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The biodiversity of marine ecosystems is diverse and a high number of species coexist side by side. However, despite the fact that most of these species share a common fertilization strategy, a high variability in terms of the size, shape, and motion of spermatozoa can be found. In this study, we have analyzed both the sperm motion parameters and the spermatozoa morphometric features of two swimmer (pufferfish and European eel) and two sessile (sea urchin and ascidian) marine species. The most important differences in the sperm motion parameters were registered in the swimming period. Sessile species sperm displayed notably higher values than swimmer species sperm. In addition, the sperm motilities and velocities of the swimmer species decreased sharply once the sperm was activated, whereas the sessile species were able to maintain their initial values for a long time. These results are linked directly to the species-specific lifestyles. Although sessile organisms, which show limited or no movement, need sperm with a capacity to swim for long distances to find the oocytes, swimmer organisms can move toward the female and release gametes near it, and therefore the spermatozoa does not need to swim for such a long time. At the same time, sperm morphology is related to sperm motion parameters, and in this study an in-depth morphometric analysis of ascidian, sea urchin, and pufferfish spermatozoa, using computer-assisted sperm analysis software, has been carried out for the first time. A huge variability in shapes, sizes, and structures of the studied species was found using electron microscopy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Ascidians (primitive chordates) are hermaphroditic animals that release spermatozoa and eggs almost simultaneously, but some species, including Halocynthia roretzi, show strict self-sterility. In H. roretzi, a 70-kDa vitelline coat (VC) protein consisting of 12 EGF-like repeats, named HrVC70, appears to be a promising candidate for the self/nonself-recognition (or allorecognition) system during gamete interaction. After spermatozoon recognizes the VC as nonself, sperm 700-kDa extracellular ubiquitin-conjugating enzyme complex appears to ubiquitinate Lys234 of HrVC70, and the ubiquitinated HrVC70 is degraded by the sperm 26S proteasome that is located on the sperm head surface. This novel ubiquitin-proteasome system enables spermatozoa to penetrate through the VC. Sperm trypsin-like proteases, acrosin and spermosin, also participate in fertilization, probably as sperm-side 'movable' binding proteins to the VC.

Egg-derived sperm-activating factors and attractants activate sperm motility and attract the sperm, respectively. These phenomena constitute the first communication signaling between males and females in the process of fertilization in many animals and plants, and in many cases, these are species-specific events. Thus, sperm motility activation and chemotaxis may act as a safety process for the authentication between conspecific egg and sperm, and help to prevent crossbreeding. Here, we examine species-specificity of sperm motility activation and chemotaxis in the ascidians belonging to the order Phlebobranchiata: Ciona intestinalis, Ciona savignyi, Phallusia mammillata, Phallusia nigra, and Ascidia sydneiensis. Cross-reactivity in both motility activation and chemotaxis of sperm was not observed between C. savignyi and P. mammillata, or between A. sydneiensis and Phallusia spp. However, there is a "one way" (no reciprocity) cross-reaction between P. mammillata and P. nigra in sperm activation, and between C. savignyi and A. sydneiensis in sperm chemotaxis. Furthermore, the level of activity is different, even when cross-reaction is observed. Thus, sperm motility activation and chemotaxis are neither "species-" nor "genus-" specific phenomena among the ascidian species. Moreover, the interaction between the sperm-activating and sperm-attracting factors (SAAFs) in the ascidian species and the SAAF receptors on the sperm cells are not all-or-none responses.

Sperm trypsin-like proteases are known to play important roles in fertilization, but their detailed functions are still unknown. We previously explored the binding partners of sperm trypsin-like proteases, HrProacrosin and HrSpermosin, in the ascidian Halocynthia roretzi, and we isolated several candidate proteins on the vitelline coat. We found that some of these proteins are identical to the C-terminal coding region (CT) and von Willebrand factor type D (vWF-D) domain of vitellogenin. We also found that CT on the vitelline coat disappears after fertilization. Vitellogenin is a large lipid transfer protein that is enzymatically processed during vitellogenesis. Although the processed domains including phosvitin and lipovitellin are known to function as yolk nutrient proteins, the roles of the CT and vWF-D domain remain elusive. Our results showed that the CT and vWF-D domain of vitellogenin are processed and attached to the vitelline coat, which in turn participate in fertilization as the binding partners of sperm proteases., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

In this paper, we report how to track quickly and vigorously swimming ascidian spermatozoa using high-speed visual feedback at a frame rate of 1 kHz. Ascidian spermatozoa swim as fast as 300 microm/s by rotating their flagella 50 times/s. This vigorous swimming style has prevented stable image observation and made it difficult to track them reliably with our previously developed visual tracking system. Here, we describe how we overcame these problems using image processing techniques to achieve stable tracking of fast, small ascidian spermatozoa for more than 180 s using high-speed visual feedback.

Solitary ascidians are hermaphrodites that release sperm and eggs simultaneously. However, many species are self-sterile, owing to a self/non-self recognition system operating at the outer surface of the chorion during sperm-egg interaction. In Ciona intestinalis, self-incompatibility is thought to have a genetic basis. Here, we report a survey of the self-fertilization potential of a Santa Barbara, California, population of Ciona savignyi, a close relative of C. intestinalis. We found that, in contrast to reports on C. intestinalis, C. savignyi is highly self-fertile. However, using two nonlethal recessive mutant strains, aimless (aim) and immaculate (imc), and a stable transgenic strain that expresses green fluorescent protein (GFP) in the notochord to follow offspring genotype, we demonstrate that non-self sperm outcompete self-sperm in fertilization competition assays. When the chorion was removed, both self- and non-self sperm performed equally well in the competition assay. Thus the non-self/self gamete recognition in C. savignyi is not absolute but relative, and is mediated by one or more components in the chorion. We discuss the significance of this finding in the context of natural populations in the wild, where individuals of C. savignyi are typically found growing in large groups that spawn in unison and where self-fertilization would be expected to be very rare.

