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.     

Chemical characterization of the component of the jelly coat from sea urchin eggs responsible for induction of the acrosome reaction.

Jelly coat, a multicomponent extracellular matrix surrounding the sea urchin egg, induces the acrosome reaction in sperm. The jelly coats of the four species studied, Arbacia punctulata, Strongylocentrotus purpuratus, Strongylocentrotus drobachiensis, and Lytechinus variegatus, were found to be very similar in chemical composition. A sialoprotein (approximately 20% of the mass of the jelly coat) and a fucose sulfate polysaccharide (approximately 80%) are the major macromolecular components of the jelly coat. The acrosome reaction inducing capacity resides solely in the fucose sulfate polysaccharide. Induction of the acrosome reaction ranges from highly species specific to nonspecific. Thus, A. punctulata and S. drobachiensis sperm are induced to undergo the acrosome reaction only with their homologous jelly coat, while S. purpuratus sperm react equally well with homologous or L. variegatus jelly coat, but not with A. punctulata jelly coat. L. variegatus sperm seem to be relatively nonspecific in response. Species-specific induction of the acrosome reaction resides solely in the fucose sulfate polysaccharide, suggesting that there must be structural differences in this polysaccharide in the various species. Therefore, in some species, fertilization appears to involve sperm-egg recognition at the level of the jelly coat as well as at the level of sperm-egg receptors.     

Bufo japonicus japonicus and Xenopus laevis laevis egg jellies contain structurally related antigens and cortical granule lectin ligands

The antigenic relationship of the egg jelly coat glycoproteins from Bufo japonicus japonicus and Xenopus laevis laevis was investigated using agar double diffusion methods. The presence of ligands in the jelly coats for the cortical granule lectin from X.l. laevis eggs was also investigated. Anti-jelly serum for both anuran species crossreacted with the jelly coat from the other species with precipitin patterns of identity. Each egg jelly coat of both species contained two ligands for the cortical granule lectin. Although the ligands in the two different jelly coats appeared to react with the lectin in a pattern of identity, the species ligands were antigenically distinguishable using anti-Xenopus jelly serum. The observations that the two anuran egg jelly coats were antigenically related and that they both contained ligands for the X.l. laevis cortical granule lectin was interpreted in terms of fertilization mechanisms in the two different species. In addition, these observations bring into question the currently accepted phylogenetic relationship of B.j. japonicus and X.l. laevis.     

Profiling the morphological distribution of O-linked oligosaccharides

The morphological distribution of oligosaccharides is determined in the egg jelly surrounding Xenopus laevis eggs. This biological system is used to illustrate a method for readily identifying and quantifying oligosaccharides in specific tissues. The extracellular matrix surrounding X. laevis eggs consists of a vitelline envelope and a jelly coat. The jelly coat contains three morphologically distinct layers designated J1, J2, and J3 from the innermost to the outermost and is composed of 9-11 distinct glycoproteins. Each jelly layer is known to have specific functions in the fertilization of the egg. We developed a rapid method to separate and identify the oligosaccharides from X. laevis egg jelly layers. Identification was based on the retention times in high-performance liquid chromatography (porous graphitized carbon column), exact masses, and tandem mass spectrometry. Over 40 neutral and 30 sulfated oligosaccharides were observed in the three jelly layers. Neutral oligosaccharide structures from different jelly layers were both unique and overlapping, while sulfated oligosaccharides were detected only in layers J1 and J2. Neutral oligosaccharides unique to jelly layer J3 and the combined layers J1+J2 had similar core structures and similar residues. However, differences between these two sets of unique oligosaccharides were also observed and were primarily due to the branching carbohydrate moieties rather than the core structures.     

The jelly envelopes and fertilization of eggs of the newt, Notophthalmus viridescens

Fertilization in Notophthalmus viridescens is internal and involves passage of the sperm through five layers of egg jelly (J5-J1, from outermost to innermost), each of which is secreted by a discrete region of the oviduct. Polyspermy is normal. Passage of the sperm through the jelly and into the egg was studied by a technique of artificial insemination similar to natural insemination, in that undiluted fluid from the vas deferens was applied directly to eggs with various layers of jelly present, followed by flooding with water three to five minutes later. In general, successful fertilization increased as the number of jelly layers increased; jellyless coelomic eggs were not fertilizable. Sperm passage through the jelly and into the egg usually occurs within one to three minutes. Upon hydration of the jelly, barriers to sperm penetration develop in layers J5 and J3. Changes in the egg jelly thus seem to be involved in the restriction of polyspermy to a low level.     

