The role of jelly coats in sperm-egg encounters, fertilization success, and selection on egg size in broadcast spawners

Sperm limitation may be an important selective force influencing gamete traits such as egg size. The relatively inexpensive extracellular structures surrounding many marine invertebrate eggs might serve to enhance collision rates without the added cost of increasing the egg cell. However, despite decades of research, the effects of extracellular structures on fertilization have not been conclusively documented. Here, using the sea urchin Lytechinus variegatus, we remove jelly coats from eggs, and we quantify sperm collisions to eggs with jelly coats, eggs without jelly coats, and inert plastic beads. We also quantify fertilization success in both egg treatment groups. We find that sperm-egg collision rates increase as a function of sperm concentration and target size and that sperm are not chemotactically attracted to eggs nor to jelly coats in this species. In fertilization assays, the presence of the jelly coat is correlated with a significant but smaller-than-expected improvement in fertilization success. A pair of optimality models predict that, despite the large difference in the energetic value of egg contents and jelly material, the presence of the jelly coat does not diminish selection for larger egg cell size when sperm are limiting.     

Components of fertilization success in the bluehead wrasse, Thalassoma bifasciatum

In many species of marine organisms, males and females releasegametes directly into the water column. Although free-spawningmarine invertebrates appear to have highly variable fertilizationsuccess, in tropical reef fishes the average fertilizationsuccess is quite high, typically over 90%; nevertheless, substantialvariation has been reported, and fertilization has a directeffect on fitness. We investigated the factors affecting fertilization success in natural spawnings of the bluehead wrasse, Thalassoma bifasciatum. During a two-year study at a site in St. Croix,we found extensive and predictable variation in fertilizationsuccess in pair spawns of this reef fish. Fertilization successaveraged 95%, but was affected by the amount of sperm released,the water velocity at a site, the mating success of the male,and the size of the female. As sperm released in a spawn increases, and as water velocity at a site decreases, sperm concentrationsshould remain higher in the vicinity of eggs for a longer periodof time, and both of these factors are correlated with increasingfertilization success. The recent history of individuals withpartners or sites did not affect the fertilization successof their spawn. In an evolutionary context, the real and predictable variance in fertilization success in this species may influencethe mating choices of males and females. However, there iscurrently no evidence that females use differences in fertilizationsuccess among males or sites in their reproductive decisions.     

Fertilization in an egg-brooding colonial ascidian does not vary with population density

The possibility that free-spawning marine organisms may be subject to fertilization failure at low population density (due to the effects of sperm dilution) has sparked much interest, but these effects have been demonstrated only in a few species that broadcast their eggs. Some egg-brooding species may overcome dilution effects by filtering low concentrations of sperm from seawater and fertilizing eggs throughout an extended period of time. We examined the effects of population density and size on fertilization in Botryllus schlosseri, a hermaphroditic colonial ascidian that free-spawns sperm, but broods eggs. We experimentally manipulated the size and density of mating groups and surveyed fertilization levels in natural populations that varied in density. Fertilization was not affected by variation in population size or density in either the experimental or natural populations. Near the end of the reproductive season, some eggs may have been fertilized too late to complete development, suggesting a temporal form of sperm limitation that has not been considered in other systems. We also detected greater variability in fertilization levels at lower population density. Nevertheless, these results suggest that caution must be used in extrapolating reported density effects on fertilization to all taxa of free-spawners; density effects may be reduced in brooders that have efficient sperm collection mechanisms.     

Does egg competition occur in marine broadcast‐spawners?

When the availability of sperm limits female reproductive success, competition for sperm, may be an important broker of sexual selection. This is because sperm limitation can increase the variance in female reproductive success, resulting in strong selection on females to compete for limited fertilization opportunities. Sperm limitation is probably common in broadcast-spawning marine invertebrates, making these excellent candidates for investigating scramble competition between broods of eggs and its consequences for female reproductive success. Here, we report our findings from a series of experiments that investigate egg competition in the sessile, broadcast-spawning polychaete Galeolaria caespitosa. We initially tested whether the order in which eggs encounter sperm affects their fertilization success at two ecologically relevant current regimes. We used a split-clutch-split--ejaculate technique to compare the fertilization success of eggs from individual females that had either first access (competition-free treatment) or second access (egg competition treatment) to a batch of sperm. We found that fertilization success depended on the order in which eggs accessed sperm; eggs that were assigned to the competition-free treatment exhibited significantly higher fertilization rates than those assigned to the egg competition treatment at both current speeds. In subsequent experiments we found that prior exposure of sperm to eggs significantly reduced both the quantity and quality of sperm available to fertilize a second clutch of eggs, resulting in reductions in fertilization success at high and low sperm concentrations. These findings suggest that female traits that increase the likelihood of sperm-egg interactions (e.g. egg size) will respond to selection imposed by egg competition.     

