Victor D. Vacquier

The rapid evolution of reproductive proteins

Many genes that mediate sexual reproduction, such as those involved in gamete recognition, diverge rapidly, often as a result of adaptive evolution. This widespread phenomenon might have important consequences, such as the establishment of barriers to fertilization that might lead to speciation. Sequence comparisons and functional studies are beginning to show the extent to which the rapid divergence of reproductive proteins is involved in the speciation process.

Protease released from sea urchin eggs at fertilization alters the vitelline layer and aids in preventing polyspermy

Abstract Sea urchin eggs release protease activity extracellularly from 30 to about 60 sec after insemination. This timing corresponds to the breakdown of the cortical granules. The evidence presented shows that the protease prevents polyspermy through its action on the vitelline layer. The protease ‘hardens’ the layer and makes it incapable of binding sperm. Sperm that are bound to the vitelline layer before cortical granule breakdown lose their attachment to the layer as the protease is excreted. By digesting the bonds between supernumerary sperm and the vitelline layer and by ‘hardening’ the layer the protease aids in establishing the block against polyspermy.

Exploring the Phylogenetic Utility of ITS Sequences for Animals: A Test Case for Abalone (Haliotis)

To evaluate the general utility of sequences of the nuclear rDNA internal transcribed spacer (ITS) regions for phylogenetic analyses of animal species groups and their broader relationships, sequences were obtained for 19 species of the genus Haliotis plus a keyhole limpet and a more distantly related gastropod, the Chilean abalone. Three subclades of Haliotis species appear consistently, each encompassing little variation. They are (A) the North Pacific species, (B) the European species, and (C) the Australia species. The one Caribbean species examined clearly groups with the North Pacific clade, not the European clade. H. midae (South Africa) and H. diversicolor supertexta (Taiwan) both diverge basal to the European and Australian species groups in the phylogenetic trees. Sequence comparisons showed that one species of Haliotis, H. iris from New Zealand, is quite distant from the remaining Haliotis species, almost as much as the more obvious outgroup, the keyhole limpet, an observation common to other DNA sequence analyses of these taxa. Using the rate of nucleotide change calculated from the sister Caribbean-Pacific pair, the length of the H. iris long branch is compatible with the suggestion that its ancestry became isolated on New Zealand at Gondwandan breakup. Use of ITS permits a totally independent estimate of the phylogenetic relationships, yet branching order was very similar to that established using other DNA regions studied previously, including those under strong positive selection. Knowledge of the RNA transcript secondary structure is particularly useful in the optimal alignment of more distantly related taxa. The RNA transcript secondary structure of Haliotis ITS2 shows conservation of features found also in ITS2 of angiosperms and algal taxa. Since ITS, particularly ITS2, is not saturated with nucleotide changes even at the family level, it should be useful for phylogenetic reconstruction of animal groups, not just at the species and genus levels but perhaps also for families and above.

Positive selection in the egg receptor for abalone sperm lysin

The mechanism of speciation is a central problem in evolutionary biology. In free-spawning animals with no complex mating behavior, prezygotic reproductive isolation (speciation) could result from the rapid divergence of genes coding for sperm and egg proteins that bind each other during fertilization. In abalone, sperm lysin evolves rapidly by positive Darwinian selection. The egg vitelline envelope receptor for lysin had previously been shown to evolve neutrally and be subjected to concerted evolution. Several mathematical simulations predict that both male and female reproductive proteins should evolve rapidly by positive selection. Here we report that the sequence diversity of the amino-terminal end of the egg vitelline envelope receptor for lysin has been promoted by positive Darwinian selection. These data provide molecular support for theoretical models showing that the two sexes are locked in a “coevolutionary chase” that could be driven by processes such as sexual selection, sexual conflict, or microbial attack (pathogen avoidance). The result of this continuous coevolution of the gamete recognition system could be the splitting of one population into two that are reproductively isolated (speciation).

Monoclonal antibody to a membrane glycoprotein inhibits the acrosome reaction and associated Ca2+ and H+ fluxes of sea urchin sperm

Monoclonal antibodies were prepared using sea urchin (Strongylocentrotus purpuratus) sperm or isolated sperm plasma membrane vesicles as immunogen. Two monoclonal antibodies, J4/4 and J10/14, react with different epitopes on external domains of the same Mr = 210,000 integral-membrane glycoprotein. At Fab fragment concentrations of equivalent cell surface binding, J10/14 inhibits the egg jelly-induced exocytotic acrosome reaction and associated Ca2+ influx and H+ efflux, whereas J4/4 does not. Immunofluorescent localization shows that both monoclonal antibodies react with a narrow collar of plasma membrane over the acrosomal complex and also with the entire flagellum.