Fertilization triggers repetitive waves of cytosolic Ca(2+) in the egg of many species. The mechanism involved in the generation of Ca(2+) waves has been studied in much detail in mature ascidian eggs, by raising artificially the level of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] or of its poorly metabolizable analogue, glycero-myo-phosphatidylinositol 4,5-bisphosphate [gPtdIns(4,5)P(2)]. Here, we use this strategy and the experimental results it provides to develop a realistic theoretical model for repetitive Ca(2+) wave generation and propagation in mature eggs. The model takes into account the heterogeneous spatial distribution of the endoplasmic reticulum. Our results corroborate the hypothesis that Ca(2+) wave pacemakers are associated with cortical accumulations of endoplasmic reticulum. The model is first tested and validated by the adequate match between its theoretical predictions and the observed effects of localized injections of massive amounts of Ins(1,4,5)P(3) analogues. In a second step, we use the model to make some propositions about the possible characteristics of the sperm factor. We find that to account for the spatial characteristics of the first series of Ca(2+) waves seen at fertilization in ascidian eggs, it has to be assumed that, if the sperm factor is a phospholipase C, it is Ca(2+)-sensitive and highly diffusible. Although the actual state of knowledge does not allow us to explain the observed relocalization of the Ca(2+) wave pacemaker site, the model corroborates the assumption that PtdIns(4,5)P(2), the substrate for phospholipase C is distributed over the entire egg. We also predict that the dose of sperm factor injected into the egg should modulate the temporal characteristics of the first, long-lasting fertilization wave.

Many benthic marine invertebrates reproduce by releasing sperm into the sea (free-spawning), but the amount of time that sperm are viable after spawning may have different consequences for fertilization, depending on the type of free-spawner. In egg-broadcasting marine organisms, gamete age is usually assumed to be irrelevant because of the low probability of contact between dilute sperm and egg. However, direct dilution effects might be reduced in egg-brooding free-spawners that filter dilute sperm out of the water column, and sperm longevity may play a role in facilitating fertilization in these taxa. We investigated the effects of time, temperature, and mixing on the viability of naturally released sperm of the colonial ascidian Botryllus schlosseri. Our data indicate that B. schlosseri sperm have a functional life span that is considerably longer than those of the sperm of many other marine invertebrate taxa (half-life of approximately 16 to 26 h), are able to fertilize eggs at extremely low external sperm concentrations (ca. 10(1) sperm ml(-1)), and have a longevity that varies with temperature. It is possible that such prolonged sperm longevity may be achieved by reductions in motility, reactivation of quiescent sperm by chemical cues, or intermittent swimming.

Chemotactic swimming behaviors of spermatozoa toward an egg have been reported in various species. The strategies underlying these behaviors, however, are poorly understood. We focused on two types of chemotaxis, one in the siphonophores and the second in the ascidians, and then proposed two models based on experimental data. Both models assumed that the radius of the path curvature of a swimming spermatozoon depends on [Ca(2+)](i), the intracellular calcium concentration. The chemotaxis in the siphonophores could be simulated in a model that assumes that [Ca(2+)](i) depends on the local concentration of the attractant in the vicinity of the spermatozoon and that a substantial time period is required for the clearance of transient high [Ca(2+)](i). In the case of ascidians, trajectories similar to those in experiments could be adequately simulated by a variant of this model that assumes that [Ca(2+)](i) depends on the time derivative of the attractant concentration. The properties of these strategies and future problems are discussed in relation to these models.

Fertilization success may be severely limited in marine invertebrates that spawn both male and female gametes. In a diverse group of aquatic organisms only sperm are released, with sperm-egg fusion occurring at the mother. Here, we report fertilization kinetics data for two such 'brooding' or 'spermcast' species--representing each major clade of the animal kingdom. High levels of fertilization were achieved at sperm concentrations of two or three orders of magnitude lower than is common with broadcast spawning species. At a concentration of 100 sperm ml(-1), fertilization rates of a bryozoan and colonial ascidian were near maximum, whereas most broadcast spawners would have displayed near complete reproductive failure. A further experiment looked at the rate of uptake of sperm under natural conditions. Results suggested that sperm released at ca. 0.9 m from an acting female could be collected at a rate of 3-12 times greater than the minimum required simply to avoid sperm limitation. Thus, evolutionary pressures on gametic and other reproductive characteristics of many species that release sperm but retain eggs may be quite different from those of broadcast spawners and may confer on the former an enhanced scope for sperm competition and female choice.

