Jelly Coat Of Eggs Research Articles

Allurin and egg jelly coat impact on in-vitro fertilization success of endangered Albanian water frog, Pelophylax shqipericus

Amphibian egg-jelly coat plays an important role in successful fertilization and development. Here, we ask whether proteins like allurin in the jelly coats of frog eggs might influence fertilization rate success. Using in vitro fertilization of Albanian water frog, Pelophylax shqipericus, we found that body cavity eggs or eggs deprived of jelly coat were not fertilized, compromising the success of in vitro fertilization procedure. When de-jellied eggs were inseminated with sperm suspension, the fertilization efficiency is dramatically decreased even inhibited, suggesting that the gel structure is one of the major factors in the achievement of fertilization in the frogs. Fertilization of de-jellied eggs with sperm pre-treated with egg jelly coat, restored the fertilization competency. Such a result suggests that egg jelly coat probably guides the sperm to the egg surface while maintaining the fertilization ability, contributing to a successful in vitro fertilization of Pelophylax shqipericus.

Proportion of Sperm and Eggs for Maximalin vitroFertilization inHaliotis asininaand the Chronology of Early Development

ABSTRACT To obtain the highest yield during in vitro fertilization of tropical abalone Haliotis asinina, optimal proportion of the gametes, the timing of sperm-egg interaction, and subsequent development were investigated. The highest yield of fertilization (75%) with fewest abnormal eggs was obtained when incubating eggs and sperm at the ratio of 1:100 in seawater with a salinity of 27.5 ppt, a pH of 7.8, and a temperature range from 27–29°C. After incubation, sperm swim through the eggjelly coat and become bound to the vitelline envelope within 30 sec, followed by an acrosomal reaction at 1 min. The fertilized egg extrudes the first and second polar bodies at 8–10 min, and then the zygote begins cleavage at 15–20 min. This is followed by the second cleavage, and development through the stages of blastula, gastrula, trochophore, veliger, and early creeping larvae, which were completed within 3 days. Noticeably, occurrence of egg jelly condensation after penetration of the first sperm would not allow othe...

Embryonic development in the bivalve Scrobicularia plana (Semelidae) using scanning electron microscopy

Summary The development of the intertidal bivalve Scrobicularia plana, from the fertilised egg to the hatched veliger stage, is described using light and scanning electron microscopy (SEM). This species exhibits planktotrophic development, which takes place within an egg jelly-coat, progressing through a trochophore stage to a typical D-shaped veliger. Oocytes have an average size diameter of 64 μm; the cleavage pattern is spiral and unequal and gives rise to a ciliated blastula stage ca. 7–9 h after fertilisation. The gastrula, which forms 10–14 h after fertilisation, possesses two depressions, the blastopore and the shell field. A typical top-shaped trochophore is formed after ca. 20 h, which rotates vigorously within the jelly-coat. This is followed by a straight hinged veliger stage, still within the jelly-coat, that rotates vigorously using the velum. The veliger hatches from the jelly-coat after ca. 48–56 h.

Species specificity of O-linked carbohydrate chains of the oviducal mucins in amphibians: structural analysis of neutral oligosaccharide alditols released by reductive β-elimination from the egg-jelly coats of Rana clamitans

The extracellular matrix (the so-called jelly coat) surrounding amphibian eggs mainly comprises highly O-glycosylated proteins. These oviducal mucins have an important role in the fertilization process, and their carbohydrate chains are remarkably species-specific. Alkaline reductive treatment of the jelly-coat material of the frog Rana clamitans led to the release of oligosaccharide alditols. The neutral oligosaccharide alditols were fractionated and purified by successive chromatographic techniques. The structures of 27 of them, ranging from three to sixteen monosaccharides, were established by a combination of NMR spectroscopy, methylation analyses and matrix-assisted laser-desorption ionization-time of flight MS. Typically, some of the neutral compounds appeared to possess the core structure: Gal(beta1-3)[GlcNAc(beta1-6)]Gal(beta1-3)[GlcNAc(beta1-6)]GalNAc-ol (where GalNAc-ol represents N-acetylgalactosaminitol). Moreover, a novel type of chain termination, characterized by an unusual sequence [Fuc(alpha1-2)Gal(alpha1-3)Gal(alpha1-4)Gal(beta1-3/4)] was observed. Indeed, the most complex representative structure of this series was found to be: Fuc(alpha1-2)Gal(alpha1-3)Gal(alpha1-4)Gal(beta1-3)[Fuc(alpha1-2)Gal(alpha1-3)Gal(alpha1-4)Gal(beta1-4)GlcNAc(beta1-6)]Gal(beta1-3)[Fuc(alpha1-2)Gal(alpha1-3)Gal(alpha1-4)Gal(beta1-4)GlcNAc(beta1-6)]GalNAc-ol.

