neutral expectation.
Because it is known that averaging dN/dS ratios across all sites and lineages is not a robust test for positive selection, we tested for variation in the dN/dS ratios between sites. Maximum likelihood was used to identify those sites in each CRD that are subjected to positive selection with a posterior probability greater than 0.95. The analysis (table 2) shows 12 sites in suREJ1CRD1, 11 sites in suREJ1CRD2, seven sites in suREJ2CRD, and nine sites in suREJ3CRD.
Table 2 Positive Darwinian Selection in CRDs of Sperm REJ Proteins
Threading suREJCRDs onto the CRD Crystal Structure
Because of the high conservation of Cys and aromatic residues, all intron-containing, calcium-dependent CRDs have the same three-dimensional structure (Taylor and Drickamer 2003). The four CRD amino acid sequences were threaded onto a known CRD crystal structure (Feinberg et al. 2000) and the sites subjected to positive selection marked on each structure (fig. 4). For three of the CRDs (suREJ1CRD1, suREJ2CRD, and suREJ3CRD), similar regions appear to be subjected to positive selection. These include several of the extended loops implicated in saccharide binding and a region near the N-terminus (Taylor and Drickamer 2003). In all four CRDs, the sites predicted to be subjected to positive selection appear to be located on the front face of the CRD. These sites are likely to be involved in the species-specific recognition of EJ carbohydrate polymers of the different species.
FIG. 4.— Threading the suREJ CRDs onto the prototypic CRD crystal structure. Amino acid sites predicted to be under positive selection are shown in spacefill. Many of the positively selected sites are in similar regions in the different CRDs.
Polymorphism in the Four CRDs from the Three S. purpuratus suREJ Sequences
The four CRDs were sequenced from five S. purpuratus from the same population. suREJ1CRD1 has three polymorphic nucleotide sites, one of which is nonsynonymous (amino acid altering). suREJ1CRD2 has two polymorphic sites, both of which are nonsynonymous. suREJ2CRD has five polymorphic sites, four of which change the amino acid. suREJ3CRD has six polymorphic sites, and five are amino acid altering. Of the 16 polymorphic sites in the five individuals, 12 are nonsynonymous and three of these are subjected to positive selection. The percent nucleotide polymorphism varies from 0.56% in suREJ1CRD2 to 1.7% in suREJ3CRD. Although none of these polymorphism levels depart from equilibrium-neutral expectations, the amino acid differences potentially result in functional differences within this species, as has been observed for polymorphisms in sea urchin sperm bindin, the protein that binds the sperm to the egg surface (Palumbi 1999).
Discussion
Phylogeny and Rate of CRD Change in the Six Species
The complete sequences of the three suREJ proteins are known only for S. purpuratus. Thus, the analysis of these CRDs reflects only the CRDs themselves (120 residues) and not the entire proteins, which vary in S. purpuratus from 1,450 (suREJ1) to 2,681 (suREJ3) residues. In pairwise comparisons of amino acid position (table 1), the four S. purpuratus CRDs are 42% to 46% identical, showing that they have diverged considerably since they arose by gene duplication. Figure 2 shows that these four CRDs were already differentiated in the common ancestor of these six species. The phylogenetic topology of the 24 CRDs agrees with trees of these species based on the sperm protein bindin (Biermann 1998) and several mitochondrial genes (Biermann, Kessing, and Palumbi 2003; Lee 2003). The major point of agreement is that S. purpuratus, S. droebachiensis, S. pallidus, and A. fragilis always fall close to each other, on a different branch from S. franciscanus and H. pulcherrimus. Thus, as previously discussed (Biermann 1998; Biermann, Kessing, and Palumbi 2003; Lee 2003), the genera Allocentrotus and Hemicentrotus should be within the genus Strongylocentrotus. The only point of disagreement between figure 2 and the previously published trees is with S. franciscanus suREJ2CRD, which is so divergent that it falls on its own branch basal to its five homologs.
Lee (2003) has estimated divergence times for these six species based on 12S rDNA. Using the averages of Lee's range of divergence times and the percent identities in table 1, we calculated the average percent amino acid change per million years in the 10 pairwise comparisons of the species in common between Lee's study and ours. Averaging the 10 comparisons yields 2.8% amino acid divergence per million years for suREJ1CRD1, 2.6% for suREJ1CRD2, 4.2% for suREJ2CRD, and 1.9% for suREJ3CRD. These data suggest that suREJ2CRD is the fastest changing and suREJ3CRD the slowest changing CRD.
Function of suREJ Proteins
The functions of suREJ2 and suREJ3 remain unknown. However, the location of suREJ3 exclusively in the plasma membrane over the acrosomal vesicle and the fact that it is physically associated with polycystin-2, suggest that it may form a cation channel that functions in the induction of the AR (Neill, Moy, and Vacquie 2004). The low cation selectivity of polycystin-2 channels would explain older data on cation transport into sea urchin sperm suggesting that calcium and sodium entered through the same ion channel during the acrosome reaction (Schackmann and Shapiro 1981).
