Comparison of the bindin proteins of Strongylocentrotus franciscanus, S. purpuratus, and Lytechinus variegatus: sequences involved in the species specificity of fertilization.

Bindin is the sea urchin sperm acrosomal protein that is responsible for the species-specific adhesion of the sperm to the egg. Two new bindin cDNA sequences that contain the entire open reading frame for the binding precursor are reported: one for Strongylocentrotus franciscanus and one for Lytechinus variegatus. Both contain inverted repetitive sequences in their 3' untranslated regions, and the S. franciscanus cDNA contains an inverted repetitive sequence match between the 5' untranslated region and the coding region. The middle third of the mature bindin sequence is highly conserved in all three species, and the flanking sequences share short repeated sequences that vary in number between the species. Cross-fertilization data are reported for the species S. purpuratus, S. franciscanus, L. variegatus, and L. pictus. A barrier to cross-fertilization exists between the sympatric Strongylocentrotus species, but there is no barrier between the allopatric Lytechinus species.     

The evolution of sea urchin sperm bindin

Sea urchins have been model organisms for the study of fertilization for more than a century. Fertilization in sea urchins happens externally, which facilitates the study of sperm-egg attachment and fusion, and means that all of the molecules involved in gamete recognition and fusion are associated with the gametes. Sea urchin sperm bindin was the first "gamete recognition protein" to be isolated and characterized (Vacquier and Moy 1977), and bindin has since been studied by developmental biologists interested in fertilization, by biochemists interested in membrane fusion and by evolutionary biologists interested in reproductive isolation and speciation. Research on bindin was last reviewed thirteen years ago by Vacquier et al. (1995) in an article titled "What have we learned about sea urchin sperm bindin?" in which the authors reviewed the identification, isolation and early molecular examinations of bindin. Research since then has focused on bindin's potential role in fusing egg and sperm membranes, comparisons of bindin between distantly related species, studies within genera linking bindin evolution to reproductive isolation, and studies within species looking at fertilization effects of individual bindin alleles. In addition, the egg receptor for bindin has been cloned and sequenced. I review this recent research here.     

Low rates of bindin codon evolution in lecithotrophic Heliocidaris sea urchins

Life-history variables including egg size affect the evolutionary response to sexual selection in broadcast-spawning sea urchins and other marine animals. Such responses include high or low rates of codon evolution at gamete recognition loci that encode sperm- and egg-surface peptides. Strong positive selection on such loci affects intraspecific mating success and interspecific reproductive divergence (and may play a role in speciation). Here, we analyze adaptive codon evolution in the sperm acrosomal protein bindin from a brooding sea urchin (Heliocidaris bajulus, with large eggs and nonfeeding or lecithotrophic larval development) and compare our results to previously published data for two closely related congeners. Purifying selection and low relative rates of bindin nonsynonymous substitution in H. bajulus were significantly different from selectively neutral bindin evolution in H. erythrogramma despite similar large egg size in those two species, but were similar to the background rate of nonsynonymous bindin substitution for other closely related sea urchins (including H. tuberculata, all with small egg size and feeding planktonic larval development). Bindin evolution is not driven by egg size variation among Heliocidaris species, but may be more consistent with an alternative mechanism based on the effects of high or low spatial density of conspecific mates.     

Evolution of bindin in the pantropical sea urchin Tripneustes: comparisons to bindin of other genera

