PATTERNS OF INTERSPECIFIC INTERACTIONS IN THE ULVA-DOMINATED INTERTIDAL COMMUNITY IN A SOUTHERN COAST OF KOREA

The reproductive success of marine species with external fertilization depends on environmental conditions during gamete release. There is special interest presently in whether water motion causes sperm limitation under natural conditions. We investigated gamete release of Fucus vesiculosus from an exposed shore to ascertain: 1) when gametes are released during the tidal cycle, 2) when fertilization occurs, and 3) what the natural sperm:egg ratios are. Water samples were collected and concentrated over five minutes every half hour off Pemaquid Point, ME from three replicate sites within each of two locations using a pump-filter device. Immunofluorescence microscopy revealed that gamete release occurred only on the two calmest spring tides. Sperm became present in the water column at the same time as oogonia (30 min⁻¹ h prior to high tide [HT]) and reached peak concentration at exactly HT. The sperm:egg ratio was 76:1 on 8 Oct 1999 and 21:1 on 8 Nov 1999 at exactly 30 min prior to HT and dropped sharply after HT. Gametes continued to be collected for several hours after HT but analysis of pronuclear position in aceto-iron-hematoxylin stained eggs revealed that all fertilization occurred at approximately HT. We modelled the total number of days when reproduction was possible using these results and wind and wave data from the National Data Buoy Center. Our research provides evidence that gamete release by F. vesiculosus occurs at slack HT on calm days and that sperm are not a limiting factor in fertilization for this species.     

Female‐induced remote regulation of sperm physiology may provide opportunities for gamete‐level mate choice

In sedentary externally fertilizing species, direct interactions between mating partners are limited and prefertilization communication between sexes occurs largely at the gamete level. Certain combinations of eggs and sperm often have higher fertilization success than others, which may be contingent on egg‐derived chemical factors that preferentially attract sperm from compatible males. Here, we examine the mechanisms underlying such effects in the marine mussel Mytilus galloprovincialis, where differential sperm attraction has recently been shown to be associated with variation in offspring viability. Specifically, we focus on the sperm surface glycans, an individually unique layer of carbohydrates that moderate self‐recognition and other cellular‐level interactions. In many species egg‐derived factors trigger remarkable changes in the sperm's glycan layer, physiology, and swimming behavior, and thus potentially moderate mate choice at the gamete level. Here, we show that sperm glycan modifications and the strength of acrosome reaction are both dependent on specific male–female interactions (male–female combination). We also find associations between female‐induced sperm glycan changes and the Ca²⁺ influx into sperm–‐a key regulator of fertilization processes from sperm capacitation to gamete fusion. Together, our results suggest that female‐induced remote regulation of sperm physiology may constitute a novel mechanism of gamete‐level mate choice.     

Ocean acidification during prefertilization chemical communication affects sperm success

Ocean acidification (OA) poses a major threat to marine organisms, particularly during reproduction when externally shed gametes are vulnerable to changes in seawater pH. Accordingly, several studies on OA have focused on how changes in seawater pH influence sperm behavior and/or rates of in vitro fertilization. By contrast, few studies have examined how pH influences prefertilization gamete interactions, which are crucial during natural spawning events in most externally fertilizing taxa. One mechanism of gamete interaction that forms an important component of fertilization in most taxa is communication between sperm and egg‐derived chemicals. These chemical signals, along with the physiological responses in sperm they elicit, are likely to be highly sensitive to changes in seawater chemistry. In this study, we experimentally tested this possibility using the blue mussel, Mytilus galloprovincialis, a species in which females have been shown to use egg‐derived chemicals to promote the success of sperm from genetically compatible males. We conducted trials in which sperm were allowed to swim in gradients of egg‐derived chemicals under different seawater CO₂ (and therefore pH) treatments. We found that sperm had elevated fertilization rates after swimming in the presence of egg‐derived chemicals in low pH (pH 7.6) compared with ambient (pH 8.0) seawater. This observed effect could have important implications for the reproductive fitness of external fertilizers, where gamete compatibility plays a critical role in modulating reproduction in many species. For example, elevated sperm fertilization rates might disrupt the eggs' capacity to avoid fertilizations by genetically incompatible sperm. Our findings highlight the need to understand how OA affects the multiple stages of sperm‐egg interactions and to develop approaches that disentangle the implications of OA for female, male, and population fitness.     