Negative frequency-dependent mating success--the rare male effect--is a potentially powerful evolutionary force, but disagreement exists as to whether previous work, focusing on copulating species, has robustly demonstrated this phenomenon. Noncopulating sessile organisms that release male gametes into the environment but retain their eggs for fertilization may routinely receive unequal mixtures of sperm. Although promiscuity seems unavoidable it does not follow that the resulting paternity obeys 'fair raffle' expectations. This study investigates frequency dependence in the mating of one such species, the colonial ascidian Diplosoma listerianum. In competition with an alternative sperm source males fathered more progeny if previously mated to a particular female than if no mating history existed. This suggests positive frequency-dependent selection, but may simply result from a mate order effect involving sperm storage. With fewer acclimation matings, separated by longer intervals, this pattern was not found. When, in a different experimental design, virgin females were given simultaneous mixtures of gametes at widely divergent concentrations, sperm at the lower frequency consistently achieved a greater than expected share of paternity--a rare male effect. A convincing argument as to why D. listerianum should favour rare sperm has not been identified, as sperm rarity is expected to correlate very poorly with ecological or genetic male characteristics in this pattern of mating. The existence of nongenetic female preferences at the level of colony modules, analogous in effect to fixed female preferences, is proposed. If visible to selection, indirect benefits from increasing the genetic diversity of a sibship appear the only likely explanation of the rare male effect in this system as the life history presents virtually no costs to multiple mating, and a near absence of direct (resource) benefits, whereas less controversial hypotheses of female promiscuity (e.g. trade up, genetic incompatibility) do not seem appropriate.

The sperm-activating and -attracting factor released from the eggs of the ascidians Ciona intestinalis and Ciona savignyi requires extracellular Ca(2+) for activating sperm motility and eliciting chemotactic behavior of the activated sperm toward the egg. Here, we show that modulators of the store-operated Ca(2+) channel, SK&F96365, Ni(2+), 2-aminoethoxydiphenylborane, and thapsigargin inhibit the chemotactic behavior of the ascidian sperm; on the other hand, blockers of voltage-dependent Ca(2+) channels did not inhibit the chemotaxis, even though they inhibited the sperm activation operated by voltage-dependent Ca(2+) channels. The blockers of store-operated Ca(2+) channels also inhibited the asymmetrical flagellar beating and turning movements of the ascidian sperm, which are typical signs of sperm chemotaxis. Depletion of internal Ca(2+) stores by thapsigargin induced capacitative Ca(2+) entry into the sperm, which was blocked by SK&F96365. These results suggest that the intracellular Ca(2+) concentration increase through the store-operated Ca(2+) channels induces asymmetrical flagellar movements to establish the chemotactic behavior of the sperm.

Sperm chemotaxis toward eggs before fertilization has been demonstrated in many animals and plants, and several peptides and small organic compounds acting as chemoattractants have been identified. We previously showed that sperm of the ascidians Ciona intestinalis and Ciona savignyi are activated and then attracted toward the egg by a common factor released from the egg. In this study, we purified sperm-activating and -attracting factor (SAAF) from the egg-conditioning medium of C. intestinalis by using several steps of column chromatography. Determination of the molecular structure by NMR and MS/MS analysis revealed that SAAF is a previously uncharacterized sulfated steroid: 3,4,7,26-tetrahydroxycholestane-3,26-disulfate. Furthermore, it was shown that the SAAF of C. savignyi was indistinguishable from that of C. intestinalis in terms of the chromatographic behavior and molecular weight, indicating that the same compound might be responsible for sperm activation and chemotaxis in both the species. Furthermore, we established a method for quantitative analysis of sperm chemotaxis and showed that the chemotactic behavior of Ciona sperm is controlled by the "chemotactic turn" associated with decrease in the concentration of SAAF.

Fertilization is a precisely controlled process involving many gamete molecules in sperm binding to and penetration through the extracellular matrix of the egg. After sperm bind to the extracellular matrix (vitelline coat), they undergo the acrosome reaction which exposes and partially releases a lytic agent called "lysin" to digest the vitelline coat for the sperm penetration. The vitelline coat sperm lysin is generally a protease in deuterostomes. The molecular mechanism of the actual degradation of the vitelline coat, however, remains poorly understood. In order to understand the lysin system, we have been studying the fertilization mechanism in ascidians (Urochordata) because we can obtain large quantities of gametes which are readily fertilized in the laboratory. Whereas ascidians are hermaphrodites, which release sperm and eggs simultaneously, many ascidians, including Halocynthia roretzi, are strictly self-sterile. Therefore, after sperm recognize the vitelline coat as nonself, the sperm lysin system is thought to be activated. We revealed that two sperm trypsin-like proteases, acrosin and spermosin, the latter of which is a novel sperm protease with thrombin-like substrate specificity, are essential for fertilization in H. roretzi. These molecules contain motifs involved in binding to the vitelline coat. We found that the proteasome rather than trypsin-like proteases has a direct lytic activity toward the vitelline coat. The target for the ascidian lysin was found to be a 70-kDa vitelline coat component called HrVC70, which is made up of 12 EGF-like repeats. In addition to the proteasome system, the ubiquitination system toward the HrVC70 was found to be necessary for ascidian fertilization. In this review, I describe recent progress on the structures and roles in fertilization of the two trypsin-like proteases, acrosin and spermosin, and also on the novel extracellular ubiquitin-proteasome system, which plays an essential role in the degradation of the ascidian vitelline coat.