Bindin, a sea urchin sperm protein, mediates sperm-egg attachment and membrane fusion and is thus important in species recognition and speciation. Patterns of bindin variation differed among three genera that had been studied previously. In two genera of the superorder Camarodonta, Echinometra and Strongylocentrotus, both of which contain sympatric species, bindin is highly variable within and between species; a region of the molecule evolves at high rates under strong positive selection. In Arbacia, which belongs to the superorder Stirodonta and whose extant species are all allopatric, bindin variation is low, and there is no evidence of positive selection. We cloned and sequenced bindin from Tripneustes, a sea urchin that belongs to the Camarodonta but whose three species are found in different oceans. Worldwide sampling of bindin alleles shows that the bindin of Tripneustes (1) contains the highly conserved core characteristic of all other bindins characterized to date, (2) has an intron in the same position, and (3) has approximately the same length. Its structure is more like that of bindin from other camarodont sea urchins than to bindin from the stirodont ARBACIA: The resemblances to other camarodonts include a glycine-rich repeat structure upstream of the core and lack of a hydrophobic domain 3' of the core, a characteristic of Arbacia bindin. Yet the mode of evolution of Tripneustes bindin is more like that of Arbacia. Differences between bindins of the Caribbean Tripneustes ventricosus and the eastern Pacific T. depressus, separated for 3 my by the Isthmus of Panama, are limited to four amino acid changes and a single indel. There are no fixed amino acid differences or indels between T. depressus from the eastern Pacific and T. gratilla from the Indo-Pacific. Bindin of Tripneustes, like that of Arbacia, also shows no evidence of diversifying selection that would manifest itself in a higher proportion of amino acid replacements than of silent nucleotide substitutions. When the rate of intrageneric bindin divergence is standardized by dividing it by cytochrome oxidase I (COI) divergence, Tripneustes and Arbacia show a lower ratio of bindin to COI substitutions between the species of each genus than exists between the species of either Echinometra or Strongylocentrotus. Thus, mode of bindin evolution is not correlated with phylogenetic affinities or molecular structure, but rather with whether the species in a genus are allopatric or sympatric. For a molecule involved in gametic recognition, this would suggest a pattern of evolution via reinforcement. However, in bindin the process that gave rise to this pattern is not likely to have been selection to avoid hybridization, because there is no excess of amino acid replacements between species versus within species in the bindins of Echinometra and Strongylocentrotus, as would have been expected if specific recognition were the driving force in their evolution. We suggest instead that the pattern of reinforcement is a secondary effect of the ability of species with rapidly evolving bindins to coexist in sympatry.     

Molecular cloning of the ets proto-oncogene of the sea urchin and analysis of its developmental expression

The locus SU(Lv)-ets-2 of the sea urchin Lytechinas variegatus related to the oncogene v-ets of avian erythroblastosis virus E26 has been molecularly cloned. The cloned DNA was found to contain a region with a high degree of homology to E26 v-ets. The sea urchin homology with v-ets starts at a consensus splice acceptor sequence and stops at the point where homology between v-ets and human c-ets ends. This region corresponds to the Hu-ets-2 homologous sequences defined by Watson et al. (1985, Proc. Natl. Acad. Sci, USA 82, 7294-7298). Ninety-one out of 97 (or 94%) predicted amino acids are identical between sea urchin c-ets and E26 v-ets over the region of homology. This degree of homology exceeds the maximum homology previously found between any oncogene and an invertebrate homolog. A somewhat weaker homology with the Hu-ets-2 sequences continues beyond, for 13 codons, ending at a common termination codon. Northern blot analysis of mature unfertilized eggs and early embryos from sea urchins of the species Strongylocentrotus purpuratus revealed a single 6.8-kb ets-related RNA that is expressed at a maximum level during the early stages of embryonic development. This RNA species is polyadenylated indicating that it is the message for the sea urchin ets-2 gene.     

Horseradish peroxidase. XXV. An electron spin resonance study of the low temperature photochemical reaction of compound I.

The insoluble acrosome granule content of sea urchin sperm consists of a single 30,500 dalton protein named bindin. Bindin mediates species-specific recognition and adhesion of sperm to the egg surface. Bindin from $\text{Strongylocentrotus purpuratus}$ (Sp) and $\text{Strongylocentrotus franciscanus}$ (Sf) have tyrosine as their single N-terminal amino acid. The pI of Sp bindin is 6.62 and of Sf 6.59. Amino acid analysis reveals almost identical composition between the two species for 16 amino acids. Only two (or three) amino acids, Pro and Asx, show large species differences. Tryptic peptide maps of the two species of bindin show very similar patterns with 24 spots of identical correspondence.     