Species specific agglutination of eggs by bindin isolated from sea urchin sperm

SPERMATOZOA adhere to eggs during sea urchin fertilisation1,2. The adhesion is between the acrosome process of the sperm and the vitelline layer covering of the egg3. During the sperm acrosome reaction, the membrane of the extending acrosome process becomes coated with protein derived from the sperm acrosome vesicle3,6. We believe4,5, like others3,6, that the acrosome vesicle protein binds the sperm to the egg by interacting with glycoprotein receptors on the egg vitelline layer4,5,7,8. We have isolated the insoluble contents of the acrosome vesicle and have shown it to be composed of a single protein of molecular weight 30,500 which we have named bindin4. In most interspecies inseminations sea urchin sperm fail to adhere to the vitelline layers of foreign eggs and fertilisation does not occur, even though sperm undergo the acrosome reaction in the presence of the foreign eggs9. Presumably this failure of gamete adhesion occurs because bindin, on the sperm acrosome process, does not bind to the glycoprotein receptors on the vitelline layers of eggs of another species. We report here the species specific agglutination of unfertilised eggs by isolated bindin. This demonstrates the preservation of the species specific recognition event between the isolated protein and its receptor which should facilitate the in vitro reconstruction and analysis of the molecular mechanism of gamete adhesion.

A protein from abalone sperm dissolves the egg vitelline layer by a nonenzymatic mechanism

Unfertilized abalone eggs (Haliotis rufescens) possess an elevated fibrous glycoproteinaceous vitelline layer (VL) about 0.6 μm in thickness. Sperm bind to the VL by the tip of a large unreacted acrosome granule. After binding, the tip of the granule opens and the soluble contents are released onto the VL. A hole about 3 μm in diameter then forms in the VL in the area of the discharging acrosome. Ultrastructural observations show the hole to be filled with attenuated VL fibers. The sperm then swims through the hole and interacts with the egg plasma membrane. The soluble contents of abalone acrosomes can be obtained by induction of the acrosome reaction in high-calcium seawater. Two major proteins of subunit molecular weights 13,000 (13K) and 15,000 (15K) are found in the supernatant after removal of the reacted sperm by centrifugation. Gel analysis of whole sperm shows these two proteins are the major components of the cell. The 13K protein can be purified on the basis of its solubility at lower ionic strength. This protein is a potent solubilizer (lysin) of egg vitelline layers. Characterization of the 13K lysin yields an isoelectric point of about 9, basic amino acids accounting for 19.6% of its weight, a negative PAS reaction, a nondenatured-molecular-weight estimate of 17,000, the presence of exposed hydrophobic regions, and a lack of enzyme activity. The lytic action of the 13K protein is rapidly inactivated by boiling, showing that the native conformation is necessary for activity. The lysin does not degrade the macromolecular components of the VL. It does not produce reducing sugars, peptides, lysophosphatides, or SH groups. A turbidometric assay for lysin activity was developed using isolated VLs and 13K lysin. When lysin is added to VLs in seawater the dissolution action occurs for only 15–30 sec before abruptly stopping. Mixing various amounts of lysin with a constant amount of VLs shows that the lysin dissolves VLs by a stoichiometric, noncatalytic (nonenzymatic) mechanism. For example, about 11 μg of lysin are required for the complete dissolution of 63 μg of VL protein (the VL is 36% protein). An identical conclusion was reached by K. Haino-Fukushima (1974, Biochim. Biophys. Acta, 352, 179–191) working with an 8.8K lysin of another archeogastropod, Tegula pfeifferi. Isolated abalone VLs are composed of about five major glycoproteins ranging in molecular weight from 32 to 44K. High ionic strength such as 2 M KCl does not solubilize VLs, but agents which destroy hydrophobic bonds between macromolecules, such as NaSCN, dimethylsulfoxide, and heat, are VL solubilizers. Exposed hydrophobic portions of the lysin might bind to the hydrophobic regions of VL glycoproteins and competitively dissociate the VL fibers from each other, thus, destroying the VLs structural integrity. Stoichiometric mechanisms for making holes in egg investments may be more biologically attractive than enzymatic mechanisms. A stoichiometric reaction would be quickly self-limiting and nondegradative to other cell surface components.

