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.     

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.     

Localization of fodrin during fertilization and early development of sea urchins and mice

Fodrin, a spectrin-like protein, is localized in gametes, zygotes, and embryos from sea urchins and mice. Mammalian fodrin comprises two polypeptides with molecular weights of approximately 240 kDa (alpha) and 235 kDa (beta). An antibody specific for mammalian alpha-fodrin cross-reacted with a 240-kDa polypeptide from sea urchin egg extracts. This indicates that sea urchins contain a protein of similar electrophoretic mobility and immunological properties to mammalian alpha-fodrin. When this antibody was used to stain the sea urchin gametes with indirect immunofluorescence, fodrin-specific fluorescence was localized to the acrosome of the sperm and was distributed over the entire egg near the surface in a punctate pattern similar to the distribution of polymeric actin. During sperm incorporation, the fodrin-specific fluorescence is found at the site of sperm incorporation, in the fertilization cone. After fertilization, the intensity of fodrin fluorescence increases. During mitosis and cytokinesis in sea urchins, the entire surface of the egg remains stained; the cleavage furrow also was stained but no more intensely than was the rest of the egg surface. Antibody labeling with colloidal gold followed by electron microscopy showed that fodrin was loated in the cytoplasm immediately beneath the plasma membrane. In unfertilized mouse oocytes, both actin and fodrin were stained most intensely beneath the membrane adjacent to the meiotic spindle. After insemination, the cell surfaces of the pronucleate egg and the second polar body were stained; however, the actin matrix surrounding the apposed pronuclei did not bind the fodrin antibody. During cytokinesis in the mouse, the cleavage furrow stained more intensely than did the rest of the egg cortex, and in embryos the cell borders were delineated. These results indicate that organisms as unrelated to mammals as sea urchins have fodrin-like proteins; the rearrangements of such proteins suggest that they participate in the actin-mediated events at the cell surface during fertilization and early development in both mice and sea urchins.     

Egg longevity and time-integrated fertilization in a temperate sea urchin (Strongylocentrotus droebachiensis)

Recent field experiments have suggested that fertilization levels in sea urchins (and other broadcast spawners that release their gametes into the water column) may often be far below 100%. However, past experiments have not considered the potentially positive combined effects of an extended period of egg longevity and the release of gametes in viscous fluids (which reduces dilution rates). In a laboratory experiment, we found that eggs of the sea urchin Strongylocentrotus droebachiensis had high viability for 2 to 3 d. Fertilization levels of eggs held in sperm-permeable egg baskets in the field and exposed to sperm slowly diffusing off a spawning male increased significantly with exposure from 15 min to 3 h. In a field survey of time-integrated fertilizations (over 24, 48, and 72 h) during natural sperm release events, eggs held in baskets accrued fertilizations over as much as 48 h and attained fairly high fertilization levels. Our results suggest that an extended period of egg longevity and the release of gametes in viscous fluids may result in higher natural fertilization levels than currently expected from short-term field experiments.     

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.     

Fusion of membranes during fertilization. Increases of the sea urchin egg''s membrane capacitance and membrane conductance at the site of contact with the sperm

The early events of fertilization that precede and cause activation of an egg have not been fully elucidated. The earliest electrophysiological change in the sea urchin egg is a sperm-evoked increase of the egg's membrane conductance. The resulting depolarization facilitates entry of the fertilizing sperm and precludes the entry of supernumerary sperm. The sequence of the increase in the egg's membrane conductance, gamete membrane fusion, egg activation, and sperm entry, including causal relationships between these events, are not known. This study reports the use of whole egg voltage clamp and loose patch clamp to monitor simultaneously changes of membrane conductance and capacitance at the site of sperm-egg contact. Measurements were made during sperm-egg interactions where sperm entry readily proceeded or was precluded by maintaining the egg's membrane potential either at large, negative values or at positive values. Whenever the sperm evoked an increase of the egg's membrane conductance, that increase initiated abruptly, was localized to the site of sperm attachment, and was accompanied by a simultaneous abrupt increase of the membrane capacitance. This increase of capacitance indicated the establishment of electrical continuity between gametes (possibly fusion of the gametes' plasma membranes). If sperm entry was blocked by large negative membrane potentials, the capacitance cut off rapidly and simultaneously with a decrease of the membrane conductance, indicating that electrical continuity between gametes was disrupted. When sperm entry was precluded by positive membrane potentials, neither conductance nor capacitance increased, indicating that sperm entry was halted before the fusion of membranes. A second, smooth increase of capacitance was associated with the exocytosis of cortical granules near the sperm in eggs that were activated. Electrical continuity between the gametes always preceded activation of the egg, but transient electrical continuity between the gametes alone was not always sufficient to induce activation.     

FLAGELLAR MOTILITY IS NOT INVOLVED IN THE INCORPORATION OF THE SPERM INTO THE EGG AT FERTILIZATION*

Cinemicrography of sea urchin fertilization reveals that the fertilizing sperm is one of the first sperm to attach to the egg. Just before the cortical reaction the fertilizing sperm ceases motility and then is incorporated into the egg without flagellar beating. The rate of incorporation is 5–11 μm/sec and is constant. Lytechinus pictus sperm rendered immotile by azide treatment can bind to and fertilize eggs but binding, and therefore fertilization, is blocked by azide treatment of Strongylocentrotus purpuratus gametes.     