Species specificity of O-linked carbohydrate chains of the oviducal mucins in amphibians: structural analysis of neutral oligosaccharide alditols released by reductive β-elimination from the egg-jelly coats of Rana clamitans

The extracellular matrix (the so-called jelly coat) surrounding amphibian eggs mainly comprises highly O-glycosylated proteins. These oviducal mucins have an important role in the fertilization process, and their carbohydrate chains are remarkably species-specific. Alkaline reductive treatment of the jelly-coat material of the frog Rana clamitans led to the release of oligosaccharide alditols. The neutral oligosaccharide alditols were fractionated and purified by successive chromatographic techniques. The structures of 27 of them, ranging from three to sixteen monosaccharides, were established by a combination of NMR spectroscopy, methylation analyses and matrix-assisted laser-desorption ionization—time of flight MS. Typically, some of the neutral compounds appeared to possess the core structure: Gal(β1–3)[GlcNAc(β1–6)]Gal(β1–3)[GlcNAc(β1–6)]GalNAc-ol (where GalNAc-ol represents N-acetylgalactosaminitol). Moreover, a novel type of chain termination, characterized by an unusual sequence {Fuc(α1–2)Gal(α1–3)Gal(α1–4)Gal(β1–3/4)} was observed. Indeed, the most complex representative structure of this series was found to be: Fuc(α1–2)Gal(α1–3)Gal(α1–4)Gal(β1–3)[Fuc(α1–2)Gal(α1–3)Gal(α1–4)Gal(β1–4)GlcNAc(β1–6)]Gal(β1–3)[Fuc(α1–2)Gal(α1–3)Gal(α1–4)Gal(β1–4)GlcNAc(β1–6)]GalNAc-ol.

Diffusible highly glycosylated protein from Bufo arenarum egg‐jelly coat: Biological activity

L-HGP is a highly glycosylated protein from Bufo arenarum egg-jelly coat that diffuses into the surrounding medium when the strings of oocytes are incubated in saline solutions. L-HGP was purified from egg water and the estimated percentage of L-HGP/total protein in egg water was estimated in 30%. In the present study we examine, by indirect immunofluorescence, the effect of L-HGP on acrosome status of homologous spermatozoa. A high percentage (77%) of sperm lost the acrosome when incubated in 10% Ringer solution buffered with 10 mM Tris-HCl, pH 7.6, during 60 min, a condition that resembles egg-jelly osmolarity. The addition of purified L-HGP to the incubation medium prevents acrosome breakdown. The acrosome integrity is maintained for at least 1 hr. This effect is specific for L-HGP at concentration ranging from 0.01 to 0.1 mg/ml since neither BSA nor fetuin seems to have similar activity at similar concentrations. The same effect was observed when spermatozoa were incubated in egg water. Preliminary results suggest that L-HGP binds to B. arenarum spermatozoan membranes. Mol. Reprod. Dev. 56:392–400, 2000. © 2000 Wiley-Liss, Inc.

Diffusible highly glycosylated protein from Bufo arenarum egg-jelly coat: biological activity.