The divergence of species-specific abalone sperm lysins is promoted by positive Darwinian selection

Recognition by sperm lysin of the egg vitelline envelope may be one component in determining the species-specificity of fertilization in abalones. The amino acid sequences of lysin proteins of seven California abalone species were deduced from the cDNA sequences. This is the first extensive comparison of a gamete recognition protein from congeneric species. Each prelysin has a highly conserved signal peptide of 18 amino acids, followed by a mature sequence of 136-138 residues. Of 136 aligned positions, 68 have the same amino acid in all seven sequences. The % identity relative to the red abalone lysin sequence is: white 90%, flat 83%, pinto 82%, pink 78%, black 71%, and green 65%. Hydropathy plots and a distance tree of the seven lysins show that red, white, and flat lysins are more closely related to each other than to the lysins of the other four species. A hypervariable, species-specific, domain exists in all sequences between positions 2-12. Amino acid replacements between any two lysins are mostly nonconservative. Analysis of the cDNA sequences shows the number of nonsynonymous substitutions (amino acid altering) exceeds the number of synonymous substitutions (silent) in 20 of the 21 pairwise comparisons of the seven sequences, indicating that positive Darwinian selection must promote the divergence of lysin sequences.

Positive darwinian selection on two homologous fertilization proteins: what is the selective pressure driving their divergence?

Most examples of positive selection inferred from nucleotide sequence data involve host-pathogen interactions. However, positive selection also promotes the divergence of proteins mediating spermegg recognition in marine invertebrates. The abalone spermatozoon has a large acrosomal vesicle containing two proteins of 16 kDa and 18 kDa. Lysin, the 16-kDa protein, exhibits species-specificity in dissolving a hole in the egg vitelline envelope through which the sperm swims to reach the egg plasma membrane. The 18-kDa protein coats the sperm acrosomal process and probably mediates fusion of the two gametes. In this review, we compare sequences of both proteins from five species of California abalones. Both proteins show extensive divergence which has been promoted by positive Darwinian selection. The ratios of nonsynonymous to synonymous nucleotide substitutions may be the highest yet discovered for full-length sequences. Although extensive divergence has occurred, there is conservation of the shape and polarity of residues in both proteins. The two acrosomal proteins arose by a gene duplication followed by their extensive divergence. Five hypotheses are presented which attempt to explain the nature of the unknown selective force responsible for the robust positive selection. The positive selection may, in some unknown way, be related to the establishment of prezygotic barriers to reproduction. Because positive selection promotes the divergence of unrelated, species-specific gamete recognition proteins in both abalones and sea urchins, we predict that positive selection may be a general phenomenon in the evolution of gamete recognition systems in marine invertebrates.

Evolution and systematics in Haliotidae (Mollusca: Gastropoda): inferences from DNA sequences of sperm lysin

Abalone taxonomy and systematics have remained unresolved: neither stable species-level nomenclature nor a cladistic hypothesis of relationships among species have been established. To infer the phylogeny of the genus Haliotis and to identify species using molecular data, we compared complementary DNA (cDNA) sequences of sperm lysin from 27 species-group taxa from California, Japan, Australia, New Zealand, Taiwan, Borneo, Madagascar, South Africa, Greece, France, Italy and the Azores. The lysin cDNA sequences reveal that 22 of the 27 taxa are clearly distinguishable by >20 nucleotide differences. Of the remaining 5, H. coccinea from the Azores may be a subspecies of H. tuberculata, if not a sibling species (10 nucleotide differences). The other four taxa are most probably the same species as one of the 22 taxa: the lysin sequences are almost identical between H. madaka and H. discus hannai, H. conicopora and H. rubra, H. diversicolor supertexta and H. diversicolor aquatilis, and H. tuberculata lamellosa and H. tuberculata tuberculata. The phylogeny of lysin cDNA suggests that there are three groups among the 27 species-group taxa: (1) all California species and 3 Japanese species (H. gigantea, H. discus hannai, and H. madaka): (2) 1 New Zealand species (H. iris); (3) 1 Japanese species (H. diversicolor aquatilis), Indo-West Pacific species and European species. These groups can be assigned to three previously recognized subgenera (Nordotis, Paua and Padollus) in the genus Haliotis. Two historical hypotheses are proposed to explain the biogeography and evolution within these abalone: (1) Tethyan distribution of the ancestral abalone, during the Cretaceous, followed by extinction in most of the habitat, but radiation in California and Southeast Asia which later spread to the other areas; (2) North Pacific rim distribution of the ancestral abalone, followed by dispersal to the other areas during the Paleogene.