Positive selection and sequence rearrangements generate extensive polymorphism in the gamete recognition protein bindin

Bindin is a gamete recognition protein of sea urchins that mediates species-specific attachment of sperm to an egg-surface receptor during fertilization. Sequences of bindin from closely related urchins show fixed species-specific differences. Within species, highly polymorphic bindin alleles result from point substitution, insertion/deletion, and recombination. Since speciation, positive selection favoring allelic variants has generated diversity in bindin polypeptides. Intraspecific bindin variation can be tolerated by the egg receptor, which suggests functional parallels between this system and other flexible recognition systems, including immune recognition. These results show that polymorphism in mate recognition loci required for rapid evolution of sexual isolation can arise within natural populations.      

The Interactions of Sea Urchin Gametes During Fertilization

The interactions between sea urchin spermatozoa and ova duringfertilization usually exhibit a high degree of species specificity.Under natural conditions and reasonable gamete concentrations,most interspecific inseminations fail to yield zygotes. Macromoleculeson the external surfaces of the apposing gametes must surelybe responsible for successful gamete recognition, adhesion andfusion. Species specific recognition between surface componentsof sperm and egg could occur during at least three events comprisingthe fertilization process. The first event is the interactionof the sperm plasma membrane with the egg jelly coat. This inducesthe sperm acrosome reaction resulting in the exocytosis of the"bindin" -containing acrosome granule and also the extrusionof the acrosome process from the anterior tip of the sperm.The second event is the adhesion of the bindin-coated acrosomeprocess to glycoprotein "bindin receptors" on the external surfaceof the egg vitelline layer. The third event is the penetrationof the vitelline layer and the fusion of sperm and egg plasmamembranes. With the isolations of the component of egg jellywhich induces the acrosome reaction, sperm bindin from the acrosomevesicle and the egg surface bindin receptor from the vitellinelayer, there is hope of discovering the molecular basis of thismost interesting intercellular interaction which results inthe activation of embryonic development.     

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.     

Speciation genes in free-spawning marine invertebrates

Research on speciation of marine organisms has lagged behind that of terrestrial ones, but the study of the evolution of molecules involved in the adhesion of gametes in free-spawning invertebrates is an exception. Here I review the function, species-specificity, and molecular variation of loci coding for bindin in sea urchins, lysin in abalone and their egg receptors, in an effort to assess the degree to which they contribute to the emergence of reproductive isolation during the speciation process. Bindin is a protein that mediates binding of the sperm to the vitelline envelope (VE) of the egg and the fusion of the gametes' membranes, whereas lysin is a protein involved only in binding to the VE. Both of these molecules are important in species recognition by the gametes, but they rarely constitute absolute blocks to interspecific hybridization. Intraspecific polymorphism is high in bindin, but low in lysin. Polymorphism in bindin is maintained by frequency-dependent selection due to sexual conflict arising from the danger of polyspermy under high densities of sperm. Monomorphism in lysin is the result of purifying selection arising from the need for species recognition. Interspecific divergence in lysin is due to strong positive selection, and the same is true for bindin of four out of seven genera of sea urchins studied to date. The differences between the sea urchin genera in the strength of selection can only partially be explained by the hypothesis of reinforcement. The egg receptor for lysin (VERL) is a glycoprotein with 22 repeats, 20 of which have evolved neutrally and homogenized by concerted evolution, whereas the first two repeats are under positive selection. Selection on lysin has been generated by the need to track changes in VERL, permitted by the redundant structure of this molecule. Both lysin and bindin are important in reproductive isolation, probably had a role in speciation, but it is hard to determine whether they meet the strictest criteria of "speciation loci," defined as genes whose differentiation has caused speciation.     

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.     

MORPHOLOGY OF GAMETE MEMBRANE FUSION AND OF SPERM ENTRY INTO OOCYTES OF THE SEA URCHIN

Sea urchin gametes predominate in molecular studies of fertilization, yet relatively little is known of the subcellular aspects of sperm entry in this group. Accordingly, it seemed desirable to make a detailed examination of sperm entry phenomena in sea urchins with the electron microscope. Gametes of the sea urchins Arbacia punctulata and Lytechinus variegatus were used in this study. Samples of eggs containing 2 to 8 per cent oocytes were selected and fixed with osmium tetroxide in sea water at various intervals after insemination. Fixed specimens were embedded in Epon 812, sectioned, and examined with an electron microscope. An apical vesicle was observed at the anterior end of the acrosome. The presence of this structure, together with other observations, suggested that initiation of the acrosome reaction in sea urchin sperm involves dehiscence of the acrosomal region with the subsequent release of the acrosomal granule. Contact and initial fusion of gamete membranes was observed in mature eggs and oocytes and invariably involved the extended acrosomal tubule of the spermatozoon. Only one spermatozoon normally enters the mature egg. The probability of locating such a sperm in ultrathin sections is exceedingly low. Several sperm do normally enter oocytes. Consequently, observations of sperm entry were primarily restricted to the latter. The manner of sperm entry into oocytes did not resemble phagocytosis. Organelles of the spermatozoon were progressively divested of their plasma membrane as they entered the ground cytoplasm of the oocyte fertilization cone. Initiation of the acrosome reaction, contact and initial fusion of gamete membranes, and sperm entry into oocytes of sea urchins conform to the Hydroides-Saccoglossus pattern of early fertilization events as described by Colwin and Colwin (13).     