Ascidian eggs are surrounded by a noncellular layer and two cellular layers, which are penetrated by sperm. Three sperm surface proteases are essential for fertilization of eggs from the stolidobranch ascidian Halocynthia: spermosin, acrosin, and the proteasome. In the phlebobranch Ciona, a chymotrypsin-like protease and the proteasome are essential in fertilization. Sperm from the phlebobranch ascidians Phallusia mammillata, Ascidia (=Phallusia) nigra, and Ascidia columbiana, all express spermosin, acrosin, and the proteasomal chymotrypsin activities on their surfaces. Chymostatin blocks cleavage in phlebobranchs, but inhibitors of spermosin and acrosin only delay it by several minutes. Protease inhibitors have little effect upon sperm binding in Phallusia but strongly affect the rate of sperm passage through the vitelline coat. Peptide substrates and inhibitors to spermosin and acrosin cause a significant decline in the number of eggs undergoing pre-meiotic contractions at 3 min after fertilization. Thus while chymotrypsin activity is essential for penetration of the vitelline coat, spermosin and acrosin both function to increase the rate of fertilization. A crucial step in the divergence of the phlebobranchs and stolidobranchs may have been the conversion of spermosin and acrosin to essential proteases in the stolidobranchs, or, perhaps, their essential function was lost in the evolution of phlebobranchs. Aplousobranch ascidians are all colonial with very small zooids. Sperm from Aplidium californicum, Aplidium solidum (Polyclinidae), and Distaplia occidentalis (Holozoidae) have acrosin and chymotrypsin activities but lack spermosin activity. This enzyme is also missing from sperm of colonial phlebobranch and stolidobranch ascidians, suggesting that spermosin is not necessary for small zooids with internal fertilization., (Copyright 2002 Wiley-Liss, Inc.)

The process of fertilization begins when sperm contact the outermost egg investment and ends with fusion of the two haploid pronuclei in the egg cytoplasm. Many steps in fertilization involve carbohydrate-based molecular recognition between sperm and egg. Although there is conservation of gamete recognition molecules within vertebrates, their homologues have not yet been discovered in echinoderms and ascidians (the invertebrate deuterostomes). In echinoderms, long sulfated polysaccharides act as ligands for sperm receptors. Ascidians employ egg coat glycosides that are recognized by sperm surface glycosidases. Vertebrate egg coats contain zona pellucida (ZP) family glycoproteins, whose carbohydrates bind to sperm receptors. Several candidate sperm receptors for vertebrate ZP proteins have been identified and are discussed here. This brief review focuses on new information concerning fertilization in deuterostomes (the phylogenetic group including echinoderms, ascidians, and vertebrates) and highlights protein-carbohydrate interactions involved in this process.

Sperm-triggered Ca(2+) oscillations occur throughout the animal kingdom. The mechanism sperm use to trigger Ca(2+) oscillations at fertilization has not been resolved in any egg. The temporal, spatial and regulatory characteristics of the Ca(2+) oscillations during fertilization in ascidians offer a unique advantage over other systems for determining the mechanism of fertilization. For example, sperm trigger two phases of Ca(2+) oscillations that are all waves in ascidians. The first of these Ca(2+) waves begins at the point of sperm-egg fusion while a second phase of Ca(2+) waves originates at a vegetal protrusion termed the contraction pole. In addition, cyclin B1-dependent kinase activity provides a form of positive feedback, maintaining the second phase of Ca(2+) waves during meiosis and thereby ensuring meiotic exit. We therefore prepared cytosolic ascidian sperm extracts or MonoQ-fractionated ascidian sperm extracts from this urochordate to investigate if a Ca(2+)-releasing sperm-borne factor was responsible for egg activation. Spatially, ascidian sperm extract induced repetitive Ca(2+) waves that mimicked the spatial pattern displayed during fertilization: all the second-phase Ca(2+) waves originated at a vegetal protrusion termed the contraction pole (thus mimicking fertilisation). We also demonstrated that ascidian sperm extract-induced Ca(2+) oscillations were maintained when CDK activity was elevated and MAP kinase activity was low, as found previously for sperm-triggered Ca(2+) oscillations. As would be predicted, large doses of ascidian sperm extract injected into prophase-stage oocytes, lacking CDK activity, failed to induce any Ca(2+) release even though they responded to microinjection of the Ca(2+)-releasing second messenger inositol 1,4,5-trisphosphate. Finally, since the Ca(2+)-releasing activity from Mono-Q fractionated ascidian sperm extract eluted predominantly as one fraction, this may imply that one factor is responsible for the Ca(2+)-releasing activity. These data support a model of egg activation whereby the sperm introduces a Ca(2+)-releasing cytosolic factor into the egg. We demonstrated that ascidian sperm contain a protein factor(s) that is regulated by the egg CDK activity and can trigger all the Ca(2+ )waves observed at fertilization with a spatial pattern that mimics those initiated by sperm.

A diverse array of sessile marine invertebrates mate by passive dispersal of sperm which fertilize the brooded eggs of neighbours. In two such species, a sea-mat (phylum Bryozoa) and an ascidian (phylum Chordata), vitellogenic egg growth is absent in reproductively isolated specimens, but is triggered by a water-borne factor released by conspecifics. In both of these colonial, hermaphroditic species, the active factor can be removed from water by filtration. The effect involves self-/non-self-recognition: water conditioned by a separate subcolony of the same genetic individual does not prompt oocyte growth. In each species, allosperm move from the surrounding water to the ovary and are then stored in close association with the growing oocytes. We concluded that sperm themselves are the water-borne factor that triggers the major phase of female reproductive investment. This mechanism is, to our knowledge, previously undescribed in animals, but has parallels with the initiation of maternal investment in flowering plants following the receipt of compatible pollen. The species studied may be representative of many other aquatic invertebrates which mate in a similar way. The stimulation of egg growth by allosperm could lead to intersexual conflict during oogenesis.

