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.     

N-Linked Oligosaccharides of Sea Urchin Egg Jelly Induce the Sperm Acrosome Reaction

The sea urchin egg jelly coat (EJ) induces the acrosome reaction (AR) of sperm. We previously demonstrated that a fraction of EJ containing two glycoproteins of 82- and 138-kDa possess the AR inducing activity (8). Here we show that Peptide-N-Glycosidase-F treatment of EJ followed by precipitation and washing in 70% ethanol results in a substantial loss of AR inducing activity in the ethanol insoluble material. When a PNGase-F digest of EJ is chromatographed on a Sepacryl-200 gel filtration column, an AR inducing fraction elutes within the partitioning volume. Acrosome reaction inducing activity of undigested EJ does not elute within the partitioning volume. The chromatographed AR inducing fraction of the PNGase-F digest reacts strongly in the phenol-sulfuric assay demonstrating carbohydrate is present; silver stained gels do not detect the presence of protein. Harsh alkaline hydrolysis of EJ in an excess of NaBH₄, preserves a substantial amount of AR inducing activity. These data show that N-linked oligosaccharides released from EJ by PNGase-F digestion are capable of inducing the sperm acrosome reaction.     

A study of factors involved in induction of the acrosomal reaction in sperm of the sea urchin, Arbacia punctulata.

Several factors involved in induction of the acrosomal reaction in sperm of the sea urchin, Arbacia punctulata, have been investigated quantitatively using a simple substrate film technique to monitor extension of the acrosomal process by electron microscopy. Verification of typical acrosomal process formation has been accomplished using thin sections. Sperm were found to undergo the acrosomal reaction in artificial sea water in the absence of egg jelly coat at pH values above 9.6. In the presence of egg jelly a high percentage of sperm react at pH 8.6. At this pH, the fraction of sperm that undergo the acrosomal reaction is directly proportional to the concentration of egg jelly. The Ca²⁺ ionophore A23187 induces the acrosomal reaction in the absence of egg jelly at pH 8.6. The proportion of sperm that react is dependent on the concentration of ionophore and on the concentration of Ca²⁺ in the medium. Pretreatment of sperm with low levels of La³⁺ ion, which is known to be a Ca²⁺ ion antagonist, results in inhibition of egg jelly induction of the acrosomal reaction. These findings suggest that there are marked similarities between the acrosomal reaction in sea urchin sperm and membrane fusion dependent secretory processes in other cell types.     

怀头鲇胚胎发育过程卵膜形态结构变化及对孵化影响

采用扫描电镜和光学解剖镜,对黑龙江水域怀头鲇(Silirus soldatovi)成熟卵膜层次构造和受精卵胚胎发育过程中卵膜形态结构变化进行观察,并比较未脱黏和人工脱黏卵受精卵膜的表面超微结构变化.结果显示,受精卵膜的胶膜表面由一层薄而致密的物质组成,上有微孔构造.未脱黏受精卵膜表面胶膜光滑致密,多孔隙,内有小梁相连,随胚胎发育逐渐膨胀、展开、变薄,破膜期自然脱落.人工脱黏几乎全部脱去鱼卵的胶膜层,从而使卵失去黏性.脱去胶膜层的受精卵膜表面由不规则的颗粒状结构紧密嵌合而成,表面粗糙,胚胎发育过程中颗粒形状变化不大,但颗粒层逐渐变薄而且疏松,直至胚胎破膜而出:胚胎发育后期颗粒层有过早脱落和破洞出现.同时对活体鱼卵进行连续比较观察,讨论了卵膜结构及动态变化与孵化效果的关系.     

Ultrastructure of detergent-resistant cytoskeletons in the noncortical domain of sea urchin eggs as revealed by the quick-freeze deep-etch technique.