Adaptive evolution of bindin in the genus Heliocidaris is correlated with the shift to direct development

Abstract Sea urchins are widely used to study both fertilization and development. In this study we combine the two fields to examine the evolution of reproductive isolation in the genus Heliocidaris . Heliocidaris tuberculata develops indirectly via a feeding larva, whereas the only other species in the genus, H. erythrogramma , has evolved direct development through a nonfeeding larva. We estimated the time of divergence between H. erythrogramma and H. tuberculata from mitochondrial DNA divergence, quantified levels of gametic compatibility between the two species in cross-fertilization assays, and examined the mode of evolution of the sperm protein bindin by sequencing multiple alleles of the two species. Bindin is the major component of the sea urchin sperm acrosomal vesicle, and is involved in sperm-egg attachment and fusion. Based on our analyses, we conclude that: the two species of Heliocidaris diverged less than five million years ago, indicating that direct development can evolve rapidly in sea urchins; since their divergence, the two species have become gametically incompatible; Heliocidaris bindin has evolved under positive selection; and this positive selection is concentrated on the branch leading to H. erythrogramma . Three hypotheses can explain the observed pattern of selection on bindin: (1) it is a correlated response to the evolution of direct development in H. erythrogramma; (2) it is the result of an intraspecific process acting in H. erythrogramma but not in H. tuberculata; or (3) it is the product of reinforcement on the species that invests more energy into each egg to avoid hybridization.     

Positive selection in the carbohydrate recognition domains of sea urchin sperm receptor for egg jelly (suREJ) proteins

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.     

Carbohydrate‐based species recognition in sea urchin fertilization: another avenue for speciation?

Spawning marine invertebrates are excellent models for studying fertilization and reproductive isolating mechanisms. To identify variation in the major steps in sea urchin gamete recognition, we studied sperm activation in three closely related sympatric Strongylocentrotus species. Sperm undergo acrosomal exocytosis upon contact with sulfated polysaccharides in the egg-jelly coat. This acrosome reaction exposes the protein bindin and is therefore a precondition for sperm binding to the egg. We found that sulfated carbohydrates from egg jelly induce the acrosome reaction species specifically in S. droebachiensis and S. pallidus. There appear to be no other significant barriers to interspecific fertilization between these two species. Other species pairs in the same genus acrosome react nonspecifically to egg jelly but exhibit species-specific sperm binding. We thus show that different cell-cell communication systems mediate mate recognition among very closely related species. By comparing sperm reactions to egg-jelly compounds from different species and genera, we identify the major structural feature of the polysaccharides required for the specific recognition by sperm: the position of the glycosidic bond of the sulfated alpha-L-fucans. We present here one of the few examples of highly specific pure-carbohydrate signal transduction. In this system, a structural change in a polysaccharide has far-reaching ecological and evolutionary consequences.     

The structure of sulfated polysaccharides ensures a carbohydrate-based mechanism for species recognition during sea urchin fertilization

The evolution of barriers to inter-specific hybridization is a crucial step in the fertilization of free spawning marine invertebrates. In sea urchins, molecular recognition between sperm and egg ensures species recognition. Here we review the sulfated polysaccharide-based mechanism of sperm-egg recognition in this model organism. The jelly surrounding sea urchin eggs is not a simple accessory structure; it is molecularly complex and intimately involved in gamete recognition. It contains sulfated polysaccharides, sialoglycans and peptides. The sulfated polysaccharides have unique structures, composed of repetitive units of alpha-L-fucose or alpha-L-galactose, which differ among species in the sulfation pattern and/or the position of the glycosidic linkage. The egg jelly sulfated polysaccharides show species-specificity in inducing the sperm acrosome reaction, which is regulated by the structure of the saccharide chain and its sulfation pattern. Other components of the egg jelly do not possess acrosome reaction inducing activity, but sialoglycans act in synergy with the sulfated polysaccharide, potentiating its activity. The system we describe establishes a new view of cell-cell interaction in the sea urchin model system. Here, structural changes in egg jelly polysaccharides modulate cell-cell recognition and species-specificity leading to exocytosis of the acrosome. Therefore, sulfated polysaccharides, in addition to their known functions as growth factors, coagulation factors and selectin binding partners, also function in fertilization. The differentiation of these molecules may play a role in sea urchin speciation.     