The compound ascidian Diplosoma listerianum releases aquatic sperm which are dispersed passively to potential mates as individual gametes prior to storage of sperm, internal fertilization and brooding of embryos. The storage of exogenous sperm enables D. listerianum to produce a lengthy series of progeny following a brief period of mating. Molecular paternity analysis following sequential mating of colonies in laboratory culture revealed a consistent pattern with a clear initial bias in paternity towards the first of two acting males. The sites of sperm storage and fertilization and the morphology of the ovary in D. listerianum suggest that this bias reflects first-in-first-out use of individual stored gametes. The proportion of second-male paternity subsequently increased with time within the progeny arrays. This may have reflected the ageing or passive loss of first-male sperm. It is also possible that the modular nature of the organism contributed to this temporal trend: any recently budded colony modules maturing in the interval between matings would have been available exclusively to second-male sperm as virgin zooids. Two sets of mating trials were run. In the first, the collection of progeny suffered an interruption of 13 days and each male gained a larger proportion of recorded paternity within the progeny analysed when mating first rather than when mating second. In one mating combination, the first male obtained almost 100% of recorded paternity. In the second set of trials, with different clonal combinations, the complete sequence of progeny was collected and the estimated overall proportion of second-male paternity (P2) was consistently > 0.5. Taken as a whole, the results suggest that the overall P2-value can vary widely within the population studied. Proposed mechanisms of mating-order effects in species with copulatory mating include several which can have no counterpart in indirect aquatic mating since they involve the active removal, sealing off, volumetric displacement or incapacitation of first-male ejaculates. It is nevertheless clear that mating-order effects can be pronounced during the type of non-copulatory mating examined here, which is widespread in marine invertebrates.

The generation of calcium oscillations at fertilisation and during mitosis appears to be controlled by the cell cycle machinery. For example, the calcium oscillations in oocytes and embryos occur during metaphase and terminate upon entry into interphase. Here we report the manipulation of sperm-triggered calcium oscillations by cyclin-dependent kinase (CDK) activity, the major component of maturation/M phase promoting factor (MPF). To control the CDK activity we microinjected mRNAs encoding full-length GFP-tagged cyclin B1 or a truncated and therefore stabilised form of cyclin B1 ((delta)90) into unfertilised oocytes. In the presence of full-length cyclin B1, the calcium oscillations terminate when cyclin B1 levels fall along with the concomitant fall in the associated CDK activity. In addition, when the CDK activity is elevated indefinitely with (delta)90 cyclin B1, the calcium oscillations also continue indefinitely. Finally, in oocytes that contain low mitogen-activated protein (MAP) kinase activity and elevated CDK activity, the sperm-triggered calcium oscillations are again prolonged. We conclude that the CDK activity of the ascidian oocyte can be regarded as a positive regulator of sperm-triggered calcium oscillations, a finding that may apply to other oocytes that display sperm-triggered calcium oscillations at fertilisation. Furthermore, these findings may have a bearing upon the mitotic calcium signals of early embryos.

Ascidians (invertebrate chordates) are very abundant in many marine subtidal areas. They often live in dense multispecies clumps; thus, interspecific competition for space may be intense. Although most noncolonial species are broadcast spawners, their eggs can be fertilized only by sperm of the same species (1). Multiple fertilization is lethal and all animals have evolved blocks to polyspermy. Ascidian eggs block polyspermy by enzymatic (2) and electrical mechanisms (3). Sperm bind to N-acetylglucosamine groups on the vitelline coat (4, 5, 6, 7). Follice cells surrounding the vitelline coat release N-acetylglucosaminidase during egg activation (8), preventing the binding of all sperm but a few (2). I show here that this interaction is not species-specific; sperm from one species can cause glycosidase release from follicle cells of a second species. Furthermore, once glycosidase release has been induced, the subsequent addition of sperm from the egg-producing species fails to fertilize a substantial proportion of these eggs. This leads to the hypothesis that sperm from one species of ascidian can interfere with fertilization of a second species. While intraspecific sperm competition has been well documented in several taxa (9, 10), this is the first record of sperm competition between species, or interspecific sperm competition.

The solitary ascidians Ciona intestinalis and Ciona savignyi co-occur in southern California harbors, but no hybrids have been recognized in nature. Numerous differences in their egg morphology were detected. Homologous (normal outcross) fertilization yielded 96-99% cleavage, where autologous (self) fertilization showed 3% and heterologous (hybrid) fertilization showed 0-1%. Acid treatment (pH 3.2) removed the block to selfing (P < 0.0001) but not hybridization for both species. Heterologous sperm bind to the vitelline coat (VC), but fail to penetrate. Enzymatic removal of the VC resulted in 91-97% cleavage with autologous and heterologous sperm (P < 0.0001). The vitelline coats of the two species differ in lectin binding to surface glycosides. Fertilization in both species is significantly inhibited by the lectins, fucose binding protein (P < 0.0001) and concanavalin A (P < 0.0001), and wheat germ agglutinin inhibits fertilization in C. intestinalis (P < 0.0001) but is without effect on C. savignyi fertilization. Self and hybrid blocks employ different mechanisms including glycoside composition and acid sensitivity., (Copyright 2000 Wiley-Liss, Inc.)