Life-history consequences of investment in free-spawned eggs and their accessory coats

The optimal trade-off between offspring size and number can depend on details of the mode of reproduction or development. In marine organisms, broadcast spawning is widespread, and external coats are a common feature of spawned eggs. Egg jelly coats are thought to influence several aspects of fertilization and early development, including the size of the target for sperm, fertilization efficiency, egg suspension time, polyspermy, embryo survival, and fecundity. These costs and benefits of investment in jelly result in trade-offs that can influence optimal reproductive allocation and the evolution of egg size. I develop an optimization model that sequentially incorporates assumptions about the function of egg coats in fertilization. The model predicts large variation in coat size and limited variation in ovum size under a broad range of conditions. Heterogeneity among spawning events further limits the range of ovum sizes predicted to evolve under sperm limitation. In contrast, variation in larval mortality predicts a broad range of optimal ovum sizes that more closely reflects natural variation among broadcast-spawning invertebrates. By decoupling physical and energetic size, egg coats can enhance fertilization, maintain high fecundity, and buffer the evolution of ovum size from variation in spawning conditions.     

Reproduction and the energy cost of defense in a Batesian mimicry complex

Summary The amount of energy invested in reproduction and in defense was examined in a Batesian mimicry complex consisting of the modelEleodes obscura and the mimicStenomorpha marginata (both Coleoptera: Tenebrionidae). Models live up to 4 y as adults while mimic adults live only 3 mo. The energy content of the eggs of the model and mimic was determined by microbomb calorimetry. The energy content of the defensive secretions produced by the model was determined by computational chemistry and MNDO computer programming. Contrary to the predictions of some life-history theory, the long-lived model annually produces many small eggs each of low energetic content, while the short-lived mimic annually produces fewer, larger eggs each of high energetic content. However, in terms of total energy, the long-lived model has an annual investment in reproduction equal to that of the short-lived mimic. During the 3 mo of co-ocurrence of models and mimics within a year, an average individual model's cost in using defensive secretions against potential predators is 12% of the amount of energy tied up in the eggs that it produces within the year. The annual cost of defense for the model is 18% of the energy contained in the mean number of eggs produced. When the energy allocated to eggs is added to that allocated to defense, the model has an annual investiment that is greater than the annual investment in reproduction by the mimic. Although the energy invested in defense by the model is small relative to the energy invested in egg production, it buys the model considerable protection from predation. Nevertheless, the cost of defense does not explain the deviations from the predictions of life-history theory.     

Structural analysis of 20 oligosaccharide-alditols released from the jelly coat of Rana palustris eggs by reductive beta-elimination characterization of the polymerized sequence [Gal(beta1, 3)GalNAc(alpha1-4)]n.

The eggs of amphibians are surrounded by three to eight layers of jelly coats. This extracellular matrix is mainly composed of hydrated mucin-type glycoproteins. These highly glycosylated molecules are synthesized by oviduct and play an important role in the fertilization process. Recent structural analyses have shown the strict species-specificity of the O-linked oligosaccharides which constitute 60-70% of these oviducal mucins. Consequently, these carbohydrate chains represent new phenotypic markers, and from a biological point of view, can influence parasite tropism or can be involved in species-specific interaction of gametes. The primary structure of 20 oligosaccharide-alditols, released by alkali/borohydride treatment from the mucin of Rana palustris egg jelly coats, was established by 1H and 13C-NMR analysis. Thirteen of these components possess new structures and the polymerization of the sequence Gal(beta1-3)GalNAc(alpha1-4) characterizes the species-specificity of R. palustris.     