Activation of follicle cell surface phospholipase by tyrosine kinase dependent pathway is an essential event in ascidian fertilization

Eggs of Ascidia ceratodes and Phallusia mammillata block polyspermy by releasing a phosphatidylinositol‐linked glycosidase from the follicle cell and egg surface that binds to and blocks all unoccupied sperm binding sites on the vitelline coat. Release of this glycosidase is thought to be under the control of a membrane‐bound phospholipase. To elucidate the mechanism of phospholipase activation, intact eggs and isolated follicle cells are activated by either sperm or the tyrosine kinase activator 9,10‐dimethyl‐1,2‐benzanthracene (DMBA). Both treatments caused release of comparable quantities of glycosidase activity, the earliest event following fertilization. A corresponding increase in phospholipase activity accompanied this glycosidase release. The tyrosine kinase inhibitor genistein blocked release by DMBA at concentrations as low as 1 μM, but had no effect on sperm‐induced release even when used up to 100 μM. Tyrphostin A23, another tyrosine kinase inhibitor, when used at 200 μM blocked glycosidase release and decreased phospholipase activity following both DMBA activation and fertilization. Western blot analysis probing for phosphotyrosine content of disrupted intact eggs with their follicle cells revealed the absence of a band in tyrphostin‐treated eggs corresponding to a 40 kDa protein that was present in both unfertilized and fertilized egg samples. Based on these results, we propose that phosphorylation of specific tyrosine residues is necessary for phospholipase activation and is sufficient to trigger subsequent glycosidase release. Mol. Reprod. Dev. 54:69–75, 1999. © 1999 Wiley‐Liss, Inc.     

RECENT ADVANCES IN FERTILIZATION ECOLOGY OF MACROALGAE1

Our understanding of natural patterns of fertilization in seaweeds has increased substantially over the last 10 years due to new approaches and methods to characterize the nature and frequency of fertilization processes in situ, to recognize the conditions and mechanisms enhancing fertilization success, and to anticipate population and community consequences of the patterns of natural fertilization. Successful reproduction in many species depends on a delicate juxtaposition of abiotic and biotic conditions. Important abiotic factors are those triggering gamete release (e.g. single or interacting effects of light quality and water movement) and those affecting gamete viability or concentrations (e.g. salinity effects on polyspermy blocks; gamete dilution due to water movement). Examples of important biotic components are synchronous gamete release, efficiency of polyspermy‐blocking mechanisms, population density of sexually fertile thalli, interparent distances, and male‐to‐female ratios. Field data indicate fertilization frequencies of 70%–100% in broadcasting‐type seaweeds (e.g. fucoids) and 30%–80% in brooding‐type (red) algae. Red algal values are higher than previously thought and challenge presently accepted explanations for their complex life histories. Important population and community questions raised by the recent findings relate to the magnitude of gene flow and exchange occurring in many micropopulations that seemingly breed during periods of isolation, the physiological basis and population effects of male‐to‐male competition and sexual selection during fertilization of brooding seaweeds, and the effects of massive gamete release, especially in holocarpic seaweeds, on benthic and planktonic communities. Comparative studies in other algal groups are now needed to test the generality of the above patterns, to provide critical pieces of information still missing in our understanding of natural fertilization processes, and to elucidate the evolutionary consequences of the different modes of reproduction (e.g. brooders vs. broadcasters).     

Spawning behavior of Arctic charr (Salvelinus alpinus): Spawning synchrony,vibrational communication,and mate guarding