The ultrastructure of detergent-resistant cytoskeletons in the noncortical cytoplasm of sea urchin eggs was studied by quick-freeze, deep-etch electron microscopy. Two different cytoskeletal organizations were identified in the detergent-treated sea urchin eggs. They were distinguished by the presence or the absence of long actin filaments and probably correspond to the cortex and the noncortical cytoplasm, respectively. The non-cortical cytoplasm was composed of a complex network (designated here as the ground network) of filaments 6 to 13 nm in diameter, that interconnected aggregates of small globular materials, yolk granules and a meshwork of uniform filaments (8-9 nm in diameter). The 6 to 13 nm filaments comprising the ground network were branched and associated with filaments of the same or other sizes, resulting in the formation of an extremely complex network. The meshwork of 8-9 nm filaments was homogeneous in composition and constitutes a novel structure which has not been previously described. The 8-9 nm filaments were connected to one another at their ends, forming a meshwork of polygons. Meshworks, ranging up to 3 microns in diameter, were distributed throughout the non-cortical cytoplasm of the egg. Similar cytoplasmic structures were also observed in fertilized eggs.     

Electron Microscopic Observations on the Cortical Reaction of the Brittle-Star Amphipholis kochii Lütken, with Special Reference to its Vitelline Coat Modification as Revealed by the Surface Replica Method

This paper describes the fine structural changes of the egg of the brittle-star Amphipholis kochii Lütken during the cortical reaction. The vitelline coat is 20 nm thick, when Ruthenium Red stain is used, and consists of a dense network of fibers. The cortical granules are large, 1.5–2.0 μm in diameter, and exist in several layers in the egg cortex, unlike the monolayer arrangement found in many other animals. The contents of the cortical granules are clearly distinguished into two components: peripheral fibrous (PF) material and central fibrous (CF) material that consists of two components differing in electron density. The PF material is densely stained by periodic acid-chromic acid-silver methenamine stain, while the CF material is stained little if at all by this technique. The vitelline coat and some PF materials form the fertilization membrane, which is about 40 nm thick and consists of three layers; the outer and the inner layer of the fertilization membrane each have a trilaminated structure. The vitelline coat substances are probably located in the upper part of the fertilization membrane. The hyaline layer, 7–8 μm thick, consists mainly of CF materials. These observations on the morphology of the ophiuroid egg are discussed in comparison with those on other echinoderms, especially echinoids and asteroids.     

Egg jelly layers of Xenopus laevis are unique in ultrastructure and sugar distribution

Jelly coats surrounding the eggs of the South African clawed toad, Xenopus laevis, consist of three transparent, gelatinous layers: the innermost layer (J1), the middle layer (J2), and the outer layer (J3). The distribution of N-acetylglucosamine within these jelly coats, as probed with FITC-conjugated wheat germ agglutinin (WGA-FITC), and the matrix ultrastructure of each layer, as visualized in platinum replicas produced by the quick-freeze, deep-etch, and rotary-shadowing technique, suggests that each layer has a unique fiber and glycoprotein composition. J1 extends nearly 200 μm from the egg surface and exhibits no WGA-FITC staining. Stereo images of platinum replicas indicates that J1 consists of a tightly knit network of 5–10 nm fibers decorated with 10–20 nm particulate components. In contrast, J2 is a relatively thin layer, extending only 25–40 μm from the outer aspect of J1. When visualized by confocal microscopy, J2 displays a multilayered WGA-FITC staining pattern. The ultrastructure of J2 consists of sheets of fine fibers that run parallel to one another and that can be identified by their ability to bind WGA-colloidal gold. The fibers of each sheet run at an oblique angle to fibers in neighboring layers. J3 extends 100 μm or more from J2. The WGA-FITC staining pattern shows high intensity in its outer region and less intensity in regions closer to J2. Like J1, the J3 ultrastructure consists of a network of 5–10 nm fibers, decorated with 10–20 nm particulate components. The results of these studies add to a growing body of information that suggests the jelly coats surrounding the eggs of many animals consist of a fibrous glycoprotein superstructure that acts as a scaffold to which globular glycoproteins are bound. © 1996 Wiley-Liss, Inc.     