Vertebrate extracellular preovulatory and postovulatory egg coats

Extracellular egg coats deposited by maternal or embryonic tissues surround all vertebrate conceptuses during early development. In oviparous species, the time of hatching from extracellular coats can be considered equivalent to the time of birth in viviparous species. Extracellular coats must be lost during gestation for implantation and placentation to occur in some viviparous species. In the most recent classification of vertebrate extracellular coats, Boyd and Hamilton (Cleavage, early development and implantation of the egg. In: Parkes AS (ed.), Marshall's Physiology of Reproduction, vol. 2, 3rd ed. London: Longmans, Green & Co; 1961:1-126) defined the coat synthesized by the oocyte during oogenesis as primary and the coat deposited by follicle cells surrounding the oocyte as secondary. Tertiary egg coats are those synthesized and deposited around the primary or secondary coat by the maternal reproductive tract. This classification is difficult to reconcile with recent data collected using modern molecular biological techniques that can accurately establish the site of coat precursor synthesis and secretion. We propose that a modification to the classification by Boyd and Hamilton is required. Vertebrate egg coats should be classed as belonging to the following two broad groups: the preovulatory coat, which is deposited during oogenesis by the oocyte or follicle cells, and the postovulatory coats, which are deposited after fertilization by the reproductive tract or conceptus. This review discusses the origin and classification of vertebrate extracellular preovulatory and postovulatory coats and illustrates what is known about coat homology between the vertebrate groups.     

Primary structure of 12 neutral oligosaccharide-alditols released from the jelly coats of the anuran Xenopus laevis by reductive {beta}-elimination

The O-linked oligosaccharides of the jelly coat surroundingthe eggs of Xenopus laevis were analysed by ¹H-NMR spectroscopy.Among the 12 neutral oligosaccharide-alditols which have beencharacterized, three of them posses the following unusual structures,As previously observed for six other amphibian species, thecarbohydrate chains of the jelly coat of Xenopus eggs displaya high species specificity which could support a biologicalrole during the fertilization processes. amphibian egg jelly coats ¹H-NMR oligosaccharide structure Xenopus laevis     

The sea urchin egg jelly coat is a three-dimensional fibrous network as seen by intermediate voltage electron microscopy and deep etching analysis

The egg jelly (EJ) coat which surrounds the unfertilized sea urchin egg undergoes extensive swelling upon contact with sea water, forming a threedimensional network of interconnected fibers extending nearly 50 μm from the egg surface. Owing to its solubility, this coat has been difficult to visualize by light and electron microscopy. However, Lytechinus pictus EJ coats remain intact, if the fixation medium is maintained at pH 9. The addition of alcian blue during the final dehydration step of sample preparation stains the EJ for visualization of resin embedded eggs by both light and electron microscopy. Stereo pairs taken of thick sections prepared for intermediate voltage electron microscopy (IVEM) produce a threedimensional image of the EJ network, consisting of interconnected fibers decorated along their length by globular jelly components. Using scanning electron microscopy (SEM), we have shown that before swelling, EJ exists in a tightly bound network of jelly fibers, 50–60 nm in diameter. In contrast, swollen EJ consists of a greatly extended network whose fibrous components measure 10 to 30 nm in diameter. High resolution stereo images of hydrated jelly produced by the quick-freeze/deep-etch/rotary-shadowing technique (QF/DE/RS) show nearly identical EJ networks, suggesting that dehydration does not markedly alter the structure of this extracellular matrix. © 1993 Wiley-Liss, Inc.     

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Histochemical studies of jelly coat of sea-urchin eggs during oogenesis