A rise in intracellular free Ca(2+) concentration ([Ca(2+)](i)) is required to activate sperm of all organisms studied. Such elevation of [Ca(2+)](i) can occur either by influx of extracellular Ca(2+) or by release of Ca(2+) from intracellular stores. We have examined these sources of Ca(2+) in sperm from the sea squirt Ascidia ceratodes using mitochondrial translocation to evaluate activation and the Ca(2+)-sensitive dye fura-2 to monitor [Ca(2+)](i) by bulk spectrofluorometry. Sperm activation artificially evoked by incubation in high-pH seawater was inhibited by reducing seawater [Ca(2+)], as well as by the presence of high [K(+)](o) or the Ca channel blockers pimozide, penfluridol, or Ni(2+), but not nifedipine or Co(2+). The accompanying rise in [Ca(2+)](i) was also blocked by pimozide or penfluridol. These results indicate that activation produced by alkaline incubation involves opening of plasmalemmal voltage-dependent Ca channels and Ca(2+) entry to initiate mitochondrial translocation. Incubation in thimerosal or thapsigargin, but not ryanodine (even if combined with caffeine pretreatment), evoked sperm activation. Activation by thimerosal was insensitive to reduced external calcium and to Ca channel blockers. Sperm [Ca(2+)](i) increased upon incubation in high-pH or thimerosal-containing seawater, but only the high-pH-dependent elevation in [Ca(2+)](i) could be inhibited by pimozide or penfluridol. Treatment with the protonophore CCCP indicated that only a small percentage of sperm could release enough Ca(2+) from mitochondria to cause activation. Inositol 1,4,5-trisphosphate (IP(3)) delivered by liposomes or by permeabilization increased sperm activation. Both of these effects were blocked by heparin. We conclude that high external pH induces intracellular alkalization that directly or indirectly activates plasma membrane voltage-dependent Ca channels allowing entry of external Ca(2+) and that thimerosal stimulates release of Ca(2+) from IP(3)-sensitive intracellular stores., (Copyright 1999 Academic Press.)

Sperm extract (SE) of the ascidian, Ciona savignyi, injected into oocytes induced repetitive intracellular Ca2+ increases with kinetics consistent with those at fertilization and caused reinitiation and progression of meiosis as in fertilized oocytes with the formation of polar bodies. The Ca2+ response comprised two sets of Ca2+ oscillations separated by 5 minutes and correlated with the first and second meiotic metaphase. The effects of SE were dose dependent and the critical dose corresponded roughly to a single spermatozoon. In the first Ca2+ transient observed by confocal microscopy, a Ca2+ wave started from the SE injection site at the peripheral region of the oocyte and propagated across the ooplasm. The similar wave was produced by injection at the central region, starting from an arbitrary cortical area after 30 seconds, probably after SE had diffused to the cortex. The sensitivity to SE is thought to be preferentially higher in the cortex. The effective component of SE was heat-unstable, and its molecular weight was estimated as in the range between 10x10(4 )and 3x10(4) using membrane filters. These results suggest that, in ascidian fertilization, a cytosolic sperm protein factor is introduced to the oocyte cortex and induces Ca2+ waves and thereby meiotic resumption, leading to cell-cycle-correlated Ca2+ oscillations.

We have injected soluble extracts of sperm from the ascidian Ciona intestinalis into oocytes of the same species to test whether these extracts can mimic the events of fertilisation. Injection of ascidian sperm extracts leads, after a delay of approximately 60 s, to a large calcium transient and repetitive pattern of calcium oscillations, mimicking the normal fertilisation response. The response was concentration-independent, suggesting a stimulatory mechanism in triggering the fertilisation response. We tested the pathway of calcium release in ascidian oocytes after injection of sperm extracts by preinjection of calcium release inhibitors. The data demonstrate that dual pathways to calcium release act at fertilisation in ascidians, as in other species. C. intestinalis oocytes are characterised by a ion channel in the plasma membrane that is gated uniquely by ADP ribose. We show that this channel is not gated by the injection of ascidian sperm extracts. Our data suggest that one metabolic pathway triggered by sperm, the release of nitric oxide, is not stimulated by sperm extracts and that several metabolic pathways are stimulated at fertilisation by more than one factor within sperm.

Many ascidians live in clumps and usually release sperm before the eggs. Consequently, eggs are often spawned into dense clouds of sperm. Because fertilization by more than a single sperm is lethal, ascidians have evolved at least two successive blocks to polyspermy: the rapid release of a glycosidase that inhibits sperm binding to the vitelline coat (VC) and a subsequent change in membrane potential that prevents supernumerary sperm-egg fusion. This paper shows that (1) these two blocks can be uncoupled by the use of suramin, and (2) most of the glycosidase appears to be from the follicle cells, which are accessory cells on the outside of the egg VC. Phallusia mammillata eggs initially bind numerous sperm but, after the glycosidase is released, only a few additional sperm bind. Intact eggs in 20 microM suramin release glycosidase, but the electrical response is inhibited; sperm swim actively and bind to the VC but fail to penetrate. Suramin treatment is completely reversible; intact eggs exhibit the electrical response an average of 11 minutes after the drug is washed out. Sperm must contact the follicle cells before passing through the VC; eggs with the VC removed and fertilized in the presence of 20 microM suramin show the electrical response 35% of the time, thus VC removal enhances sperm entry. Like the intact eggs, 100% of the naked eggs respond electrically to fertilization after the drug is washed out. Follicle cells that are isolated by calcium magnesium free seawater and then returned to complete seawater release N-acetylglucosaminidase activity in response to sperm. Thus, these eggs have two blocks to polyspermy that operate in sequence: an early first block resulting from enzymatic modification of the VC by N-acetylglucosaminidase released primarily from follicle cells and a second electrical block operating at the egg plasma membrane level and requiring sperm-egg fusion.