A mismatch in synchrony between male and female gamete release in external fertilizers can result in reduced or failed fertilization, sperm competition, and reduced paternity. In Arctic charr (Salvelinus alpinus), males can adopt either a guard or sneak tactic resulting in both pre‐ and postcopulatory competition between males with alternative reproduction tactics. Here, spawning behavior of free‐living Arctic charr was video‐recorded, and their reproductive behavior was analyzed. From evaluating 157 spawning events, we observed that females mainly spawned with a guarding male and that the female and the guarding male synchronized timing of gamete release under sperm competition. Although sneakers spawned with higher synchrony than the guarding male in single‐male spawning events, the average sneaker released his milt less synchronized with the female than the guarding male under sperm competition. Approximately 50% of the recorded spawning events occurred under sperm competition, where each event included an average of 2.7 males. Additionally, sneakers were more exposed to sperm competition than guarding males. An influx of males, in close proximity to the female, occurred during the behavioral sequences leading up to egg release, but this influx seemed not dependent on egg release, suggesting that something else than gonadal product attracts sneaker males to the spawning female. Just before and during the actual release of gametes, the spawning couple vibrates their bodies in close contact and it seems likely that this vibrational communication between the spawning couple, which results in a larger amplitude sound wave than seen under regular courting, reveals time of gamete release to sneaker males. Thus, vibrational communication may enable synchrony between the guarding male and the female, and this might be traded against the cost of higher detectability from surrounding sneaker males, eavesdropping in close proximity.     

SEXUAL REPRODUCTION IN EPHEDRA NEVADENSIS (EPHEDRACEAE): FURTHER EVIDENCE OF DOUBLE FERTILIZATION IN A NONFLOWERING SEED PLANT

Since the initial discovery of double fertilization in angiosperms in 1898, a number of reports of double fertilization-like events in the genus Ephedra have appeared. Until recently, convincing documentation of double fertilization in Ephedra had not been presented. In Ephedra nevadensis, following entry of a single binucleate sperm cell into the egg cell, one sperm nucleus migrates in a chalazal direction to fuse with the egg nucleus. Contemporaneous with this first fertilization event, the ventral canal nucleus regularly migrates from its initially apical position within the egg cell to a more central position within the egg cytoplasm, where it fuses with a second sperm nucleus. Based on quantitative microspectrofluorometric analysis, occasional supernumerary nuclei within the egg cell (derived by migration through pores in the cell walls between jacket cells and the central cell or egg cell) can be ruled out as participating in the second fertilization event. The evolutionary establishment of double fertilization in Ephedra (or its ancestors) was dependent on a number of specific developmental preconditions: 1) persistence of the ventral canal nucleus (which is degenerate in many groups of nonflowering seed plants) through the time of normal fertilization; 2) regular displacement of the ventral canal nucleus from its initially apical position within the egg cell to a position within the egg cytoplasm where fusion of the egg nucleus with the first sperm nucleus earlier occurred; 3) acquisition of egg-like features by the ventral canal nucleus that allow it to attract and fuse with a sperm nucleus; and 4) consistent entry of a second sperm nucleus into the archegonial cavity to participate in a second fertilization event. Although it cannot be determined definitively whether double fertilization in Ephedra is evolutionarily homologous with double fertilization in flowering plants, comparative evidence is consistent with the hypothesis that double fertilization arose in a common ancestor of the Gnetales and angiosperms.     

Sperm incorporation,the pronuclear migrations,and their relation to the establishment of the first embryonic axis: Time-lapse video microscopy of the movements during fertilization of the sea urchin Lytechinus variegatus

The movements during fertilization have been investigated with differential interference optics and recorded by time-lapse video microscopy of the clear egg of the sea urchin Lytechinus variegatus. Sperm-egg binding occurs rapidly, and following a time when the sperm gyrates on the egg surface, gamete fusion occurs. A rapid cortical contraction radiates from the fusion site and is succeeded by the elevation of the fertilization coat. Sperm incorporation occurs in two stages: the fertilization cone enlarges around and above the erect and immotile sperm and then the sperm head, midpiece, and tail are displaced along the subsurface region of the egg at an average rate of 3.5 μm/min. The formation of the sperm aster moves the male pronucleus from the subsurface region of the egg toward the egg center at a rate of 4.9 μm/min. When the rays of the radial sperm aster appear to contact the female pronucleus, the female pronucleus migrates at a rate of 14.6 μm/min to the center of the sperm aster. The now adjacent pronuclei are moved to the egg center by the continuing enlargement of the sperm aster at a rate of 2.6 μm/min. Syngamy is usually preceded by the disassembly of the sperm aster. The centripetal migration of the pronuclei appears involved in the establishment of the first embryonic axis; cleavage occurs within 8° of the direction of this centering motion.     