Summary Jelly coat of sea-urchin eggs consists of polysaccharides and glycoproteins. Some properties of jelly coat have already been investigated, but not histochemically. The oogenesis in Paracentrotus lividus was studied histologically and the oocytes were classified into six different stages. The extracellular jelly appeared first around the growing oocytes II which remained attached to the germinal epithelium. The jelly became thicker when the oocyte approached maturation. Histochemical analysis revealed that the jelly consists of mucopolysaccharide-protein-complexes. The polysaccharide component is composed of both neutral and acid mucopolysaccharides. The former are amylase-resistant. The acid mucopolysaccharides contain both carboxyl and sulfate groups, which are in close proximity to vicinal hydroxyl groups. Sulfated mucopolysaccharide is hyaluronidase-resistant. Sialic acid could not be clearly demonstrated, because it seems to be resistant to neuraminidase. Pepsin digestion indicated the masking of acidic groups by proteins which compete with basic dyes (Alcian blue, Azure A, coriphosphine etc.). Proteolytic digestion enhanced dye-binding ability of jelly, but removed also some of the periodate-reactive mucosubstances. Also a protein component could be demonstrated histochemically. No histochemical difference between jelly coat of oocytes and that of eggs has been found. The possible molecular structure of jelly coat is discussed.     

Structure and Macromolecular Composition of the Egg and Embryo Jelly Coats of the Anuran Lepidobatrachus laevis

Eggs and cleavage-stage embryos of the frog Lepidobatrachus laevis are encased by 3 μm thick vitelline/fertilization envelope and two jelly layers, termed J₁ (innermost) and J₂ (outermost). Based on light and transmission electron microscopy, J₁ had a dense reticular appearance whereas J₂ had a laminar structure. Direct dissolution of the jelly coats was accomplished by reduction of disulfide bonds with 0.08 M 2-mercaptoethanol at pH 10. Soluble jelly preparations were uncontaminated with nucleic acid (A²⁸⁰/A²⁶⁰=1.44) and yielded an average of 150 μg protein/egg or embryo (n=5). The biochemical composition of the jelly coats in unfertilized eggs was different from that in embryos. When examined via gel permeation chromatography, soluble jelly from unfertilized eggs contained macromolecules which were markedly larger and more heterogeneous (earlier eluting and broader peaks) than jelly from embryos. Differences in the components of jelly from unfertilized eggs and embryos were also observed by electrophoresis, however, a 29,700 molecular weight glycoprotein chain was common to both jelly preparations. The electrophoretic pattern of jelly obtained from parthenogenetically activated eggs was identical to that of unfertilized eggs, therefore the fertilization-associated changes are not due to the exclusive action of cortical granule products.     

Structural features of the abalone egg extracellular matrix and its role in gamete interaction during fertilization

Abalone eggs are surrounded by a complex extracellular coat that contains three distinct elements: the jelly layer, the vitelline envelope, and the egg surface coat. In this study we used light and electron microscopy to describe these three elements in the red abalone (Haliotis rufescens) and ascribe function to each based on their interactions with sperm. The jelly coat is a spongy matrix that lies at the outermost margin of the egg and consists of variably sized fibers. Sperm pass through this layer with their acrosomes intact and then go on to bind to the vitelline envelope. The vitelline envelope is a multilamellar fibrous layer that appears to trigger the acrosome reaction after sperm binding. Next, sperm release lysin from their acrosomal granules, a nonenzymatic protein that dissolves a hole in the vitelline envelope through which the sperm swims. Sperm then contact the egg surface coat, a network of uniformly sized filaments lying directly above the egg plasma membrane. This layer mediates attachment of sperm, via their acrosomal process, to the egg surface. © 1995 Wiley-Liss, Inc.     

Identification of a calsequestrin-like protein from sea urchin eggs

Following studies on calcium transport by isolated smooth endoplasmic reticulum from unfertilized sea urchin eggs (Oberdorf, J. A., Head, J. F., and Kaminer, B. (1986) J. Cell Biol. 102, 2205-2210) we have purified and partially characterized a calsequestrin-like protein from this organelle isolated from eggs from Strongylocentrotus droebachiensis and Arbacia punctulata. Muscle calsequestrin from sarcoplasmic reticulum is well characterized as a calcium storage protein. The egg protein resembles calsequestrin in its behavior in purification steps, electrophoretic mobility, blue staining with Stains-all on polyacrylamide gels, and its calcium binding and amino acid composition. Purification was attained with DEAE-cellulose and hydroxyapatite chromatography. The egg protein Mr of 58,000 in the Laemmli gel system is reduced to 54,000 under Weber-Osborn (neutral) conditions, thus showing a pH dependence in its mobility, although less than occurs with muscle calsequestrins. 25% of its amino acids are acidic and 10% basic. It binds 309 nmol of Ca2+/mg of protein, within the range reported for cardiac calsequestrin. Antigenically, the sea urchin egg protein is related to cardiac calsequestrin capable of binding anti-cardiac calsequestrin antibody.     