Although it has not been experimentally demonstrated, the existence of at least one CRD in the three suREJ proteins suggests that their function involves binding saccharide residues (Taylor and Drickamer 2003). Agarose beads with covalently immobilized suREJ1 bind the FSP component of EJ. FSP is a pure polysaccharide with no amino acid content and a molecular mass greater than 1 million Da (Vacquier and Moy 1997; Hirohashi and Vacquier 2002a). FSP is indispensable for the induction of the sperm AR, and it can be very species specific as an AR inducer. This specificity of molecular recognition resides in the pattern of sulfation of the fucosyl residues and the nature of the glycosidic bond (Vilela-Silva et al. 2002). With the exception of suREJ1CRD2, many of the sites under positive selection in the other three CRDs are located on the external faces of the extended loops that are known to bind saccharide residues (Taylor and Drickamer 2003). The evolution of CRDs on sperm receptor proteins could be one way in which the species specificity of gamete recognition is encoded.
Monoclonal antibody to suREJ1 induces the AR and competes with EJ for AR induction (Moy et al. 1996), showing that suREJ1 plays a major role in AR induction. Both CRDs of suREJ1 show positive selection, but selection is more intense on suREJ1CRD2 (fig. 3). This CRD could play a more significant role in recognizing FSP as the sperm swims into the hydrated EJ layer. Although FSP is an indispensable inducer of the AR, another saccharide, a polysialic acid, is a strong potentiator of the FSP-induced AR. By itself, the polysialic acid has no AR-inducing activity; however, in the presence of FSP, it causes an elevation of intracellular pH. This additional biologically active saccharide component of EJ must bind to a CRD carried by a sperm receptor protein (Hirohashi and Vacquier 2002b).
Adaptive Evolution at Several Levels of the Fertilization Cascade
Sperm-egg interaction proceeds through a series of steps that are common to most animals (Vacquier 1998). Sperm may be chemotactically attracted to the egg. Depending on the species, sperm binds to the egg envelope either before or after induction of the AR. After exocytosis of the acrosomal contents, a hole is created in the egg envelope through which the sperm passes to fuse with the egg cell membrane. In different animal species, the genes mediating these steps have been shown to be subjected to positive selection. Before this study, acrosomal bindin was the only sea urchin fertilization protein known to be subjected to positive selection (Metz and Palumbi 1996). Here, we demonstrate positive selection acting on at least one gene (suREJ1) that is known to bind an egg carbohydrate (FSP), mediating AR induction. It will be important to study the pattern of evolution of all genes mediating the steps in the fertilization cascade to gain insight into the number of loci that may be involved in establishing prezygotic reproductive isolation (Coyne and Orr 2004).
Sexual Antagonism As a Reason for Positive Selection on suREJ CRDs
Our favored hypothesis to explain positive selection in the evolution of suREJCRDs combines sexual antagonism (Rice 1996; Gavrilets 2000; Civetta 2003) and sperm competition (Parker 1970; Birkhead 1996; Frank 2000). In sexual antagonism (as applied to this paper), sperm and egg have different "interests" in the efficiency of their interaction leading to fusion. In presenting this hypothesis, we must discuss the problem of more than one sperm fusing with the egg, the biochemistry of the egg jelly coat, the physical properties of the egg jelly coat, sperm competition, and positive selection on sperm suREJ proteins.
In sea urchins and mammals, if more than one sperm fuses with the egg, the result is pathological polyspermy, which arrests development. Eggs must evolve ways to decrease the probability of fusion with more than one sperm. In sea urchins, there is both a fast electrical block to prevent sperm fusion and a slow physical block that involves the elevation of a fertilization envelope to prevent sperm passage. As discussed below, the egg may also regulate sperm entry through molecular interactions involving gamete recognition proteins.
When sea urchin eggs are spawned into seawater, the egg jelly coat (EJC) swells to approximately one egg diameter and is dense enough to exclude colloidal particles of India ink. The EJC contains two types of carbohydrate molecules that are known to be involved in AR induction; one is FSP and the other is the polysialic acid. In S. purpuratus (Vacquier and Moy 1997; Alves et al. 1998) and S. droebachiensis (Vilela-Silva et al. 2002), FSP comes in two isoforms, each isoform being female specific and both isoforms having equal potency as AR inducers. (Roughly 64% of S. purpuratus females have one FSP isoform, 31% have another FSP isoform, and 5% have both FSP isoforms [Vacquier, unpublished data]). Of the four other species studied in this report, the structure of FSP is known for S. franciscanus and S. pallidus. Females of these two species synthesize only one, species-specific, type of FSP (Vilela-Silva et al. 2002). The polysialic acid molecule of the EJC must bind to a sperm-surface receptor. In the presence of FSP, it greatly potentiates the FSP-induced AR by raising sperm intracellular pH (Hirohashi and Vacquier 2002b). Thus, in S. purpuratus and S. droebachiensis, there are at least three types of carbohydrate polymers in the EJC that interact with sperm-membrane carbohydrate-recognition proteins (suREJ proteins) to regulate sperm ion channels. The EJC also contains at least 10 other glycoproteins of unknown function that could bind suREJ and other unknown sperm-surface receptors (Vacquier and Moy 1997).