L-HGP is a highly glycosylated protein from Bufo arenarum egg-jelly coat that diffuses into the surrounding medium when the strings of oocytes are incubated in saline solutions. L-HGP was purified from egg water and the estimated percentage of L-HGP/total protein in egg water was estimated in 30%. In the present study we examine, by indirect immunofluorescence, the effect of L-HGP on acrosome status of homologous spermatozoa. A high percentage (77%) of sperm lost the acrosome when incubated in 10% Ringer solution buffered with 10 mM Tris-HCl, pH 7.6, during 60 min, a condition that resembles egg-jelly osmolarity. The addition of purified L-HGP to the incubation medium prevents acrosome breakdown. The acrosome integrity is maintained for at least 1 hr. This effect is specific for L-HGP at concentration ranging from 0.01 to 0.1 mg/ml since neither BSA nor fetuin seems to have similar activity at similar concentrations. The same effect was observed when spermatozoa were incubated in egg water. Preliminary results suggest that L-HGP binds to B. arenarum spermatozoan membranes.

Influence of creep phenomenon and surface roughness on the adhesion of Xenopus laevis eggs to different substrates

The adhesion of fertilized and nonfertilized Xenopus laevis eggs to various surfaces differing in texture, roughness, and chemical composition was examined. Techniques regularly used in the fields of coating and adhesives were adopted. The stresses for the disconnection of eggs from different surfaces were evaluated by a custom-made apparatus, which disconnects the eggs by applying a regulated water pressure. The strength of adhesion depends, among others, on the roughness of the surfaces. A commercial roughness tester evaluated this parameter. It was observed that the rougher the surface, the better the adhesion between the egg and the surface. Maximal adhesion between the eggs and rough surfaces was detected 1 h after the first contact. For smooth surfaces (hydrogels), such a maximum was recognized only after 24 h had elapsed. In addition to the character of the surfaces, the creep of the jelly coat with time significantly influenced the strength of adhesion. The creep of the jelly coat was also dependent on the degree of swelling. The swelling of an egg's jelly coat during the first 8.5 h of the process was monitored by weight. Major changes in weight were observed during the first 100 min. After longer periods, the weight reached an asymptotic level. Incorporating polyvinyl pyrrolidone (PVP) into the medium eliminates the adhesion and stickiness of Xenopus eggs in the laboratory. Tensile bond stress and peel techniques were used to estimate the egg's adhesion to several surfaces. These tests revealed that the first contact between the egg and substrate resulted in a stronger adhesion than when the egg was disconnected and re-glued (re-adhered) to the same surface. Adhesion to natural surfaces (pebbles and Nimephea leaves) was better than to synthetic smooth surfaces. The shorter the distance between the female toad and any surface, the better the adhesion of the eggs.

Inhibition of the sea urchin sperm acrosome reaction by a lignin-derived macromolecule

The major organic components of effluents from commercial pulping processes are lignin-derived macromolecules (LDMs), which have recently been shown to inhibit fertilization and embryonic development in a variety of marine organisms, as well as to exhibit immunostimulating activity in mammalian cells. We conducted studies on the effects of an isolated LDM from bleached kraft mill effluent (BKME), and its sub-components, at the cellular level utilizing the purple sea urchin ( Strongylocentrotus purpuratus) sperm acrosome reaction (AR) as an experimental system. The AR is an event that is induced by the egg's jelly coat and is prerequisite for successful fertilization. Sperm were preincubated with increasing concentrations of isolated LDM or electrophoretically purified LDM sub-components, followed by addition of isolated egg jelly to induce the AR in vitro. These LDM preparations significantly inhibited the AR as assessed by fluorescence (utilizing the rhodamine-conjugated phallicidin) and transmission electron microscopy. Preincubation of sperm with LDM did not have any effect on sperm motility. The level of AR inhibition was comparable to that observed in experiments assessing successful fertilization. The ability of LDM to inhibit jelly induced AR was overcome by the calcium ionophores A23187 and ionomycin. In addition, LDM was shown to inhibit the normal increase in intracellular calcium (Ca ++) associated with induction of the AR. When eggs were preincubated with LDM prior to addition of unexposed sperm, no effect on fertilization was observed, indicating that LDM specifically affects sperm function during fertilization. Fine structural studies, utilizing biotinylated LDM, confirmed LDM's specificity and revealed that its binding was restricted to the plasma membrane domain of the sperm head. The present observations on the inhibition of the AR by LDM is consistent with our hypothesis that this macromolecule inhibits the AR by blocking egg jelly interaction with the sperm surface, thus inhibiting ionic events such as increases in intracellular calcium. Our present approach also demonstrates that echinoderm sperm functions can be used as a model system for the investigation of the mode of action of toxicants at the sub-cellular level.