250 million years of bindin evolution

Bindin plays a central role in sperm-egg attachment and fusion in sea urchins (echinoids). Previous studies determined the DNA sequence of bindin in two orders of the class Echinoidea, representing 10% of all echinoid species. We report sequences of mature bindin from five additional genera, representing four new orders, including the distantly related sand dollars, heart urchins, and pencil urchins. The six orders in which bindin is now known include 70% of all echinoids, and indicate that bindin was present in the common ancestor of all extant sea urchins more than 250 million years ago. Over this span of evolutionary time there has been (1). remarkable conservation in the core region of bindin, particularly in a stretch of 29 amino acids that has not changed at all; (2). conservation of a motif of basic amino acids at the cleavage site between preprobindin and mature bindin; (3). more than a twofold change in length of mature bindin; and (4). emergence of high variation in the sequences outside the core, including the insertion of glycine-rich repeats in the bindins of some orders, but not others.     

Phylogeography and bindin evolution in Arbacia, a sea urchin genus with an unusual distribution

Among shallow water sea urchin genera, Arbacia is the only genus that contains species found in both high and low latitudes. In order to determine the geographical origin of the genus and its history of speciation events, we constructed phylogenies based on cytochrome oxidase I and sperm bindin from all its species. Both the mitochondrial and the nuclear gene genealogies show that Arbacia originated in the temperate zone of the Southern Hemisphere and gave rise to three species in the eastern Pacific, which were then isolated from the Atlantic by the Isthmus of Panama. The mid-Atlantic barrier separated two additional species. The bindin data suggest that selection against hybridization is not important in the evolution of this molecule in this genus. Metz et al. in a previous publication found no evidence of selection on bindin of Arbacia and suggested that this might be due to allopatry between species, which obviated the need for species recognition. This suggestion formed the basis of the conclusion, widely spread in the literature, that the source of selection on sea urchin bindin (where it does occur) was reinforcement. However, the range of Arbacia spatuligera overlaps with that of two other species of Arbacia, and our data show that it is hybridizing with one of them. We found that even in the species that overlap geographically, there are no deviations from selective neutrality in the evolution of bindin.     

A regulatory sequence near the 3'' end of sea urchin histone genes.

The 3' flanking sequences of all five histone genes have been sequenced in the histone DNA clone h19 of the sea urchin Psammechinus miliaris. A large (23 bp) and a small (10 bp) conserved sequence was found by sequence comparison, some 29-40 bp downstream from the termination codon. 12 bases of the larger homology block show a dyad symmetry. The available sequences of clone h22 of the same species and those of the histone clones pSp2 and pSp17 of Strongylocentrotus purpuratus, another sea urchin species, fit well into this comparison. Two types of sequences are involved in the dyad symmetry; one is H1, H3 and H4 specific, the other is H2A and H2B specific. If these conserved sequences are transcribed, a hairpin loop could form in the RNA molecules. This secondary structure might serve as a recognition signal for a regulatory protein.     

Genetic structure of populations of the red sea urchin, Strongylocentrotus franciscanus

Population subdivision was evaluated in the red sea urchin, Strongylocentrotus franciscanus, using DNA sequence data from 134 adult individuals collected in 1995 and 1996. On average 22 individuals were sequenced from six geographic locations between Alaska and Baja California (N=134), nearly the full extent of the species range. DNA sequence data was obtained from direct sequencing of a 273 base pair region of the bindin gene, which encodes a sperm fertilization protein. Results indicate that bindin is sufficiently polymorphic to serve as a genetic marker. We identified 14 unique alleles present in the entire range sampled with a maximum of eight alleles at a specific site. Mean pairwise comparison of the 14 unique alleles indicates moderate sequence diversity (p-distance=1.06). Although there is conflicting evidence to suggest that Alaska populations may deviate from the Hardy-Weinberg expectations, analysis of bindin genotype frequencies indicate that it is not possible to reject the null hypothesis of random mating throughout the species range. The results of a chi-square test with pooling conform to Hardy-Weinberg expectations for all populations (P>0.05) except for the Alaska population (P=0.037). Inbreeding coefficients are consistent with this result and suggest that for the bindin locus, there is high gene flow. These results are compared with previously published results of genetic substructuring in sea urchins to examine relationships among population structure, dispersal potential and biogeography.     