The physical function of the hydrated EJC is to prevent eggs from sticking together and also to prevent microbes from binding to the egg surface. If the EJC is removed from S. franciscanus eggs, the eggs irreversibly clump together and abnormal development occurs (Vacquier, unpublished data). However, this is not true of S. purpuratus eggs. In some species of free-spawning echinoderms, the hydrated EJC increases the effective diameter of the egg, creating a larger target for the sperm to hit. Thus, in some species, the EJC increases the rate of fertilization by being of greater diameter than the egg's cytoplasmic mass (Levitan and Irvine 2001; Podolsky 2002). In S. purpuratus, eggs in which the bulk of the EJC has been removed bind more sperm and are more susceptible to higher levels of polyspermy than eggs with intact EJC. In these "dejellied" eggs, the optimal amount of EJ needed to induce the sperm AR remains tightly bound to the egg surface (Vacquier, Brandriff, and Glabe 1979).
From the egg's side of the sexual antagonism theory, the hydrated EJC provides three positive things for the egg: it protects the egg from physical abuse and microbes, it holds an excess of molecules needed to induce the sperm AR, and it slows sperm as they approach the egg surface, by inducing premature AR, which decreases the frequency of polyspermy (Vacquier, Brandriff, and Glabe 1979; Frank 2000). Premature induction of the AR decreases sperm fertilizability with a half-life of 23 seconds (Vacquier 1979). The EJC slowing the frequency of sperm fusion is of positive benefit to the egg, but is a negative benefit for sperm, supporting the hypothesis of sexual antagonism acting during sperm-egg interaction.
Competition among individual sperm cells will continuously select for sperm that most efficiently fuse with eggs (Frank 2000). One ml of undiluted S. purpuratus semen contains 40 billion sperm cells, and one large male can spawn 5 ml of semen (Vacquier 1986). Thus, the large numbers of sperm available enhance the probability that favorable mutants (in suREJ proteins) will enjoy increased fertilization success. The egg must evolve ways to retard these potentially supersuccessful sperm and optimize the chance for monospermy.
Finally, the suREJ protein variants that optimize sperm fusion rates with eggs could be selected for on the basis of changes in their CRDs. Theory suggests that positive selection should be found in both the sperm protein and its cognate egg-surface receptor (Gavrilets 2000). Such is the case with abalone sperm lysin and its egg receptor VERL, where both cognate binding partners are subjected to positive selection (Galindo, Vacquier, and Swanson 2003b). The only molecules we know of in sea urchin EJ that induce the AR in seawater at pH 8.0 are the carbohydrate polymers of FSP and polysialic acid. Although we do not yet know how to detect positive selection in a carbohydrate polymer, it could be that the enzymes involved in the biosynthesis of these EJ molecules might show positive selection.
Avoiding polyspermy is a multistep process for the sea urchin egg. The first level of blockade could be the interaction of sperm suREJ proteins with egg FSP and polysialic acid to induce premature AR and retard the rate of sperm reaching the egg surface. The egg would be selecting for a sperm that undergoes the AR at exactly the correct distance from the egg surface (cryptic female choice). The second level of polyspermy blockade is the well-known electrical block, where, after fusion with the first sperm, the egg membrane potential changes in 100 milliseconds from –70 mV to +20 mV, which prevents further sperm fusion by an unknown mechanism (Gould-Somero and Jaffe 1984). The third polyspermy blockade is the complete block provided by the fertilization protease and the formation of the fertilization envelope (Vacquier, Tegner, and Epel 1973; Haley and Wessel 2004).
The demonstration of positive selection in the CRDs of suREJ proteins shows once again that the evolution of the molecules and mechanisms involved in sperm-egg interaction is extremely complex yet always fascinating.
Acknowledgements
We thank Gary W. Moy and Anna T. Neill for helpful advice. This work was supported by an NSF Minority Graduate Fellowship to S.A.M., NIH Grants HD12986 to V.D.V., and HD42563 to W.J.S.
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Correspondence: E-mail: vvacquier@ucsd.edu.
Abstract
A wealth of evidence shows that protein-carbohydrate recognition mediates the steps of gamete interaction during fertilization. Carbohydrate-recognition domains (CRDs) comprise a large family of ancient protein modules of approximately 120 amino acids, having the same protein fold, that bind terminal sugar residues on glycoproteins and polysaccharides. Sea urchin sperm express three suREJ (sea urchin receptor for egg jelly) proteins on their plasma membranes. suREJ1 has two CRDs, whereas suREJ2 and suREJ3 both have one CRD. suREJ1 binds the fucose sulfate polymer (FSP) of egg jelly to induce the sperm acrosome reaction. The structure of FSP is species specific. Therefore, the suREJ1 CRDs could encode molecular recognition between sperm and egg underlying the species-specific induction of the acrosome reaction. The functions of suREJ2 and suREJ3 have not been explored, but suREJ3 is exclusively localized on the plasma membrane over the sperm acrosomal vesicle and is physically associated with sea urchin polycystin-2, a known cation channel. An evolutionary analysis of these four CRDs was performed for six sea urchin species. Phylogenetic analysis shows that these CRDs were already differentiated in the common ancestor of these six sea urchins. The CRD phylogeny agrees with previous work on these species based on one nuclear gene and several mitochondrial genes. Maximum likelihood shows that positive selection acts on these four CRDs. Threading the suREJ CRDs onto the prototypic CRD crystal structure shows that many of the sites under positive selection are on extended loops, which are involved in saccharide binding. This is the first demonstration of positive selection in CRDs and is another example of positive selection acting on the evolution of gamete-recognition proteins.