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.     

Sperm environment affects offspring quality in broadcast spawning marine invertebrates

The provisioning of offspring can have far-reaching consequences for later life in a wide range of organisms and generally this provisioning is thought to be under maternal influence or control. In experiments with a broadcast-spawning ascidian, we found that the size of offspring was determined by egg size and the abundance of sperm present during fertilization. Larger eggs were fertilized at low sperm concentrations, whilst smaller eggs were successfully fertilized at high sperm concentrations. These differences in fertilized egg size resulted in differences in the development rate, hatching success and mean size of the subsequent larvae. Our results suggest that, in contrast to females that reproduce by other mating systems, free-spawning mothers lack some control over the provisioning of offspring. Furthermore, because males can alter the sperm environment, they can exert paternal (non-genetic) control over key offspring characteristics.     

DOES BATEMAN'S PRINCIPLE APPLY TO BROADCAST-SPAWNING ORGANISMS? EGG TRAITS INFLUENCE IN SITU FERTILIZATION RATES AMONG CONGENERIC SEA URCHINS

Evolutionary biologists generally invoke male competition and female choice as mechanisms driving sexual selection. However, in broadcast-spawning organisms sperm may be limiting and females may compete, in the Darwinian sense, for increased mating success. In this study, I investigate how species differences in egg and sperm traits result in different patterns of fertilization among three closely related sea urchins (Strongylocentrotus purpuratus, S. franciscanus, and S. droebachiensis). Field studies demonstrate that all three species achieve similar percentages of eggs fertilized when eggs and sperm are released simultaneously. However, when sperm must disperse before encountering eggs, differences arise among species such that those with the smaller eggs and faster but shorter-lived sperm achieve relatively fewer fertilizations than do species with larger eggs and slower but longer-lived sperm. A field hybridization experiment, field estimates of sperm dispersal, correlations of egg size to field rates of fertilization, laboratory studies of fertilization kinetics, and a simulation model all suggest that it is attributes of the egg (probably egg size) that are responsible for the differences. These patterns of fertilization match the species' patterns of dispersion; species that do well only when sperm and eggs are released in close proximity are more aggregated, species that do relatively well when sperm and eggs are released farther apart are more dispersed. These results are consistent with the notion that eggs of different species are adapted to maximize reproductive success under different degrees of sperm limitation and suggest that male competition and female choice may not be an appropriate dichotomy in broadcast-spawning organisms.     

Remarkable longevity of dilute sperm in a free-spawning colonial ascidian

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.     

The early sperm gets the good egg: mating order effects in free spawners

Mating order can have important consequences for the fertilization success of males whose ejaculates compete to fertilize a clutch of eggs. Despite an excellent body of literature on mating-order effects in many animals, they have rarely been considered in marine free-spawning invertebrates, where both sexes release gametes into the water column. In this study, we show that in such organisms, mating order can have profound repercussions for male reproductive success. Using in vitro fertilization for two species of sea urchin, we found that the 'fertilization history' of a clutch of eggs strongly influenced the size distribution of unfertilized eggs, and consequently the likelihood that they will be fertilized. Males that had first access to a batch of eggs enjoyed elevated fertilization success because they had privileged access to the largest and therefore most readily fertilizable eggs within a clutch. By contrast, when a male's sperm were exposed to a batch of unfertilized eggs left over from a previous mating event, fertilization rates were reduced, owing to smaller eggs remaining in egg clutches previously exposed to sperm. Because of this size-dependent fertilization, the fertilization history of eggs also strongly influenced the size distribution of offspring, with first-spawning males producing larger, and therefore fitter, offspring. These findings suggest that when there is variation in egg size, mating order will influence not only the quantity but also the quality of offspring sired by competing males.     

The kinetics of monospermic and polyspermic fertilization in free-spawning marine invertebrates

The equation of Vogel et al. (1982) is widely used in fertilization studies of free-spawning marine invertebrates to predict the percentage of viable eggs that will be fertilized at any specified levels of gamete concentration and contact time. Here, the random collision model that underlies the Vogel et al. equation is extended to distinguish between monospermic and polyspermic fertilization, and separate equations for the percentages of monospermic and polyspermic fertilization are obtained. These equations provide an explanation for empirical observations which have shown a decreased percentage of successful egg development at high sperm concentrations. Comparison is made with an earlier heuristic attempt (Styan, 1998) to predict the extent of polyspermic fertilization, and it is found that this earlier method can underestimate the percentage of polyspermic fertilization by up to 10 percent. Moreover, the approach used here retains the flexibility to model changes in sperm concentration due to dispersal mechanisms, and is able to model different mechanisms for the block to polyspermy.     