An inhibitor against the egg beta-D-N-acetylglucosaminidase (GlcNAcase) was purified from the vitelline coat of eggs of the ascidian, Halocynthia roretzi. The purified inhibitor gave a single band with an apparent molecular mass of 70 kDa on SDS-PAGE under non-reducing conditions. The GlcNAcase inhibitor potently inhibited the ascidian egg GlcNAcase activity, and also substantially inhibited the GlcNAcase activity in the ascidian sperm extract, although to lesser extent. In contrast, it showed little inhibitory effect on the activity of GlcNAcase from Jack beans. This inhibitor was found to block the binding of sperm to the vitelline coat of the eggs in a concentration-dependent manner. These results strongly suggest that the GlcNAcase inhibitor isolated from the vitelline coat functions in the binding of sperm to the egg coat, as a possible sperm receptor.

During fertilization, species-specific gamete binding must be followed by sperm penetration of egg vestments before gamete fusion can occur. Sperm proteases, called lysins, aid this process. Sperm from Ascidia ceratodes, Ascidia callosa, and Ascidia paratropa were found to have a surface-mounted chymotrypsin-like protease when studied by enzymology, biotinylated, immunolabeling, and histochemistry. Chymotrypsin substrates and inhibitors blocked fertilization in a concentration-dependent manner in A. ceratodes and decreased the number of sperm heads which penetrated the egg's vitelline coat, but had no effect on sperm binding to follicle cells. Sperm bound to agarose beads coated with the chymotrypsin inhibitor alpha 2-macroglobulin. Chymotrypsin-like enzyme activity, assayed fluorimetrically using N-succinyl-leucinyl-leucinyl-valinyl-tyrosinyl-7 -amido-4-methyl-coumarin as the substrate, was associated with head fractions prepared by differential centrifugation. Biotinylation of live sperm followed by detergent extraction showed that chymotrypsin-like activity could be removed from the detergent extract using avidin-agarose beads. Indirect immunofluorescence of unreacted and reacted sperm heavily labeled membrane domains overlying the mitochondrion and at the base of the head with occasional labeling of the sperm tip. Histochemical studies, which used N-succinyl-alanyl-alanyl-prolyl-phenylalanyl-beta-naphthylamide as the substrate, colocalized enzyme activity in head regions of unreacted and near the mitochondrion of reacted sperm. Thus, we conclude that in ascidian sperm a chymotrypsin-like protease is exposed on the external surface of the plasma membrane of the head, is required for fertilization, and plays a role in sperm penetration but not binding.

The fertilization potential of the Phallusia mammillata egg has been found to consist, in natural or standard artificial sea water (NSW and ASW), of an initial sperm-triggered rapid depolarization comprising two distinct successive components, followed by a phase of membrane depolarization comprising a plateau and two series of membrane potential oscillations. These oscillations occur during meiotic divisions and they end just before formation of the second polar body. In the present work, the effects of removing external Ca2+ on the fertilization potential time course on the rate of polyspermy and on the resumption of meiosis of the P. mammillata egg were explored. Eggs inseminated in Ca(2+)-free ASW usually responded by several successive sperm-induced electrical responses strikingly different from that obtained in standard ASW: removing external Ca2+ during insemination and the initial depolarizing shift (free (Ca2+), 5-11 microM in Ca(2+)-free ASW) and during the subsequent period of the electrical responses (free (Ca2+), 0.2 microM in Ca(2+)-free ASW), completely suppressed both the second component of the initial depolarizing shift, the plateau, and membrane oscillations of the depolarization phase, did not impair sperm entry, but always blocked the resumption of meiosis. Under these conditions eggs were polyspermic (91%, n = 22, c.l. 69-99%). Lastly, only a short period of contact (approximately 20 sec at minimum) with a physiological concentration of external Ca2+ (12 mM) at the onset of the fertilization potential was necessary for the egg to resume meiosis and then to undergo complete embryonic development. These results indicate that in the P. mammillata egg, the second component of the initial depolarizing shift involves voltage-gated Ca2+ channels. The possibility is discussed that external Ca2+ ions might contribute to the activation of the P. mammillata egg by providing a source for an increase of (Ca2+)i and/or for the reloading of the Ca2+ internal stores.

N-Acetylglucosaminidase (GlcNAc'ase), which possesses by far the highest activity of all Phallusia mammillata sperm glycosidases, was isolated and purified using DEAE-cellulose, phenyl-Sepharose, and concanavalin A affinity chromatography. The molecular size of the native enzyme estimated by G-200 gel permeation was 158 kDa. On SDS-PAGE, the denatured enzyme migrated as a single band with a Mr of 78 kDa. This indicates that under nondenaturing conditions the GlcNAc'ase prevails as a dimer. The molecular activity of the enzyme was determined to be 3.7 x 10(5) U/mumole, the Km for p-NP-GlcNAc was 0.65 mM, and the Ki for GlcNAc was 5.5 mM. It has been suggested that gamete binding in ascidians might be mediated by an enzyme-substrate complex established between a sperm glycosidase and corresponding glycosides on the vitelline coat. Thus, the GlcNAc'ase should be present as an exoenzyme at the proper place on the sperm surface membrane, i.e., on the sperm tip and possibly over the mitochondrial region. We localized the enzyme with fluorescence and electron microscopy using the neoglycoprotein BSA-p-aminophenyl-N-acetyl-beta-D-glucosaminide (BSA-GlcNAc) or concanavalin A coupled either to fluorochromes or gold particles. Labeling of unreacted and activated sperm revealed three distinct binding sites, namely at the sperm tip, over the mitochondrion, and at the head-tail junction. In reacted sperm strong labeling was observed over the translocated mitochondrion as well as at the sperm tip. An intensive binding was observed along the rim which borders the cap-like structure at the sperm tip. The distribution of the enzyme reflected by these binding patterns accounts well for the suggested function. Using N-acetylglucosaminono-1,5-lactone oxime, a novel, highly specific inhibitor of GlcNAc'ase, we were able to show that this enzyme is indispensable for fertilization of intact eggs, but not of eggs deprived of their vitelline coat. These observations are discussed in terms of functional relationships which may exist between this enzyme, sperm binding, gamete recognition, and penetration of the vitelline coat.