Key Words: positive selection ? fertilization ? acrosome reaction ? sperm lectins ? sperm receptors ? C-type lectins ? maximum likelihood ? sexual antagonism
Introduction
Sea urchin spermatozoa (sperm) are model cells for studying flagellar motility (Gibbons 1996; Brokaw 2002; Imai and Shingyoji 2003), the acrosome reaction (Darszon et al. 2001; Neill and Vacquier 2004), chemotaxis toward egg-released molecules (Ward et al. 1985; Garbers 1989; Kaupp et al. 2003), and species-specific binding to eggs (Glabe and Lennarz 1979; Metz et al. 1994; Vacquier, Swanson, and Hellberg 1995; Kamei and Glabe 2003). Although many of the proteins mediating these processes are known in some detail, relatively little is known about the evolution of sperm proteins involved in the underlying signal-transduction events.
The acrosome reaction (AR) is a general feature of animal sperm that renders the sperm capable of penetrating egg investments and fusing with the egg plasma membrane. We are studying the sea urchin sperm plasma membrane proteins that bind egg jelly (EJ) molecules to induce the AR. The interaction of EJ with sperm causes ion channels to activate, resulting in the influx of Ca2+ and Na+ and the efflux of K+ and H+. In addition to these ion fluxes, there is the depolarization of the sperm plasma membrane, an increase in intracellular pH of approximately 0.25 units, and increases in cAMP, cGMP, and protein kinase activity (Garbers 1989; Darszon et al. 2001; Neill and Vacquier 2004).
Monoclonal antibodies (Mabs) were produced that react with a 210-kDa glycoprotein present on the flagellar plasma membrane and also on the plasma membrane covering the sea urchin sperm acrosomal vesicle (Trimmer 1987). Mabs to this protein induce the AR and compete with EJ for AR induction (Moy et al. 1996). When this sperm protein was purified and attached to agarose beads, the fucose sulfate polymer (FSP) of EJ was the only EJ component binding the beads (Vacquier and Moy 1997). The FSP component of EJ is an indispensable inducer of the AR (Vacquier and Moy 1997; Hirohashi and Vacquier 2002a). This 210-kDa protein was named suREJ1 (sea urchin receptor for egg jelly–1), and cloning it showed that its NH2-terminal region possessed two carbohydrate recognition domains (CRDs) of the intron-containing, calcium-dependent (C-type lectin) variety (Taylor et al. 1990; Drickamer 1999; Drickamer and Fadden 2002; Taylor and Drickamer 2003).
When suREJ1 was cloned, two additional homologous cDNAs were obtained and named suREJ2 and suREJ3. suREJ2 has one N-terminal CRD and appears to be an intracellular plasma membrane protein located over the entire cell but concentrated over the giant sperm mitochondrion (Galindo, Moy, and Vacquier 2003a). Functional studies have not been done on suREJ2. suREJ3 has one N-terminal CRD and is localized exclusively on the sperm plasma membrane covering the acrosomal vesicle, suggesting it may be involved in AR signal transduction (Mengerink, Moy, and Vacquier 2002; Neill and Vacquier 2004). suREJ3 is physically associated with the sea urchin homolog of human polycystin-2 (Neill, Moy, and Vacquier 2004), an important human disease protein known to form nonselective cation channels in mammalian cells (Cantiello 2004; Delmas et al. 2004).
Collectively, the three suREJ plasma membrane proteins possess a total of four CRDs, which suggests that they function by binding specific terminal saccharide residues (Taylor and Drickamer 2003). CRDs are ancient protein domains (King, Hittinger, and Carroll 2003) of approximately 120 amino acids, which form a major portion of the C-type lectin superfamily (Ebner, Sharon, and Ben-Tal 2003) and are important in cell-cell adhesion, innate immune recognition, and cell signaling. Crystallographic studies show that all CRDs have the same protein fold (Taylor and Drickamer 2003).
Many eukaryotic gamete recognition proteins exhibit rapid evolution, which can be driven by positive Darwinian selection (Swanson and Vacquier 2002). Positive selection results from fixation of mutations that increase the fitness of the organism. To determine whether positive selection has acted on the evolution of the four CRDs of the three suREJ proteins, we analyzed the four CRD sequences from six sea urchin species.
Materials and Methods
RNA Isolation
Fresh testes (0.8 g) from Strongylocentrotus purpuratus, S. franciscanus, and Allocentrotus fragilis (all three are Northeast Pacific species) and testes fixed in 80% ethanol and stored at –20°C of S. pallidus, S. droebachiensis (both circumpolar Arctic species), and Hemicentrotus pulcherrimus (Northwest Pacific), were extracted with 8 ml TRIZOL reagent (Invitrogen) following the manufacturer's high-salt procedure. For polymorphism studies of one species, RNA was isolated from testes of five male S. purpuratus from the same population.
cDNA Synthesis
First-strand cDNA synthesis was performed using the Invitrogen Superscript protocol. Reactions contained 2.5 mg total RNA, 200 μg random primers, 4 μl 10 mM dNTP, and DEPC-treated water to a total volume of 12 μl. Reactions were heated 3 min at 70°C and frozen in dry ice–ethanol. Tubes were thawed and 4 μl of 5x first-strand buffer, 2 μl 100 μM DTT and 1 μl RNase OUT added and the tubes incubated 10 min at 23°C, and then 2 min at 42°C. One ml of Superscript II RT was added and the tubes incubated 42°C for 1 h, followed by 70°C for 15 min. One microliter of RNase H (2U/μl) was added and the tubes incubated 37°C for 20 min before storage at –20°C.