On the macromolecular composition of the jelly coat from Strongylocentrotus purpuratus eggs

The number of macromolecular components present in the egg jelly coat of Strongylocentrotus purpuratus was investigated. The material was prepared free from egg contaminants and examined for homogeneity by cellulose acetate electrophoresis, disc gel electrophoresis, ultracentrifugation and immunological studies. The electrophoretic and ultracentrifugal studies showed the presence of at least two components. The two-dimensional immunodiffusion tests using rabbit antiserum against whole jelly coat, exhibited four and immunoelectrophoresis five different antigens. In addition, it was possible to separate two components from gel filtration experiments with Sepharose 4B. It is concluded therefore that the S. purpuratus egg jelly coat contains minimally from two to five macromolecules.     

Molecular Reproduction & Development Volume 79, Issue 4, April 2012 cover image

In sea urchins, a soluble coat of egg jelly induces morphological and physiological changes in spermatozoa. This colorized scanning electron micrograph is a snapshot of such activated sperm. In this issue, Kazama et al. identify the reactive oxygen species that are generated by this process and that may influence sperm behavior.     

The apparent absence of a cortical reaction after fertilization in a sea anemone

Eggs, embryos and larvae of the intertidal sea anemone Actinia fragacea were obtained from spontaneous spawnings in the laboratory and have been examined by scanning and transmission electron microscopy. The eggs average 150 micron in diameter and are covered by tufts of large microvilli known as cytospines, but are not surrounded by a jelly layer or a vitelline coat. The cortical layer of the egg contains large numbers of dense, homogeneous cortical granules. The surface layers of cleavage and blastula stage embryos are similar in composition to those of unfertilized eggs in that the cytospine tufts remain intact and the number of cortical granules remains apparently undiminished. No major discharge of cortical granules indicative of a cortical reaction can have occurred. During gastrulation, many embryos take up large numbers of sperm by a process resembling phagocytosis. These sperm undergo breakdown in the superficial regions of the embryos. The cortical granules persist well into larval life, and their function is unknown.     

Rho-kinase (ROCK) in sea urchin sperm: its role in regulating the intracellular pH during the acrosome reaction

Sperm must undergo the acrosome reaction (AR) in order to fertilize the egg. In sea urchins, this reaction is triggered by the egg jelly (EJ) which, upon binding to its sperm receptor, induces increases in the ion permeability of the plasma membrane and changes in protein phosphorylation. Here, we demonstrated that the sperm expresses ROCK (∼135 kDa), which is a serine/threonine protein kinase. ROCK localized, as RhoGTPase (Rho), in the acrosomal region, midpiece and flagellum. H-1152, a ROCK antagonist, inhibited the two cellular processes defining the AR: the acrosomal exocytosis and the actin polymerization. The ionophores nigericin and A23187 reversed the AR inhibition induced by H-1152, suggesting that ROCK functions at the level of the EJ-induced ion fluxes. Accordingly, H-1152 blocked 70% the intracellular alkalinization induced by EJ. These results indicate that EJ activates a Na⁺-H⁺ exchanger (NHE) in the sperm through a Rho/ROCK-dependent signaling pathway that culminates in the AR.     

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.     

Biochemical Studies on the Acrosome Reaction of the Starfish, Asterias Amurensis I. Factors Participating in the Acrosome Reaction

In contrast with the case in sea urchin sperm, in starfish the acrosome reaction is not spontaneously induced by simply increasing the extracellular Ca²⁺ concentration or pH. At higher pHs, starfish sperm undergo morphological changes accompanied by exocytosis of the acrosomal vacuole, but they do not form acrosomal filaments. Nomarski-microscopic observation confirmed that spermatozoa undergo the acrosome reaction within the jelly coat. Acrosome reaction-inducing substance, a glycoprotein from the egg jelly, required a diffusible cofactor(s) present in the egg jelly for full activity. Several lines of evidence showed that this diffusible factor(s) is not merely Ca²⁺.     