Gamete Release at Low Tide in Fucoid Algae: Maladaptive or Advantageous?

This review discusses three questions pertaining to gamete releaseby fucoid algae at low tide: 1) Are gametes viable and doesfertilization occur at low tide?, 2) How many gametes are releasedat low tide versus at high tide? and 3) Is gamete release atlow tide maladaptive or is it selectively advantageous? Gameterelease at low tide (LT) in fucoid algae is observed commonlyin monoecious species from the lower and mid-intertidal zones(e.g., Fucus distichus, F. evanescens, F. gardneri, Pelvetiacompressa); in dioecious species, intact antheridia (containingsperm) are commonly released at low tide (e.g., in Ascophyllumnodosum, F. vesiculosus). Fertilization at low tide can be determinedwith a calcofluor white assay and occurs in at least three species(F. distichus, F. gardneri, P. compressa). In general, fucoidalgae have high levels of fertilization success, but substantialmortality occurs during early embryogenesis in some intertidalzones due to physical stresses. The agarose bead assay is usefulto assess desiccation on an egg-sized scale. Constitutive dehydrin-likeproteins are present in sperm, eggs, and embryos. The releaseof gametes at low tide appears to be a consequence of a mechanismselected to permit gamete release under calm conditions in seawater;this mechanism is described. The timing of adhesion by zygotesat low and high tide is unknown. Such information and additionaldata on the relative proportions of zygotes produced by moneciousspecies at low tide versus high tide are required in order toassess effects of gamete release at low tide upon dispersaland population structure.     

Fertilization in landlocked sea lamprey: storage of gametes, optimal sperm: egg ratio, and methods of assessing fertilization success

Ovulated, unfertilized eggs of sea lamprey Petromyzon marinus could be stored for 1 day at 15° C without significant loss of fertilizing ability. After 2 days storage most eggs could still be fertilized. Lamprey semen could be stored up to 1 day. Thereafter, a decrease in sperm fertilizing ability occurred, accompanied with a decrease in sperm motility. Unlike teleost fish, sea lamprey eggs could still be fertilized after 1 h contact with water. This extended time of gamete fertility after release into water may help to account for the reproductive success of this species. Maximal fertilization rates were obtained at a sperm: egg ratio of 50 000, a ratio recommended for studies on fertility of individual males. Assessing fertilization success 3 min after fertilization (at cytoplasmic bleb stage) or 5 h after fertilization (at two–cell embryo) was strongly correlated ( r =0·92 and 0·98) with estimation and fertilization success at hatching. These results offer improvement in artificial fertilization techniques under laboratory conditions and provide new information on the biology of fertilization in sea lamprey.     

EFFECTS OF MICROTUBULE INHIBITORS ON PRONUCLEAR MIGRATION AND EMBRYOGENESIS IN FUCUS DISTICHUS(PHAEOPHYTA)1

Pronuclear migration in Fucus distichus spp. edentatus (de la Pyl.) Powell is blocked by incubation of fertilized eggs in colchicine (1 mg/ml) and Nocodazole (2 μg/ ml). Rhizoids form prior to decondensation of the sperm chromatin in eggs in which pronuclear fusion is blocked. This occurs during continuous colchicine incubation as well as in eggs recovering from a short treatment with either drug following fertilization. During recovery of the cells, the sperm and egg chromosomes condense, and the sperm chromosomes migrate toward the egg pronucleus. The delay in migration following removal of colchicine is as much as 24 h and is even slower following removal of Nocodazole. The egg chromosomes form a metaphase plate in treated cells while the sperm chromosomes are still distant in the cytoplasm. This suggests that egg centrioles are important in the mitotic division of the zygote, not sperm centrioles. The effect of colchicine treatment on the mitotic plane and cytokinesis is also discussed.     