Molecular phenotyping of maternally mediated parallel adaptive divergence within Rana arvalis and Rana temporaria

When similar selection acts on the same traits in multiple species or populations, parallel evolution can result in similar phenotypic changes, yet the underlying molecular architecture of parallel phenotypic divergence can be variable. Maternal effects can influence evolution at ecological timescales and facilitate local adaptation, but their contribution to parallel adaptive divergence is unclear. In this study, we (i) tested for variation in embryonic acid tolerance in a common garden experiment and (ii) used molecular phenotyping of egg coats to investigate the molecular basis of maternally mediated parallel adaptive divergence in two amphibian species (Rana arvalis and Rana temporaria). Our results on three R. arvalis and two R. temporaria populations show that adaptive divergence in embryonic acid tolerance is mediated via maternally derived egg coats in both species. We find extensive polymorphism in egg jelly coat glycoproteins within both species and that acid‐tolerant clutches have more negatively charged egg jelly – indicating that the glycosylation status of the jelly coat proteins is under divergent selection in acidified environments, likely due to its impact on jelly water balance. Overall, these data provide evidence for parallel mechanisms of adaptive divergence in two species. Our study highlights the importance of studying intraspecific molecular variation in egg coats and, specifically, their glycoproteins, to increase understanding of underlying forces maintaining variation in jelly coats.     

Energy content of spider eggs

Summary The energy content of spider eggs was determined on samples from 12 species representing 6 families. These values ranged from 26.32 to 29.00 joules per mg ash-free dry mass with a mean of 27.30±SE of 0.27. The higher values were found in those species that overwinter as developmental stages within the egg sac. Rates of energy expenditure of developing eggs and spiderlings within the egg sac were only 7 to 19% of those of emerged spiderlings. The energy conserved by this reduction in rate of metabolism may facilitate survival without feeding during the potentially long periods of aerial dispersal by ballooning, a characteristic activity of most newly emerged spiderlings. The variation in mass-specific energy content is less than variation in clutch size and individual egg size on an intra-and interspecific basis. There was no correlation between energy content per unit egg mass and size of the female parent, egg size, or clutch size. Further analysis indicated that no single measure such as clutch size accurately represents the proportional amount of energy invested in reproduction in these animals.     

Fertilization of the eggs ofCynops pyrrhogaster (japanese newt) after immersion in water

Summary Cynops pyrrhogaster oviducal eggs with and without jelly envelopes (jelly egg and dejellied egg respectively) were immersed in water, and then inseminated artificially. After 1 h of immersion in water, more than half the dejellied eggs were fertilized and developed, but no jelly eggs developed. The rapid decrease in the ability of jelly eggs to be fertilized after immersion in water is not due to a deficiency in the egg. Our results make it clear that hydrated jelly envelopes prevent the eggs from fertilizing. The ability of the egg to be fertilized decreases for a long time in water and this decrease proceeds more slowly in De Boer's solution or Holtfreter's balanced salt solution than in water.     

Structural analysis of hexa to dodecaoligosaccharide-alditols released by reductive {beta}-elimination from oviducal mucins of Bufo bufo

Hexa to dodecasaccharide-alditols of the jelly coat surroundingthe eggs of the toad Bufo bufo were studied by methylation analysis,MALDI-TOF mass spectrometry, and ¹H-NMR spectroscopy. Thesehighly species-specific carbohydrate chains exhibit new structuralfeatures, such as the elongation of the blood group A determinantwith an external -1,3-linked galactose unit, or ramificationbelonging from a fucosylated galactose. The most representativeoligosaccharide-alditol of the series was defined as following: Since the jellies surrounding amphibian eggs are involved inegg-sperm interactions, these structural investigations canprovide biochemical support for exploring the fertilizationprocess. amphibian egg jelly coats Bufo bufo NMR oligosaccharides