Reproductive character displacement and the genetics of gamete recognition in tropical sea urchins

Reproductive character displacement occurs when sympatric and allopatric populations of a species differ in traits crucial to reproduction, and it is commonly thought of as a signal of selection acting to limit hybridization. Most documented cases of reproductive character displacement involve characters that are poorly understood at the genetic level, and rejecting alternative hypotheses for biogeographic shifts in reproductive traits is often very difficult. In sea urchins, the gamete recognition protein bindin evolves under positive selection when species are broadly sympatric, suggesting character displacement may be operating in this system. We sampled sympatric and allopatric populations of two species in the sea urchin genus Echinometra for variation in bindin and for the mitochondrial cytochrome oxidase I to examine patterns of population differentiation and molecular evolution at a reproductive gene. We found a major shift in bindin alleles between central Pacific (allopatric) and western Pacific (sympatric) populations of E. oblonga. Allopatric populations of E. oblonga are polyphyletic with E. sp. C at bindin, whereas sympatric populations of the two species are reciprocally monophyletic. There is a strong signal of positive selection (P(N)/P(S) = 4.5) in the variable region of the first exon of bindin, which is associated with alleles found in sympatric populations of E. oblonga. These results indicate that there is a strong pattern of reproductive character displacement between E. oblonga and E. sp. C and that the divergence is driven by selection. There is much higher population structure in sympatric populations at the bindin locus than at the neutral mitochondrial locus, but this difference is not seen in allopatric populations. These data suggest a pattern of speciation driven by selection for local gamete coevolution as a result of interactions between sympatric species. Although this pattern is highly suggestive of speciation by reinforcement, further research into hybrid fitness and egg-sperm interactions is required to address this potential mechanism for character displacement.     

Characterization of the Sperm Molecule Bindin in the Sea Urchin Genus <Emphasis Type="Italic">Paracentrotus</Emphasis>

Bindin is a sea urchin gamete-recognition protein that plays an essential role in the specificity of egg–sperm interactions and thus may be evolving under sexual selection and be related to speciation. Bindin has been found to evolve under strong selection in some sea urchin genera and neutrally in others. In this study, we characterized bindin in the two extant species of the genus Paracentrotus: P. lividus from the Atlanto-Mediterranean region and P. gaimardi from Brazil. The structure of the bindin molecule in Paracentrotus is similar to that of other genera studied thus far, consisting of a conserved core flanked by two variable regions and an intron of variable length located at the same conserved position as in other genera. Polymorphism in P. lividus is caused mainly by point substitutions and insertions/deletions, and length variations are caused mainly by the number of repeated motifs in the flanking regions. There is no evidence of recombination. Positive selection is acting on amino acid sites located in two regions flanking the conserved core.     

The interaction of boar sperm proacrosin with its natural substrate, the zona pellucida, and with polysulfated polysaccharides

Boar sperm acrosin is an acrosomal protease with trypsin-like specificity, and it functions in fertilization by assisting sperm passage through the zona pellucida by limited hydrolysis of this extracellular matrix. In addition to a proteolytic active site domain, acrosin binds the zona pellucida at a separate binding domain that is lost during proacrosin autolysis. In this study, we quantitate the binding of proacrosin to the physiological substrate for acrosin, the zona pellucida, and to a non-substrate, the polysulfated polysaccharide fucoidan. Binding was analogous to sea urchin sperm bindin that binds egg jelly fucan and the vitelline envelope of sea urchin eggs. Proacrosin was found to bind to fucoidan and to the zona pellucida with binding affinities similar to bindin interaction with egg jelly fucan. These interactions were competitively inhibited by similar relative molecular mass polysulfated polymers. Since bindin and proacrosin have distinctly different amino acid sequences, their interaction with acidic sulfate esters demonstrates an example of convergent evolution wherein different macromolecules localized in analogous sperm compartments have the same biological function. From cDNA sequence analysis of proacrosin, this binding may be mediated through a consensus sequence for binding sulfated glycoconjugates. Proacrosin binding to the zona pellucida may serve as both a recognition or primary sperm receptor, as well as maintaining the sperm on the zona pellucida once the acrosome reaction has occurred.     