Amplification and Sequencing of suREJ CRDs
Forty-five exact-match primers to the four S. purpuratus CRD sequences were used to amplify the CRDs from the three suREJ proteins of the other five sea urchin species. PCR reactions contained 50 ng template cDNA or genomic DNA, 1.5 μl 50 mM MgCl2, 5 μl 10X Taq buffer, 2 μl 10 mM dNTPs (2.5 mM each nucleotide), 0.25 μl Taq polymerase (Bioline), and water to 50 μl total volume. Temperature cycling used a hot start of 3 min at 95°C, followed by 35 cycles of 94°C for 1 min, 42°C to 60°C for 1 to 4 min, and 72°C for 1 to 4 min and a final extension of 72°C for 5 min. PCR products were TA cloned (Invitrogen) or gel extracted (Qiagen) for sequencing. Big dye sequencing (ABI) reactions were performed, precipitated with ethanol, and PCR products were dried and sequenced by the UCSD AIDS Center or Sequegene Inc. (www.sequegene.com). GenBank accession numbers for the three suREJ proteins of S. purpuratus are U40832 (suREJ1), AY346376 (suREJ2), and AF422153 (suREJ3). The 20 CRDs of the three suREJ proteins of the other five species are GenBank numbers AY620378 to AY620397.
Sequence Analysis
Sequencher and MacVector were used to align DNA and translated sequences. ClustalW was used for multiple alignments. The PHYLIP program dnamL was used for phylogenetic trees (Felsenstein 2004). A transition/transversion ratio (= 1.9) and other parameters were estimated from the data. Blast (Altschul et al. 1997) was used to search the Prosite, Pfam, InterPro, CCD, and SMART databases. Nonsynonymous (dN) and synonymous (dS) nucleotide substitutions were calculated using CODEML from the PAML software package (Yang 1997). For the analysis of the variation in the dN/dS ratio between sites, we calculated the likelihood of a neutral model where no codons could have a dN/dS ratio greater than 1 (L0) and compared it with the likelihood of a model in which a subset of sites could have a dN/dS ratio greater than 1 (L1). The negative of twice the difference in the log-likelihood obtained from these two models (–2[log(L0) – log(L1)]) was compared with the 2 distribution with degrees of freedom equal to the difference in number of estimated parameters. The variation in the dN/dS ratio between sites was modeled using both a discrete (PAML models M0 and M3) and beta (PAML models M7 and M8) distributions. The M0 versus M3 results were consistent with the M7 versus M8 results, but only the latter are presented because it is a more robust test of adaptive evolution. We checked for convergence by performing the analyses from different initial dN/dS ratios (Bielawski and Yang 2003).
Results
Phylogeny of the 24 suREJ CRDs
Alignment of the 24 CRDs of the four suREJ proteins of the six sea urchin species shows that 18 out of 120 amino acid positions (15%) are identical (fig. 1). The five Cys residues are completely conserved, as are many positions occupied by aromatic residues. Sequencing genomic DNA shows the presence of two introns in all CRDs at positions 49 and 84. The consensus residues of the 28 positions that define intron-containing, calcium-dependent CRDs (Taylor and Drickamer 2003) are shown in bold letters above the alignment. Fifteen of these positions agree with the consensus in at least 16 of the CRDs, supporting the classification of these CRDs in this large protein domain family. The largest single block of conserved residues is between positions 34 and 47.
FIG. 1.— Alignment of all 24 CRDs of the three suREJ proteins of the six sea urchin species. The consensus sequence for the intron-positive, calcium-dependent variety of CRDs is shown in bold above the sequence. Arrow heads mark the conserved positions of the two introns. Dashes are inserted for optimal alignment. Dark gray boxed residues indicate identity in at least 12 sequences. Spu, S. purpuratus; Afr, A. fragilis; Spa, S. pallidus; Sdr, S. droebachiensis; Hpu, H. pulcherrimus; Sfr, S. franciscanus; R1C1, suREJ1CRD1; R1C2, suREJ1CRD2; R2C, suREJ2CRD; R3C, suREJ3CRD. These designations are used for all figures.
A neighbor-joining tree of the 24 CRD sequences (fig. 2) shows that with the exception of the S. franciscanus suREJ2CRD, 23 of the CRDs cluster together as to the type of CRD and not as to species. The tree topology shows that the four suREJ CRDs were already differentiated from each other in the common ancestor of these six species. This is also supported by the fact that in the six pairwise comparisons of the four CRDs of S. purpuratus the percent identity ranges from 40% to 46%, whereas pairwise comparisons of the same CRD between any two species range from 69% to 97% in suREJ1CRD1, 65% to 97% in suREJ1CRD2, 45% to 95% in suREJ2CRD, and 67% to 94% in suREJ3CRD (table 1).