In vitro fertilization experiments using sockeye salmon reveal that bigger eggs are more fertilizable under sperm limitation

Although theory and widespread evidence show that the evolution of egg size is driven primarily by offspring and maternal fitness demands, an additional explanation invokes sperm limitation as a selective force that could also influence egg size optima. Levitan proposed that constraints from gamete encounter in external fertilization environments could select for enlargement of ova to increase the physical size of the fertilization target. We test this theory using in vitro fertilization experiments in an externally fertilizing fish. Sockeye salmon (Onchorhyncus nerka) females show considerable between-individual variation in ovum size, and we explored the consequences of this natural variation for the fertilization success of individual eggs under conditions of sperm limitation. By engineering consistent conditions where in vitro fertilization rate was always intermediate, we were able to compare the sizes of fertilized and unfertilized eggs across 20 fertilization replicates. After controlling for any changes in volume through incubation, results showed that successfully fertilized eggs were significantly larger than the eggs that failed to achieve fertilization. Under conditions without sperm limitation, fertility was unaffected by egg size. Our findings therefore support Levitan''s theory, demonstrating empirically that some element of egg size variation could be selected by fertilization demands under sperm limitation. However, further research on sperm limitation in natural spawnings is required to assess the selective importance of these results.     

Gamete traits influence the variance in reproductive success, the intensity of sexual selection, and the outcome of sexual conflict among congeneric sea urchins

Sea-urchin species differ in susceptibility to sperm limitation and polyspermy, but the influences of gamete traits on reproductive variance, sexual selection, and sexual conflict are unknown. I compared male and female reproductive success of two congeners at natural densities in the sea. The eggs of the species occurring at higher densities, Strongylocentrotus purpuratus, require higher sperm concentrations for fertilization but are more resistant to polyspermy compared to S. franciscanus. Both species show high variance in male fertilization success at all densities and high variance in female success at low densities, but they differ in female variance at high densities, where only S. franciscanus shows high female variance. The intensity of sexual selection based on Bateman gradients is high in males of both species, variable in S. franciscanus females, and low in S. purpuratus females. Strongylocentrotus franciscanus females experience sexual selection at low densities and sexual conflict at high densities. Strongylocentrotus purpuratus may rarely experience sperm limitation and may have evolved to ameliorate sexual conflict. This reduces the variance in female fertilization, providing females with more control over fertilization. Sperm availability influences sexual selection directly by determining sperm-egg encounter probabilities and indirectly through selection on gamete traits that alter reproductive variances.     

Sexual conflict and polyspermy under sperm-limited conditions: in situ evidence from field simulations with the free-spawning marine echinoid Evechinus chloroticus

For free-spawning organisms that release gametes into the sea, sperm limitation (too few sperm to fertilize all eggs) is a major factor limiting reproductive success. Given such circumstances, the presence of several mechanisms to prevent polyspermy (too many sperm) may seem paradoxical; however, a growing body of data suggests that natural fertilization levels, though variable, can routinely be high. Under such conditions, polyspermy is much more likely. The tension between sperm limitation and polyspermy represents sexual conflict because males, in competing to fertilize as many eggs as possible, can impose lethal costs on eggs if multiple sperm gain entry. Here we present data for a marine invertebrate indicating high levels of polyspermy under sperm-limited conditions. When the sea urchin Evechinus chloroticus was induced to spawn in situ, mean rates of polyspermy were [Formula: see text], and polyspermy was recorded at rates as high as 62.7%. Polyspermy was nearly always present, even when fertilization rates were <50%, confirming predictions that it should be present under sperm-limited conditions. Both sperm limitation and polyspermy imposed substantial reproductive costs, and we conclude that both sexual conflict related to polyspermy and sperm limitation have been simultaneous strong selective forces shaping the evolution of reproductive traits in the sea.     

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.     

Sperm limitation in the sea

Because sperm outnumber eggs, it is often assumed that variation in female reproductive success has little to do with male or sperm availability. Similarly for males, access to viable eggs and sperm competition are thought to drive variation in male fertilization success. These assumptions result from empirical studies on organisms with internal fertilization. However, recent evidence from free-spawning organisms suggests that sperm can often be limiting. This finding may alter our perspective on mating-system evolution, especially in externally fertilizing organisms.     

Influence of sperm density and contact time on herring egg fertilization

In order to standardize fish egg incubation techniques for bioassay application, fertilization procedures need to be included into the protocols. Nothing is known about the necessary sperm concentrations required to achieve optimal fertilization rates. The tests described here for the herring Clupea harengus L. include trials with 10 different sperm densities and 4 contact times (15, 30, 60 and 120 seconds). The variability of fertilization rates in eggs from different females was also investigated. Fertilization success was mainly influenced by sperm density and less by the actual contact time between unfertilized eggs and sperm containingmedia. Dilution in sperm density to 9.6 × 10⁶ cells ml^-1 or less resulted in reduced fertilization success. There was considerable variability in fertilization rates between females.     