Latrunculin inhibits the microfilament-mediated processes during fertilization, cleavage and early development in sea urchins and mice

Latrunculin A, a marine toxin from a Red Sea sponge, is a potent inhibitor of the microfilament-mediated processes of fertilization and early development in sea urchins and in mice. Sperm from sea urchins, but not those from Limulus or mice, were affected by latrunculin, and fertilization in both sea urchins and in mice was arrested but at different stages. Sea urchin sperm treated with 2.6 microM latrunculin are unable to assemble acrosomal processes and their ability to fertilize eggs is impaired. The unwinding of the Limulus sperm acrosomal process occurs in the presence of latrunculin. Treated mouse sperm are able to fertilize mouse oocytes in vitro, suggesting that microfilaments may not be required in this mammalian sperm. In sea urchin eggs, sperm incorporation, microvillar elongation and cytokinesis are inhibited. Microtubule-mediated motility occurs normally. 20 nM latrunculin prevents the morphogenetic movements during gastrulation. It reduces the viscosity of actin gels from sea urchin egg homogenates. In unfertilized mouse oocytes, it prevents the colcemid-induced dispersion of the meiotic chromosomes; accumulations of cortical actin are noted adjacent to the scattered chromosomes. Sperm incorporation during mouse fertilization in vitro is unaffected suggesting that sperm entry may occur independent of microfilament activity in mammals. However, the apposition of the pronuclei at the center of the egg cytoplasm does not occur, providing evidence that cytoplasmic microfilaments may be required for the motions leading to pronuclear union during mouse fertilization. It inhibits the second polar body formation and cytokinesis. These results indicate that latrunculin is a potent inhibitor of microfilament-mediated processes in sperm, eggs and embryos, and that it may prove to be a powerful new drug for exploring the cellular behavior of microfilaments in the maintenance of cell shape and during motility.     

Motility and centrosomal organization during sea urchin and mouse fertilization

Motility and the behavior and inheritance of centrosomes are investigated during mouse and sea urchin fertilization. Sperm incorporation in sea urchins requires microfilament activity in both sperm and eggs as tested with Latrunculin A, a novel inhibitor of microfilament assembly. In contrast the mouse spermhead is incorporated in the presence of microfilament inhibitors indicating an absence of microfilament activity at this stage. Pronuclear apposition is arrested by microfilament inhibitors in fertilized mouse oocytes. The migrations of the sperm and egg nuclei during sea urchin fertilization are dependent on microtubules organized into a radial monastral array, the sperm aster. Microtubule activity is also required during pronuclear apposition in the mouse egg, but they are organized by numerous egg cytoplasmic sites. By the use of an autoimmune antibody to centrosomal material, centrosomes are detected in sea urchin sperm but not in unfertilized eggs. The sea urchin centrosome expands and duplicates during first interphase and condenses to form the mitotic poles during division. Remarkably mouse sperm do not appear to have the centrosomal antigen and instead centrosomes are found in the unfertilized oocyte. These results indicate that both microfilaments and microtubules are required for the successful completion of fertilization in both sea urchins and mice, but at different stages. Furthermore they demonstrate that centrosomes are contributed by the sperm during sea urchin fertilization, but they might be maternally inherited in mammals.     

Inhibition of sea urchin fertilization by jaspisin, a specific inhibitor of matrix metalloendoproteinase

Jaspisin, originally isolated from a marine sponge as an inhibitor of the hatching of the sea urchin (Hemicentrotus pulcherrimus) embryo, causes inhibition of sea urchin fertilization. Electron microscopic examination revealed that the acrosome reaction was induced in jaspisin-treated sperm when they were incubated with an intact egg. The acrosome-reacted sperm bound to the vitelline layer by the acrosomal material surrounding the acrosomal process. However, fusion of the acrosomal process and the egg plasma membrane failed to take place. Membrane potential changes were monitored using eggs preloaded with a membrane potential-sensitive fluorochrome, di-8-ANEPPS. Depolarization of the membrane potential, normally observed in the fertilized egg was not observed in the egg inseminated in the presence of jaspisin, indicating the absence of electrical continuity between the jaspisin-treated egg and sperm. Jaspisin inhibited the activities of matrix metallo-endoproteinase members but not of other types of proteinases. These results provide strong, albeit indirect, evidence that a matrix metallo-endoproteinase(s) is involved in the process of gamete fusion during sea urchin fertilization.     