FIG. 2.— Neighbor-joining tree of the 24 suREJ CRDs (500 replicas). Bootstrap values are shown on nodes. The CRDs were already differentiated from each other before the speciation of these six sea urchins.
Table 1 Percent Identities and dN/dS in Pairwise Comparisons of CRDs
Positive Selection in suREJ CRDs
Two methods were used to test for positive selection (adaptive evolution). First, the average dN/dS ratios across the entire CRD sequences in all 15 pairwise comparisons of the six species were calculated (table 1). Plots of dN versus dS show that for suREJ1CRD2, all 15 comparisons fall above the line of neutral expectation (fig. 3). Although none of the pairwise comparisons are significantly different from 1 after correction for multiple testing (Bonferroni 1936), these results do indicate the likely action of positive Darwinian selection driving the divergence of this domain. For the majority of the other CRD comparisons, the dN/dS ratios are high (0.5) compared with average dN/dS ratios of most genes (0.2). Many genes with a ratio of 0.5 or higher are in fact subjected to positive selection (Swanson et al. 2004).
FIG. 3.— Plots of dN (vertical axis) versus dS (horizontal axis) for the 15 possible pairwise comparisons of the four CRDs. The method is an average across the CRD. In suREJ1CRD2, all comparisons fall above the line of neutral expectation.
Because it is known that averaging dN/dS ratios across all sites and lineages is not a robust test for positive selection, we tested for variation in the dN/dS ratios between sites. Maximum likelihood was used to identify those sites in each CRD that are subjected to positive selection with a posterior probability greater than 0.95. The analysis (table 2) shows 12 sites in suREJ1CRD1, 11 sites in suREJ1CRD2, seven sites in suREJ2CRD, and nine sites in suREJ3CRD.
Table 2 Positive Darwinian Selection in CRDs of Sperm REJ Proteins
Threading suREJCRDs onto the CRD Crystal Structure
Because of the high conservation of Cys and aromatic residues, all intron-containing, calcium-dependent CRDs have the same three-dimensional structure (Taylor and Drickamer 2003). The four CRD amino acid sequences were threaded onto a known CRD crystal structure (Feinberg et al. 2000) and the sites subjected to positive selection marked on each structure (fig. 4). For three of the CRDs (suREJ1CRD1, suREJ2CRD, and suREJ3CRD), similar regions appear to be subjected to positive selection. These include several of the extended loops implicated in saccharide binding and a region near the N-terminus (Taylor and Drickamer 2003). In all four CRDs, the sites predicted to be subjected to positive selection appear to be located on the front face of the CRD. These sites are likely to be involved in the species-specific recognition of EJ carbohydrate polymers of the different species.
FIG. 4.— Threading the suREJ CRDs onto the prototypic CRD crystal structure. Amino acid sites predicted to be under positive selection are shown in spacefill. Many of the positively selected sites are in similar regions in the different CRDs.
Polymorphism in the Four CRDs from the Three S. purpuratus suREJ Sequences
The four CRDs were sequenced from five S. purpuratus from the same population. suREJ1CRD1 has three polymorphic nucleotide sites, one of which is nonsynonymous (amino acid altering). suREJ1CRD2 has two polymorphic sites, both of which are nonsynonymous. suREJ2CRD has five polymorphic sites, four of which change the amino acid. suREJ3CRD has six polymorphic sites, and five are amino acid altering. Of the 16 polymorphic sites in the five individuals, 12 are nonsynonymous and three of these are subjected to positive selection. The percent nucleotide polymorphism varies from 0.56% in suREJ1CRD2 to 1.7% in suREJ3CRD. Although none of these polymorphism levels depart from equilibrium-neutral expectations, the amino acid differences potentially result in functional differences within this species, as has been observed for polymorphisms in sea urchin sperm bindin, the protein that binds the sperm to the egg surface (Palumbi 1999).
Discussion
Phylogeny and Rate of CRD Change in the Six Species
The complete sequences of the three suREJ proteins are known only for S. purpuratus. Thus, the analysis of these CRDs reflects only the CRDs themselves (120 residues) and not the entire proteins, which vary in S. purpuratus from 1,450 (suREJ1) to 2,681 (suREJ3) residues. In pairwise comparisons of amino acid position (table 1), the four S. purpuratus CRDs are 42% to 46% identical, showing that they have diverged considerably since they arose by gene duplication. Figure 2 shows that these four CRDs were already differentiated in the common ancestor of these six species. The phylogenetic topology of the 24 CRDs agrees with trees of these species based on the sperm protein bindin (Biermann 1998) and several mitochondrial genes (Biermann, Kessing, and Palumbi 2003; Lee 2003). The major point of agreement is that S. purpuratus, S. droebachiensis, S. pallidus, and A. fragilis always fall close to each other, on a different branch from S. franciscanus and H. pulcherrimus. Thus, as previously discussed (Biermann 1998; Biermann, Kessing, and Palumbi 2003; Lee 2003), the genera Allocentrotus and Hemicentrotus should be within the genus Strongylocentrotus. The only point of disagreement between figure 2 and the previously published trees is with S. franciscanus suREJ2CRD, which is so divergent that it falls on its own branch basal to its five homologs.