The evolution of gametic compatibility and compatibility groups in the sea urchin Mesocentrotus franciscanus: An avenue for speciation in the sea

The generation of reproductive incompatibility between groups requires a rare genotype with low compatibility to increase in frequency. We tested the hypothesis that sexual conflict driven by the risk of polyspermy can generate compatibility groups in gamete recognition proteins (GRPs) in the sea urchin Mesocentrotus franciscanus. We examined variation in the sperm (bindin) and egg (EBR1) GRPs, how this variation influences fertilization success and how allele frequencies shift in these GRPs over time. The EBR1 gene is a large, 4595 amino acid protein made up of 27 thrombospondin type 1 domain (TSP) and 20 C1s/C1r, uEGF and bone morphogenic protein subdomain (CUB) repeats. Two TSP and two CUB repeats each demonstrate two common non‐synonymous haplotypes (alleles). Sperm bindin and one of these EBR1 repeats (TSP8) shift allele frequencies from one common to two common types over an approximate 200 year interval associated with the removal of predatory sea otters and rising sea urchin abundances; the egg receptor shifts first, followed by the sperm ligand. Laboratory crosses indicate that the historically common sperm and egg gamete recognition proteins have high compatibility as do the new common proteins, with mismatches having lower compatibility. This process of creating compatibility groups sets the stage for reproductive isolation and speciation.     

The sequence of the Arbacia punctulata bindin cDNA and implications for the structural basis of species-specific sperm adhesion and fertilization

Bindin is the major protein component of the acrosome granule of sea urchin sperm which mediates the species-specific adhesion of sperm to the egg surface during fertilization. Bindin isolated from both Arbacia punctulata and Strongylocentrotus purpuratus sperm demonstrate a distinct adhesive preference for eggs of the same species although a significant amount of cross-species reactivity is observed. Here we describe the isolation and sequence of A. punctulata bindin cDNA clones and a comparison of the predicted protein sequence with the sequence previously reported for S. purpuratus bindin (Gao et al., 1986, Proc. Natl. Acad. Sci., USA 83, 8634-8638). Bindins from these genera show substantial sequence similarity in both the mature bindin domain and the probindin precursor region. The most striking identity is a region of 42 conserved amino acids in the central part of the mature bindins. This conserved domain may be responsible for conserved functions of bindin. Regions flanking this conserved element on both the amino and carboxyl side are more highly divergent, suggesting that they are responsible for the species-specific properties of bindin. The mature A. punctulata sequence contains a putative transmembrane segment between residues 431 and 451 that is absent from S. purpuratus bindin. This structural element may account for the previous observation that isolated A. punctulata bindin uniquely forms multilamellar structures reminiscent of lipid bilayers and binds significant amounts of phospholipid and detergent. The structure of this hydrophobic segment also displays a number of similarities to viral fusion peptides.     

A conserved region in the sea urchin U1 snRNA promoter interacts with a developmentally regulated factor.

The expression of the sea urchin L. variegatus U1 snRNA gene is temporally regulated during embryogenesis. Using a microinjection assay we show that a region between 203 and 345 nts 5' of the gene is required for expression. There are four conserved regions between two sea urchin species in the 345 nts 5' to the U1 gene. One region, located at about -300, binds a protein factor which is present in blastula but not gastrula nuclei. Three other potential protein binding sites within the first 200 nts 5' to the gene have been identified using a mobility shift assay and/or DNase I footprinting. Two of these regions bind factors which are not developmentally regulated and one binds a factor which is developmentally regulated. It is likely that the factor which binds at -300 is involved in expression and developmental regulation of the sea urchin U1 snRNA gene.