SIMULTANEOUS POSITIVE AND NEGATIVE FREQUENCY‐DEPENDENT SELECTION ON SPERM BINDIN,A GAMETE RECOGNITION PROTEIN IN THE SEA URCHIN STRONGYLOCENTROTUS PURPURATUS

Gamete‐recognition proteins often, but not always, evolve rapidly. We explored how variation in sperm bindin influences reproductive success of the sea urchin Strongylocentrotus purpuratus during group spawning in the sea. Despite large variation in male and female abundance and neighbor distances, males with common genotypes had higher reproductive success than males with rare genotypes. However, males with a relatively uncommon proline‐for‐serine substitution were the most successful. Females also showed a fitness consequence of sperm‐bindin genotype, suggesting linkage disequilibrium between the sperm‐bindin locus and the egg receptor locus. Females with common genotypes had higher reproductive success than rare genotypes, but females with relatively uncommon insertions were most successful. Overall, these results suggest that rare male proteins are selected against, as supported by molecular evidence of purifying selection and probably caused by poor matches to the female receptor protein. Within the pool of moderately common to common alleles, however, individuals with less‐common functional variants were favored and probably maintained by negative frequency‐dependent selection. These results support the hypothesis that sperm availability and sexual conflict influence the evolution of gamete recognition systems in broadcast spawners and highlight the benefits of combining fitness measures with molecular signatures for estimation of patterns of selection.     

Molecular Reproduction & Development Volume 78, Issue 8 cover image

Transmission electron micrograph of a sea urchin (Strongylocentrotus purpuratus) spermatozoon (purple) bound to the tip of an egg microvillus (yellow) by the bindin protein (diffuse purple), which was released during exocytosis of the sperm acrosomal vesicle. Egg microvilli are approximately 0.25 µm in diameter. More on observing these events on the cellular level in a classroom laboratory setting is presented by VD Vacquier (this issue).     

Laboratory on sea urchin fertilization

Since about 1880, the eggs and sperm of sea urchins have been used for the study of fertilization, the metabolic activation of development and gene regulatory mechanisms governing embryogenesis. Sea urchin gametes are a favorite material for observations of the process of fertilization in advanced high school, community college, and university biology laboratory courses. This article is a laboratory handout, designed for the student to follow in learning about fertilization. In addition to observations of sperm-egg interaction, simple experiments are described that demonstrate some mechanisms involved in the process. The hope is that by making simple observations of fertilization, the student will gain an appreciation for the fact that successive generations of higher organisms are bridged by the fusion of egg and sperm, two very different single cells.     

Correlative ultrastructural and electrophysiological studies of sperm-egg interactions of the sea urchin, Lytechinus variegatus

The sequence of ultrastructural events following the onset of the sperm-induced conductance increase in eggs of the sea urchin, Lytechinus variegatus, was investigated. Eggs voltage clamped at -20 mV were fixed 1 to 20 sec after onset of the conductance increase caused by single sperm. Continuity between the plasma membranes of the sperm and egg was first detected 5 sec after onset of the conductance increase. The earliest stages of formation of the fertilization cone coincided with the establishment of continuity of the gamete plasma membranes. At 6 to 8 sec after the initial conductance increase cortical granule dehiscence was first observed in the immediate vicinity where continuity of the gamete plasma membranes had occurred. These observations are consistent with the conclusion that opening of ion channels at fertilization precedes fusion of the sperm and egg plasma membranes, while exocytosis of cortical granules is initiated following fusion of the sperm and egg plasma membranes.     

Evaluation of Sequence Variation and Selection in the Bindin Locus of the Red Sea Urchin, Strongylocentrotus franciscanus

Recent evidence suggests that gamete recognition proteins may be subjected to directed evolutionary pressure that enhances sequence variability. We evaluated whether diversity enhancing selection is operating on a marine invertebrate fertilization protein by examining the intraspecific DNA sequence variation of a 273-base pair region located at the 5′ end of the sperm bindin locus in 134 adult red sea urchins (Strongylocentrotus franciscanus). Bindin is a sperm recognition protein that mediates species-specific gamete interactions in sea urchins. The region of the bindin locus examined was found to be polymorphic with 14 alleles. Mean pairwise comparison of the 14 alleles indicates moderate sequence diversity (p-distance = 1.06). No evidence of diversity enhancing selection was found. It was not possible to reject the null hypothesis that the sequence variation observed in S. franciscanus bindin is a result of neutral evolution. Statistical evaluation of expected proportions of replacement and silent nucleotide substitutions, observed versus expected proportions of radical replacement substitutions, and conformance to the McDonald and Kreitman test of neutral evolution all indicate that random mutation followed by genetic drift created the polymorphisms observed in bindin. Observed frequencies were also highly similar to results expected for a neutrally evolving locus, suggesting that the polymorphism observed in the 5′ region of S. franciscanus bindin is a result of neutral evolution. Received: 19 June 1998 / Accepted: 2 August 2000     

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.