Lee (2003) has estimated divergence times for these six species based on 12S rDNA. Using the averages of Lee's range of divergence times and the percent identities in table 1, we calculated the average percent amino acid change per million years in the 10 pairwise comparisons of the species in common between Lee's study and ours. Averaging the 10 comparisons yields 2.8% amino acid divergence per million years for suREJ1CRD1, 2.6% for suREJ1CRD2, 4.2% for suREJ2CRD, and 1.9% for suREJ3CRD. These data suggest that suREJ2CRD is the fastest changing and suREJ3CRD the slowest changing CRD.
Function of suREJ Proteins
The functions of suREJ2 and suREJ3 remain unknown. However, the location of suREJ3 exclusively in the plasma membrane over the acrosomal vesicle and the fact that it is physically associated with polycystin-2, suggest that it may form a cation channel that functions in the induction of the AR (Neill, Moy, and Vacquie 2004). The low cation selectivity of polycystin-2 channels would explain older data on cation transport into sea urchin sperm suggesting that calcium and sodium entered through the same ion channel during the acrosome reaction (Schackmann and Shapiro 1981).
Although it has not been experimentally demonstrated, the existence of at least one CRD in the three suREJ proteins suggests that their function involves binding saccharide residues (Taylor and Drickamer 2003). Agarose beads with covalently immobilized suREJ1 bind the FSP component of EJ. FSP is a pure polysaccharide with no amino acid content and a molecular mass greater than 1 million Da (Vacquier and Moy 1997; Hirohashi and Vacquier 2002a). FSP is indispensable for the induction of the sperm AR, and it can be very species specific as an AR inducer. This specificity of molecular recognition resides in the pattern of sulfation of the fucosyl residues and the nature of the glycosidic bond (Vilela-Silva et al. 2002). With the exception of suREJ1CRD2, many of the sites under positive selection in the other three CRDs are located on the external faces of the extended loops that are known to bind saccharide residues (Taylor and Drickamer 2003). The evolution of CRDs on sperm receptor proteins could be one way in which the species specificity of gamete recognition is encoded.
Monoclonal antibody to suREJ1 induces the AR and competes with EJ for AR induction (Moy et al. 1996), showing that suREJ1 plays a major role in AR induction. Both CRDs of suREJ1 show positive selection, but selection is more intense on suREJ1CRD2 (fig. 3). This CRD could play a more significant role in recognizing FSP as the sperm swims into the hydrated EJ layer. Although FSP is an indispensable inducer of the AR, another saccharide, a polysialic acid, is a strong potentiator of the FSP-induced AR. By itself, the polysialic acid has no AR-inducing activity; however, in the presence of FSP, it causes an elevation of intracellular pH. This additional biologically active saccharide component of EJ must bind to a CRD carried by a sperm receptor protein (Hirohashi and Vacquier 2002b).
Adaptive Evolution at Several Levels of the Fertilization Cascade
Sperm-egg interaction proceeds through a series of steps that are common to most animals (Vacquier 1998). Sperm may be chemotactically attracted to the egg. Depending on the species, sperm binds to the egg envelope either before or after induction of the AR. After exocytosis of the acrosomal contents, a hole is created in the egg envelope through which the sperm passes to fuse with the egg cell membrane. In different animal species, the genes mediating these steps have been shown to be subjected to positive selection. Before this study, acrosomal bindin was the only sea urchin fertilization protein known to be subjected to positive selection (Metz and Palumbi 1996). Here, we demonstrate positive selection acting on at least one gene (suREJ1) that is known to bind an egg carbohydrate (FSP), mediating AR induction. It will be important to study the pattern of evolution of all genes mediating the steps in the fertilization cascade to gain insight into the number of loci that may be involved in establishing prezygotic reproductive isolation (Coyne and Orr 2004).
Sexual Antagonism As a Reason for Positive Selection on suREJ CRDs
Our favored hypothesis to explain positive selection in the evolution of suREJCRDs combines sexual antagonism (Rice 1996; Gavrilets 2000; Civetta 2003) and sperm competition (Parker 1970; Birkhead 1996; Frank 2000). In sexual antagonism (as applied to this paper), sperm and egg have different "interests" in the efficiency of their interaction leading to fusion. In presenting this hypothesis, we must discuss the problem of more than one sperm fusing with the egg, the biochemistry of the egg jelly coat, the physical properties of the egg jelly coat, sperm competition, and positive selection on sperm suREJ proteins.
In sea urchins and mammals, if more than one sperm fuses with the egg, the result is pathological polyspermy, which arrests development. Eggs must evolve ways to decrease the probability of fusion with more than one sperm. In sea urchins, there is both a fast electrical block to prevent sperm fusion and a slow physical block that involves the elevation of a fertilization envelope to prevent sperm passage. As discussed below, the egg may also regulate sperm entry through molecular interactions involving gamete recognition proteins.