Chemically moderated gamete preferences predict offspring fitness in a broadcast spawning invertebrate

Sperm chemoattraction, where sperm locate unfertilized eggs by following a concentration gradient of egg-derived chemoattractants, has been widely documented across numerous taxa. While marine invertebrates are favoured models for understanding the underlying mechanisms of sperm chemoattraction, the evolutionary forces underpinning the process remain enigmatic. Here, we show that in mussels (Mytilus galloprovincialis), chemically moderated gamete preferences promote assortative fertilizations between genetically compatible gametes. When offered the choice of egg clutches from two females, sperm exhibited consistent but differential ‘preferences’ for chemical cues secreted from conspecific eggs. Critically, our data reveal that the preferences shown by sperm during the egg-choice trials are highly predictive of early embryonic viability when eggs and sperm from the same individuals are mixed during standard (no-choice) fertilization assays. Moreover, we demonstrate that by experimentally separating chemoattractants from eggs, sperm swimming behaviour is differentially regulated by egg-derived chemoattractants, and that these changes in sperm behaviour are highly consistent with observed patterns of gamete preferences, fertilization and larval survival. Together, this integrated series of experiments reveals that the behaviour of sperm is fine-tuned to respond differentially to the chemical signals emitted from different conspecific eggs, and that these choices have measurable fitness benefits.     

Mating behavior in the black hamlet — gamete trading or egg trading?

Synopsis The black hamlet (Hypoplectrus nigricans, Serranidae) is a simultaneous hermaphrodite, like many other serranines. It has external fertilization and planktonic eggs and engages in a kind of reciprocal spawning consisting of three components: (1) The clutch is divided into sequentially spawned parcels. (2) Partners regularly alternate release of parcels in a spawning bout. (3) Courtship is associated with the female spawning role. Two hypotheses have been proposed to account for this pattern. The egg trading hypothesis states that the pattern results from competition for fertilizations and assumes that reproductive success (RS) as a male is limited by access to eggs and that female RS is not limited by access to sperm. The gamete trading hypothesis states that the pattern results from eggs being at substantial risk of not being fertilized — i.e. female RS is limited by access to sperm. An analysis was performed of data from the black hamlet and three other serranines to determine whether significant sperm limitation occurs. The evidence fails to support the hypothesis that access to sperm limits female RS. Unspawned eggs were not found in fish collected outside the spawning period, but fish without eggs always had milt (sperm). The percentage of eggs fertilized did not decrease over the course of a spawning bout. There was also a slight positive correlation between the number of eggs released in a spawning and the percentage that were fertilized, and serranines that divide the clutch into parcels do not tend to have higher fecundities than those that do not. The gamete trading hypothesis can therefore be rejected for H. nigricans.     

A profile of fertilization in mammals

Fertilization is defined as the process of union of two gametes, eggs and sperm. When mammalian eggs and sperm come into contact in the female oviduct, a series of steps is set in motion that can lead to fertilization and ultimately to development of new individuals. The pathway begins with species-specific binding of sperm to eggs and ends a relatively short time later with fusion of a single sperm with each egg. Although this process has been investigated extensively, only recently have the molecular components of egg and sperm that participate in the mammalian fertilization pathway been identified. Some of these components may participate in gamete adhesion and exocytosis, whereas others may be involved in gamete fusion. Here we describe selected aspects of mammalian fertilization and address some of the latest experimental evidence that bears on this important area of research.     

Density-dependent sexual selection in external fertilizers: variances in male and female fertilization success along the continuum from sperm limitation to sexual conflict in the sea urchin Strongylocentrotus franciscanus

Sperm competition and female choice are fundamentally driven by gender differences in investment per offspring and are often manifested as differences in variance in reproductive success: males compete and have high variance; most females are mated and have low variance. In marine organisms that broadcast spawn, however, females may encounter either sperm limitation or sperm competition. I measured the fertilization success of male and female Strongylocentrotus franciscanus over a range of population densities using microsatellite markers. Female fertilization success first increased and then decreased with mate density, limited at low density by sperm limitation and at high density by polyspermy. Mate density affected variance in fertilization success in both males and females. In males, the variance in fertilization success increased with mate density. In females, the pattern was more complex. The variance in female success increased similarly to males with increased mate density but then decreased to low levels at intermediate densities, where almost all eggs were fertilized. As density increased further, the female variances again increased as polyspermy lowered average fertilization success. Male and female variances differed only at intermediate densities. At low densities, both sexes may be under selection to increase fertilization success; at intermediate densities, males may compete; and at high densities, both sexes may be under selection to increase success by increasing (males) or decreasing (females) likelihood of fertilization during sexual conflict. Only within a narrow range of densities do patterns of sexual selection mirror those typically noted in internally fertilizing taxa.