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.     

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.     

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.     

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.     

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.     

Peptides from sperm acrosomal protein that initiate egg development

How sperm initiate egg development is being investigated with gametes of the marine worm Urechis. Sperm acrosomal protein, previously shown to activate eggs (Gould et al., 1986, Dev. Biol. 117, 306-318; Gould and Stephano, 1987, Science 235, 1654-1656), was enzymatically cleaved into soluble peptide fragments. When this mixture was added to eggs they activated, and parthenogenetic cleavage often occurred. An active peptide (P23) was purified from the mixture and its sequence was determined to be Val-Ala-Lys-Lys-Pro-Lys. Synthetic peptide had the same biological activity. P23 induced eggs to undergo the complete sequence of changes that normally follows fertilization, including the fertilization potential, completion of meiosis, and DNA replication. When a sperm centrosome was introduced into eggs by prefertilization without activation, and the eggs were subsequently activated by P23, they developed normally to trochophore larvae (the contribution of another sperm component is not ruled out by this experiment). P23 covalently coupled to bovine serum albumin also activated eggs, showing that it acted on the external surface of the egg. The peptide did not activate sea urchin eggs, but did cause oyster eggs to undergo germinal vesicle breakdown.     

Isolation and characterization of a plasma membrane fraction from sea urchin sperm exhibiting species specific recognition of the egg surface

A method is described for isolating preparative quantities of plasma membranes from sea urchin sperm. The final membrane fraction is homogeneous by sucrose density sedimentation and is enriched in adenylate cyclase as well as in the four glycoproteins accessible to radioiodination of intact sperm. The electrophoretic profiles of sperm membranes from three sea urchin species are very similar. The membrane preparation consists primarily of sealed vesicles which release carboxyfluorescein when exposed to detergents or distilled water. Ninety-two percent of the 125I-labeled vesicle material binds to wheat germ lectin columns, suggesting a right-side-out orientation. The isolated sperm membrane vesicles exhibit species specific adhesion to the surfaces of sea urchin eggs; this adhesion is blocked by pretreatment of the vesicles with trypsin or egg jelly. This method will be useful for isolating biologically active sperm membrane components involved in sperm-egg recognition during fertilization.     

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.     

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).     

Fertilization triggers activation of Fyn kinase in the zebrafish egg

Fertilization results in the tyrosine phosphorylation of several egg proteins and studies have shown that tyrosine protein kinase activity is required for successful fertilization. The Fyn protein kinase has been detected in eggs of the sea urchin, frog and rat, although measurement of fertilization-induced changes in Fyn kinase activity have only been successful in the sea urchin system. The present study demonstrates the presence of Fyn kinase in the zebrafish egg and the stimulation of this enzyme at fertilization. Activation of Fyn was detected as early as 30 seconds post-fertilization and increased approximately six-fold by 2 minutes post-insemination. The activation of Fyn in the zebrafish egg required sperm and was not observed in spontaneously activated eggs.     

Bioelectric responses of sea urchin eggs inseminated with oyster spermatozoa: a sperm evoked potential without egg activation

Multiple oyster spermatozoa can enter sea urchin eggs with or often without fertilization membrane formation (Osanai and Kyozuka, 1982). In the present work, electrical responses of sea urchin (Temnopleurus hardwicki) eggs inseminated with oyster (Crassostrea gigas) sperm were examined and correlated to the failure of monospermy and egg activation. With diluted sperm, a transient depolarization of the membrane with a constant pattern appeared repeatedly and discretely, and the depolarizations (sperm evoked potentials, SEPs) were not associated with fertilization membrane elevation. With dense sperm, the SEPs occurred consecutively, and sometimes an assembled consecutive depolarization was followed by an activation potential associated with cortical granule discharge. When the membrane potential was artificially held at positive levels, the frequency of SEPs was strongly suppressed but not completely blocked. The present results indicate that an individual heterologous spermatozoon neither produces a depolarization sufficient to block additional sperm entry, nor stimulates egg activation, and that simultaneous entries of multiple heterologous spermatozoa, as possibly reflected by the assembled consecutive depolarizations, induce cortical granule discharge and egg activation.     

Tandem mass spectrometry identifies proteins phosphorylated by cyclic AMP-dependent protein kinase when sea urchin sperm undergo the acrosome reaction

The exocytotic acrosome reaction (AR), which is required for fertilization, occurs when sea urchin sperm contact the egg jelly (EJ) layer. Among other physiological changes, increases in adenylyl cyclase activity, cAMP and cAMP-dependent protein kinase (PKA) activity occur coincident with the AR. By using inhibitors of PKA, a permeable analog of cAMP and the phosphodiesterase inhibitor IBMX, we show that PKA activity is required for AR induction by EJ. A minimum of six sperm proteins are phosphorylated by PKA upon exposure to EJ, as detected by a PKA substrate-specific antibody. The phosphorylation of these proteins and the percentage of acrosome reacted sperm can be regulated by PKA modulators. The fucose sulfate polymer (FSP), a major component of EJ, is the molecule that triggers sperm PKA activation. Extracellular Ca(2+) is required for PKA activation. Six sperm proteins phosphorylated by PKA were identified by tandem mass spectrometry (MS/MS) utilizing the emerging sea urchin genome. Based on their identities and localizations in sperm head and flagellum, the putative functions of these proteins in sperm physiology and AR induction are discussed.