Egg size in relation to fertilization dynamics in free-spawning tropical reef fishes

In marine invertebrates that spawn by simply releasing their gametes into the water (free-spawning), fertilization success likely is often limited by low sperm concentrations, due to dispersion of mates and dispersal of gametes by water movements. Production of large, low density eggs might be advantageous when sperm concentrations consistently are low, because large target size might increase egg/sperm encounters, and more low than high density eggs could be produced per clutch. Although average fertilization success in the labrid Thalassoma bifasciatum is 95% in both group spawns (in which multiple males compete for fertilizations by producing large quantities of sperm) and pair (mono-male) spawns, it is slightly lower in pair spawns, due to low level sperm limitation that arises because pair-spawning males release near the minimum number of sperm necessary for maximum fertilization. I examined whether variation in egg size and content in T. bifasciatum and other free-spawning fishes is related to variation in spawning mode, to assess whether compensatory production of large, low-density eggs might be contributing to high fertilization success in pair spawns. I found no difference between the volume or density of eggs of (1) pair- and group-spawning females of T. bifasciatum, or (2) pair-and group-spawning congeneric species of labrids, scarids, and serranids, or (3) labrids and scarids with vigorous, rapid spawning movements (which could turbulently diffuse gamete clouds) and those with slow movements. Further, egg density does not decline with increasing egg volume among those fishes. Assuming that egg size can affect fertilization success, then sperm limitation seems unlikely to represent a significant problem for pair-spawning T. bifasciatum, probably because mates place their vents close together during gamete release. The situation regarding sperm limitation in other fishes, and effects of environmentally generated water turbulence on it, are less clear. Interspecific variation in the size and content of these fishes' eggs may relate to provisioning of offspring for different larval life-histories.     

Ultrastructural and experimental investigations of sperm-egg interactions in fertilization ofHydra carnea

Summary Fertilization in the freshwater hydrozoanHydra carnea has been examined by light, scanning and transmission electron microscopy. Sperm penetrate the jelly coat which covers the entire egg surface only at the site of the emission of the polar bodies. The egg surface exhibits a small depression, the so called fertilization pit at this site. Sperm-egg fusion takes place only at the bottom of the fertilization pit.Hydra sperm lack a structurally distinct acrosome and in most of the observed cases, fusion was initiated by contact between the membrane of the lateral part of the sperm head and the egg surfacce. Neither microvilli nor a fertilization cone are formed at the site of gamete fusion. The process of membrane fusion takes only a few seconds and within 1 to 2 min sperm head and midpiece are incorporated in the egg.Electron dense material is released by the egg upon insemination but cortical granule exocytosis does not occur and a fertilization envelope is not formed. The possible polyspermy-preventing mechanisms in hydrozoans are discussed. Hydra eggs can be cut into halves whereupon the egg membranes reseal at the cut edges and the fragments assume a spherical shape. Fragments containing the female pronucleus can be inseminated and exhibit normal cleavage and development. The observation that in such isolated parts the jelly coat will not fuse along the cut edges was used to determine its role in site-specific gamete fusion. These experiments indicate that site-specificity of gamete fusion can be attributed to special membrane properties at the fertilization pit.     

Gamete membrane dynamics during double fertilization in Arabidopsis

In the double fertilization of angiosperms, one sperm cell fertilizes an egg cell to produce a zygote, whereas the other sperm cell fertilizes a central cell to give rise to an endosperm. There is little information on gamete membrane dynamics during double fertilization even though the cell surface structure is critical for male and female gamete interactions. In a recent study, we analyzed gamete membrane behavior during double fertilization by live-cell imaging with Arabidopsis gamete membrane marker lines. We observed that the sperm membrane signals occasionally remained at the boundary of the female gametes after gamete fusion. In addition, sperm membrane signals entering the fertilized female gametes were detected. These findings suggested that plasma membrane fusion between male and female gametes occurred with the sperm internal membrane components entering the female gametes, and this was followed by plasmogamy.     

Mechanism of Ca2+ release at fertilization in mammals.