When sea urchin eggs are spawned into seawater, the egg jelly coat (EJC) swells to approximately one egg diameter and is dense enough to exclude colloidal particles of India ink. The EJC contains two types of carbohydrate molecules that are known to be involved in AR induction; one is FSP and the other is the polysialic acid. In S. purpuratus (Vacquier and Moy 1997; Alves et al. 1998) and S. droebachiensis (Vilela-Silva et al. 2002), FSP comes in two isoforms, each isoform being female specific and both isoforms having equal potency as AR inducers. (Roughly 64% of S. purpuratus females have one FSP isoform, 31% have another FSP isoform, and 5% have both FSP isoforms [Vacquier, unpublished data]). Of the four other species studied in this report, the structure of FSP is known for S. franciscanus and S. pallidus. Females of these two species synthesize only one, species-specific, type of FSP (Vilela-Silva et al. 2002). The polysialic acid molecule of the EJC must bind to a sperm-surface receptor. In the presence of FSP, it greatly potentiates the FSP-induced AR by raising sperm intracellular pH (Hirohashi and Vacquier 2002b). Thus, in S. purpuratus and S. droebachiensis, there are at least three types of carbohydrate polymers in the EJC that interact with sperm-membrane carbohydrate-recognition proteins (suREJ proteins) to regulate sperm ion channels. The EJC also contains at least 10 other glycoproteins of unknown function that could bind suREJ and other unknown sperm-surface receptors (Vacquier and Moy 1997).
The physical function of the hydrated EJC is to prevent eggs from sticking together and also to prevent microbes from binding to the egg surface. If the EJC is removed from S. franciscanus eggs, the eggs irreversibly clump together and abnormal development occurs (Vacquier, unpublished data). However, this is not true of S. purpuratus eggs. In some species of free-spawning echinoderms, the hydrated EJC increases the effective diameter of the egg, creating a larger target for the sperm to hit. Thus, in some species, the EJC increases the rate of fertilization by being of greater diameter than the egg's cytoplasmic mass (Levitan and Irvine 2001; Podolsky 2002). In S. purpuratus, eggs in which the bulk of the EJC has been removed bind more sperm and are more susceptible to higher levels of polyspermy than eggs with intact EJC. In these "dejellied" eggs, the optimal amount of EJ needed to induce the sperm AR remains tightly bound to the egg surface (Vacquier, Brandriff, and Glabe 1979).
From the egg's side of the sexual antagonism theory, the hydrated EJC provides three positive things for the egg: it protects the egg from physical abuse and microbes, it holds an excess of molecules needed to induce the sperm AR, and it slows sperm as they approach the egg surface, by inducing premature AR, which decreases the frequency of polyspermy (Vacquier, Brandriff, and Glabe 1979; Frank 2000). Premature induction of the AR decreases sperm fertilizability with a half-life of 23 seconds (Vacquier 1979). The EJC slowing the frequency of sperm fusion is of positive benefit to the egg, but is a negative benefit for sperm, supporting the hypothesis of sexual antagonism acting during sperm-egg interaction.
Competition among individual sperm cells will continuously select for sperm that most efficiently fuse with eggs (Frank 2000). One ml of undiluted S. purpuratus semen contains 40 billion sperm cells, and one large male can spawn 5 ml of semen (Vacquier 1986). Thus, the large numbers of sperm available enhance the probability that favorable mutants (in suREJ proteins) will enjoy increased fertilization success. The egg must evolve ways to retard these potentially supersuccessful sperm and optimize the chance for monospermy.
Finally, the suREJ protein variants that optimize sperm fusion rates with eggs could be selected for on the basis of changes in their CRDs. Theory suggests that positive selection should be found in both the sperm protein and its cognate egg-surface receptor (Gavrilets 2000). Such is the case with abalone sperm lysin and its egg receptor VERL, where both cognate binding partners are subjected to positive selection (Galindo, Vacquier, and Swanson 2003b). The only molecules we know of in sea urchin EJ that induce the AR in seawater at pH 8.0 are the carbohydrate polymers of FSP and polysialic acid. Although we do not yet know how to detect positive selection in a carbohydrate polymer, it could be that the enzymes involved in the biosynthesis of these EJ molecules might show positive selection.
Avoiding polyspermy is a multistep process for the sea urchin egg. The first level of blockade could be the interaction of sperm suREJ proteins with egg FSP and polysialic acid to induce premature AR and retard the rate of sperm reaching the egg surface. The egg would be selecting for a sperm that undergoes the AR at exactly the correct distance from the egg surface (cryptic female choice). The second level of polyspermy blockade is the well-known electrical block, where, after fusion with the first sperm, the egg membrane potential changes in 100 milliseconds from –70 mV to +20 mV, which prevents further sperm fusion by an unknown mechanism (Gould-Somero and Jaffe 1984). The third polyspermy blockade is the complete block provided by the fertilization protease and the formation of the fertilization envelope (Vacquier, Tegner, and Epel 1973; Haley and Wessel 2004).
The demonstration of positive selection in the CRDs of suREJ proteins shows once again that the evolution of the molecules and mechanisms involved in sperm-egg interaction is extremely complex yet always fascinating.
Acknowledgements
We thank Gary W. Moy and Anna T. Neill for helpful advice. This work was supported by an NSF Minority Graduate Fellowship to S.A.M., NIH Grants HD12986 to V.D.V., and HD42563 to W.J.S.
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