Using patch electrodes and the whole-cell recording technique to study fertilization currents in ascidian oocytes under voltage clamp, this paper shows that between -85 and 0 mV the currents are inward with an initial peak ranging from 50 to 600 pA. Voltages more positive than 0 mV inhibit initiation of the fertilization current, but by allowing the oocyte to return briefly to its resting potential fertilization occurs and fertilization currents are outward at positive potentials. By comparison with previous single-channel work, a fertilizing spermatozoon opens about 300 large-conductance channels with zero reversal potential.

In the Ascidian, Ciona intestinalis, sperm alpha-L-fucosidase is concluded to be a recognition protein for the sperm receptor in the vitelline envelope. The spermatozoa bind to the vitelline envelope probably by forming rather stable substrate (alpha-L-fucoside)-enzyme complex at many spots. Experimental evidence indicates a good correspondence in the properties between receptor binding activity and alpha-L-fucosidase activity such as the effects of saccharides, of pH and of monoclonal antibodies raised against sperm alpha-L-fucosidase, and locus in the sperm. A general mechanism involving an enzyme as a recognition protein for the cell to cell binding is also discussed in relation to the molecular evolution of the recognition proteins.

Sperm bind to vitelline coat (VC) glycosides of ascidian eggs by means of a sperm surface glycosidase (Hoshi et al.: Zool Sci 2:65, 1985). In the genus Ascidia, N-acetylglucosamine (NAG) is the VC ligand. After initial binding by the tip of the head, sperm pass through the VC and perivitelline space leaving the single mitochondrion outside. This process can also be followed in vitro on a coverslip. Analysis of recorded video images shows that the sperm moves away from the anchored mitochondrion. Our model for sperm penetration suggests that mitochondrial translocation is responsible for driving the sperm into the egg. In the work presented here, we have demonstrated that ascidian sperm have N-acetyl-beta-D-glucosaminidase (NAGase) activity with an acidic pH optimum. This enzyme, which can be removed from the sperm with Triton X-100, binds to concanavalin A, demonstrating that it is glycosylated. Histochemical methods disclose that the enzyme is originally located at the tip of the head but subsequently remains with the surface overlying the mitochondrion during translocation. Fluorescent Con A was used as a second label for localization of the enzyme on the cell surface during translocation. Colocalization of both probes of the enzyme support a crucial facet of our model; the sperm surface VC binding site remains over the mitochondrion during translocation. This would couple mitochondrial translocation with sperm penetration and drive the sperm into the egg.

Inhibitory effects of three peptidyl phenylalaninals on fertilization and on chymotrypsin-like enzyme activity of sperm in three species of ascidians were examined. The results suggest that a sperm chymotrypsin-like enzyme is indispensable for the fertilization in each of the ascidians, and that these enzymes have different susceptibilities to inhibitors.

Ten kinds of argininal-containing compounds were examined for their inhibitory effects on the fertilization of the solitary ascidian and on the activities of acrosin and spermosin, trypsin-like proteases isolated from spermatozoa of this animal. Benzyloxycarbonyl-Val-Pro-argininal (I) and benzyloxycarbonyl-Phe-Leu-argininal (II) showed the strongest inhibition on the fertilization. Leupeptin (acetyl-Leu-Leu-argininal, III) was ranked next (I, II greater than III). The activity of ascidian acrosin was susceptible to most of the compounds, among which II was the best inhibitor and followed with I and III (II greater than I, III). Spermosin suffered significant inhibition only with I and II (I greater than II). These results suggest that not only acrosin but also spermosin is involved in fertilization of the ascidian.

In this paper we describe a mild procedure which results in the extraction of a glycoprotein fraction from the vitelline coat (VC) of Ciona intestinalis while leaving behind the bulk of the VC components. When acting upon the spermatozoa this fraction inhibits sperm binding to the VC and fertilization and elicits sperm activation including the acrosome reaction. SDS-PAGE shows that it contains the same (fucosyl) glycoprotein components previously recognized in the total extracts of VC. It is suggested that this material contains the sperm receptors or those components of the receptors that are essential for their Chinese function.

Fertilization-related sperm mitochondrial movement occurs at a rate comparable to other actin-myosin-driven movements and is inhibited by cytochalasin B and N-ethyl maleimide in Ascidia ceratodes sperm. F-actin was demonstrated in the tails and mitochondria using NBD-phallacidin fluorescence. Both actin and myosin were also detected on the mitochondrion and in the tail by indirect immunofluorescence. Western blot analysis verified the presence of these proteins. Boltenia villosa and Cnemidocarpa finmarkiensis also have mitochondrion and tail localized actin and myosin. In the tails of all 3 species the fluorescence takes the form of discrete spots 0.25-0.5 micron apart. Boltenia and Cnemidocarpa sperm have additional actin at the tip of the head and additional myosin at the base of the head. The presence of actin and myosin on the mitochondrion and in the tail supports a means by which the force for mitochondrial movement is generated.