At fertilization in mammals the sperm triggers a series of oscillations in intracellular Ca2+ within the egg. These Ca2+ oscillations activate the development of the egg into an embryo. It is not known how the sperm triggers these Ca2+ oscillations. There are currently three different theories for Ca2+ signaling in eggs at fertilization. One idea is that the sperm acts as a conduit for Ca2+ entry into the egg after membrane fusion. Another idea is that the sperm acts upon plasma membrane receptors to stimulate a phospholipase C (PLC) within the egg which generates inositol 1,4, 5-trisphosphate (InsP(3)). We present a third idea that the sperm causes Ca2+ release by introducing a soluble protein factor into the egg after gamete membrane fusion. In mammals this sperm factor is also referred to as an oscillogen because, after microinjection, the factor causes sustained Ca2+ oscillations in eggs. Our recent data in sea urchin egg homogenates and intact eggs suggests that this sperm factor has phospholipase C activity that leads to the generation of InsP(3). We then present a new version of the soluble sperm factor theory of signaling at fertilization. J. Exp. Zool. (Mol. Dev. Evol.) 285:267-275, 1999.     

Fertilisation and sperm competition dynamics in salmon

Fishes have evolved an enormous range of mating patterns and sperm competition is important in the majority of these. Detailed mechanisms of fertilization and sperm competition dynamics at the gamete level are poorly understood. We have been using salmon models to conduct in vitro fertilization trials that: (1) allow us experimental control, (2) eliminate confounding effects from the whole organism, and (3) enable detailed measures of sperm form and function in the natural fertilization medium to which gametes are adapted. Using both Atlantic and sockeye salmon, we present results on experiments exploring: (1) the importance of relative sperm number, size, % motility, longevity, and velocity for fertilization and sperm competition success, (2) the importance of natural variation in egg size for 'fertilizability' under sperm limited conditions and (3) the influence of ovarian fluid in fertilization dynamics.     

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis

Angiosperms have a unique sexual reproduction system called “double fertilization.” One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.     

SED1 function during mammalian sperm-egg adhesion

A prerequisite for successful fertilization is the species-specific binding of sperm to the extracellular coat of the egg. Gamete binding triggers the release of sperm hydrolytic enzymes that digest a path through the egg coat, thus bringing sperm into proximity with the egg plasma membrane where gamete fusion occurs. Although some components of the sperm membrane and the egg coat that participate in sperm-egg interactions have been identified, results from targeted deletions and gene substitutions indicate that other, as yet unidentified, gamete receptors must contribute to sperm-egg binding. Recent studies implicate the bi-motif protein, SED1, as being required for successful sperm-egg adhesion in mouse. SED1 contains Notch-like EGF repeats as well as discoidin/F5/8 complement domains--motifs that mediate a variety of cell-cell and cell-matrix interactions. SED1's ability to promote gamete adhesion resides within its two discoidin/F5/8C domains, which are able to dock to substrates as diverse as phospholipid membranes and extracellular matrices. SED1 is also expressed in a wide range of tissues and epithelia, where it may function similarly as an adhesive protein facilitating cell-cell and/or cell-matrix interactions.     

Polyspermy barriers in plants: from preventing to promoting fertilization

Barriers to polyspermy (fertilization of a female gamete by more than one sperm) are essential to successful reproduction in a wide range of organisms including mammals, echinoderms, fish, molluscs, and algae. In animals and fucoid algae, polyspermy results in early death of the zygote due to transmission of extra centrioles from the sperm and consequent disruptions to the mitotic spindle. Accordingly, a variety of mechanisms have evolved to prevent penetration of an egg by more than one sperm, or more than one sperm nucleus from fusing with an egg nucleus. The evolution of internal fertilization has also provided an opportunity to limit the number of sperm that gain access to each egg, as occurs in the mammalian female reproductive tract. Polyspermy and polyspermy barriers in plants have received much less attention. Plants lack centrioles and therefore, polyspermy would not be expected to cause lethal aberrant spindle organization. However, we find evidence from cytological, genetic and in vitro fertilization studies for polyspermy barriers in plants. Angiosperms, like mammals, are internally fertilized, and exert a high level of control over the number of sperm that have access to each female gamete. In particular, regulation of pollen tube growth ensures that in general only two sperm enter each embryo sac, where one fertilizes the egg and the other the central cell. Despite this 1:1 ratio of sperm to gametes within the embryo sac, angiosperms still require a mechanism to ensure that each female gamete is fertilized by one and only one sperm. Here, we present evidence suggesting that a polyspermy block on the egg may be part of the mechanism that promotes faithful double fertilization.