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.     

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.     

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

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

Involvement of an acrosin-like enzyme in the acrosome reaction of sea urchin sperm

Extracts of the jelly coat of eggs of several marine invertebrates are known to induce in homologous sperm morphological changes known as the acrosome reaction. When sperm of the sea urchin Strongylocentrotus purpuratus are treated with low concentrations (0.2 μg fucose/ml) of egg jelly coat or 30 mM CaCl₂ in artificial seawater the acrosome reaction does not occur. However, either of these treatments causes the exposure of an acrosin-like enzyme to exogenous substrate and inhibitors. Subsequent addition of jelly coat to 3.7 μg fucose/ml to sperm in this “initial stage” induces the acrosome reaction (as judged by the appearance of an acrosomal filament). This concentration is also effective for untreated sperm. If inhibitors of the enzyme (diisopropylphosphofluoridate or phenylmethanesulfonyl fluoride) are added to sperm in the initial stage, no acrosomal filaments are observed when the high concentration of jelly coat is added. Whether other morphological changes occur in these sperm has not been examined. If phenylmethanesulfonyl fluoride is added 4 sec after the jelly coat, the acrosomal filaments are observed, but the sperm still fail to fertilize eggs. These results suggest a dual role for the acrosin-like enzyme(s), first in the mechanism of the acrosomal filament formation and then in a subsequent event in the fertilization process.     

ELECTRON MICROSCOPIC DEMONSTRATION OF A DITHIOTHREITOL-LABILE VITELLINE COAT SURROUNDING THE UNFERTILIZED EGG OF COMANTHUS JAPONICA (ECHINODERMATA: CRINOIDEA)*

In Comanthus, the unfertilized egg is surrounded by a vitelline coat, which is separated from the underlying plasma membrane by a space several hundred Ångstroms wide. By electron microscopy, the vitelline coat is a distinct layer 100 to 150 Å thick, which consists of finely granular material of moderate electron density. Treatment for 3 min in 0.01 M dithiothreitol in sea water buffered to pH 9.2 almost completely removes the vitelline coat and causes the irregularly shaped egg to become spherical. After such DTT-treated eggs have been washed for 2 min in sea water, they cannot be fertilized, but they can undergo a cortical reaction when treated with ionophore A23187. This cortical reaction consists of the exocytosis of cortical granule material directly into the surrounding sea water. By several hours after DTT treatment, most of the eggs, whether exposed to ionophore or not, fragment into spheres of diverse sizes.     

Mouse egg extracellular coat is a matrix of interconnected filaments possessing a structural repeat

As the result of a combined biochemical and electron microscopic investigation, hitherto unrecognized structural features of the mouse egg extracellular coat, or zona pellucida, have been revealed. Specimens were prepared for electron microscopy by spraying individually isolated zonae pellucidae onto a substrate and were observed by both rotary shadowing and negative staining techniques. Results of these experiments suggest that the three zona pellucida glycoproteins, ZP1 (200,000 Mr), ZP2 (120,000 Mr) and ZP3 (83,000 Mr), are organized into long filaments. Negatively stained zona pellucida filaments resemble "beads-on-a-string", with each bead (9.5 nm in diameter) located every 17 nm or so (center-to-center distance) along the axis of the filament. The filaments, in turn, appear to be interconnected by one of the three zona pellucida glycoproteins, ZP1, giving rise to a three-dimensional matrix. Proteolysis of ZP1 by chymotrypsin or reduction of intermolecular disulfides of ZP1 by dithiothreitol results in both solubilization of zonae pellucidae and disruption of interconnections between individual zona pellucida filaments. These observations suggest that the zona pellucida, which plays important roles both during and after fertilization of mammalian eggs, is a highly organized extracellular coat in which glycoproteins are assembled into filaments possessing a recognizable structural repeat.     

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.