The Sea Urchin Egg Jelly Coat Consists of Globular Glycoproteins Bound to a Fibrous Fucan Superstructure

Intact egg jelly (EJ) coats surrounding eggs of the sea urchin Strongylocentrotus purpuratus were visualized in stereo images of platinum replicas produced by the quick-freeze, deep-etch, rotary-shadowing technique. The hydrated EJ coat forms an extensive fibrous network that makes contact with the vitelline layer at the egg surface. Fibers are decorated along their length with particles, particle density being highest in the interior regions of the coat. The macromolecular components making up the EJ network were visualized by rotary-shadowing of mica-adsorbed EJ samples. Whole EJ coats solubilized in pH 5 seawater and spread on the mica surface consist of complex networks of branching fibers decorated with large patches of amorphous material. As we have previously shown (Keller and Vacquier, 1994), EJ boiled in a dissolution buffer containing SDS and β-mercaptoethanol and applied to a Sephacryl-500 gel filtration column can be separated into three fractions: a 380-kDa fucose sulfate polymer (FSP), which elutes in the void volume, and two column-included fractions consisting of intermediate (300 kDa) and low-molecular-weight (30- to 138-kDa) glycoproteins. Rotary-shadowing of the FSP fraction reveals branched fibrous components similar in appearance to that of solubilized whole EJ but devoid of any particulate decoration. In contrast, intermediate- and low-molecular-weight EJ components are strictly globular in appearance but are distinguishable on the basis of size. Ion-exchange purification of whole EJ yields two glycoproteins, of 82 and 138 kDa, having AR-inducing activity (Keller and Vacquier, 1994). Platinum replication shows these active components to be small spherical molecules about 8 nm in diameter. The above fractionation scheme requires harsh dissociation conditions. Indeed, if EJ is not boiled in SDS buffer before fractionation, the 300-kDa fraction and the FSP appear together in the void volume. Rotary-shadowing of this complex reveals a multistranded polymer, decorated with glycoproteins at specific kink points. Taken together, our data suggest that the EJ network is composed of a fucose sulfate polymer superstructure to which glycoproteins are bound.

Evidence for a cannabinoid receptor in sea urchin sperm and its role in blockade of the acrosome reaction

Delta-9-tetrahydrocannabinol ((-)delta 9 THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [3H]CP-55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [3H]CP-55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time-dependent manner. Specific binding of [3H]CP-55,940 to sperm, defined as total binding displaced by (-)delta 9THC, was saturable: KD 5.16 +/- 1.02 nM; Hill coefficient 0.98 +/- 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 +/- 122 cannabinoid receptors per cell. Binding of [3H]CP-55,940 to sperm was reduced in a dose-dependent manner by increasing concentrations of CP-55,940, (-)delta 9THC, and (+)delta 9THC. The rank order of potency to inhibit binding of [3H]CP-55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP-55,940 > (-)delta 9THC > (+)delta 9THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [3H]CP-55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation.

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 three-dimensional network of interconnected fibers extending nearly 50 microns 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 three-dimensional 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.

Cannabinoids inhibit fertilization in sea urchins by reducing the fertilizing capacity of sperm

Delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) inhibit fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of the sperm. Sperm fertility depends upon their motility, and their capacity to undergo the acrosome reaction upon encountering a specific ligand derived from the egg's jelly coat. The acrosome reaction involves exocytosis of the acrosomal granule at the apex of the sperm head and elongation of the acrosomal filament. This process exposes the sperm membrane that will attach to and fuse with the egg. Pretreatment of sperm with THC prevents the triggering of the acrosome reaction by solubilized egg jelly in a dose and time dependent manner. Motility of THC-treated sperm is not reduced compared to control sperm in sea water or vehicle dissolved in sea water. The adverse effects of THC on the acrosome reaction and sperm-fertilizing capacity are reversible. Studies with ionophores suggest that THC blocks the acrosome reaction by affecting event(s) in the stimulation-secretion coupling mechanism in the sperm preceding the opening of ion channels. Ultrastructural studies show that THC, CBD and CBN block the membrane fusion reaction between the sperm's plasma membrane and the acrosomal membrane that normally is elicited in response to stimulation by egg jelly to initiate the acrosome reaction. However, lipid deposits are found in the subacrosomal and centriolar fossae of cannabinoid treated sperm. The nuclear envelope is fragmented in close proximity to the lipid deposits within the subacrosomal fossa. These morphological observations suggest that cannabinoids may activate phospholipase(s) within the sperm. Biochemical studies show that THC activates phospholipase A 2 activity in sperm homogenates. Our studies show that cannabinoids reduce the fertilizing capacity of sea urchin sperm by preventing the induction of the acrosome reaction by egg jelly. The localized fragmentation of cellular membranes and the formation of lipid deposits within the sperm may be due to activation of phospholipase A 2 by cannabinoids. Metabolites derived from this membrane perturbation may inhibit triggering of the acrosome reaction by egg jelly and thereby inhibit fertilization.

Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. I. Inhibition of the acrosome reaction induced by egg jelly.

delta 9-Tetrahydrocannabinol (THC) and two other major cannabinoids derived from marihuana--cannabidiol (CBD) and cannabinol (CBN)--inhibit fertilization in the sea urchin Strongylocentrotus purpuratus by reducing the fertilizing capacity of sperm (Schuel et al., 1987). Sperm fertility depends on their motility and on their ability to undergo the acrosome reaction upon encountering the egg's jelly coat. Pretreatment of S. purpuratus sperm with THC prevents triggering of the acrosome reaction by solubilized egg jelly in a dose (0.1-100 microM) and time (0-5 min)-dependent manner. Induction of the acrosome reaction is inhibited in 88.9 +/- 2.3% of sperm pretreated with 100 microM THC for 5 min, while motility of THC-treated sperm is not reduced compared to solvent (vehicle) and seawater-treated controls. The acrosome reaction is inhibited 50% by pretreatment with 6.6 microM THC for 5 min and with 100 microM THC after 20.8 sec. CBN and CBD at comparable concentrations inhibit the acrosome reaction by egg jelly in a manner similar to THC. THC does not inhibit the acrosome reaction artificially induced by ionomycin, which promotes Ca2+ influx, and nigericin, which promotes K+ efflux. THC partially inhibits (20-30%) the acrosome reaction induced by A23187, which promotes Ca2+ influx, and NH4OH, which raises the internal pH of the sperm. Addition of monensin, which promotes Na+ influx to egg jelly or to A23187, does not overcome the THC inhibition. Inhibition of the egg jelly-induced acrosome reaction by THC produces a corresponding reduction in the fertilizing capacity of the sperm. The adverse effects of THC on the acrosome reaction and sperm fertility are reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

A Novel Sialic Acid and Fucose-containing Disaccharide Isolated from the Jelly Coat of Sea Urchin Eggs

Abstract A new disaccharide was isolated from the jelly coat of the eggs of the sea urchin Pseudocentrotus depressus. Chemical and chromatographical assays suggest it is fucopyranosyl-(1 → 4)-N-glycolylneuraminic acid.

Isolation and Identification of Two Sialic Acids from the Jelly Coat of Sea Urchin Eggs

Two sialic acids were isolated from the jelly coat of the eggs of the sea urchin Pseudocentrotus depressus. Both were subjected to chemical assay, thin layer chromatography, infrared absorption study, and enzymatic treatment. The results indicate that one of the sialic acids is identical with N-glycolylneuraminic acid. The other sialic acid is a completely new type and was deduced to be N-acetoglycolyl-4-methyl-4,9-dideoxy neuraminic acid from the following data. (a) Elemental analyses inferred an empirical formula of C14H23NO9. (b) From each mole of the isolated sialic acid, 2.14 moles of C-methyl, 1.14 moles of glycolyl, and 0.93 mole of acetyl group (0.87 mole of O-acetyl group) were derived. (c) Uptake of periodate was observed to be 2 moles per mole of the sialic acid, whereas 3 moles were taken up per mole of N-glycolylneuraminic acid under the same conditions. Oxidation of 1 mole of the sialic acid liberated 0.88 mole of acetaldehyde. (d) Its reaction with thiobarbituric acid yielded only 39.6% of the color obtained by the direct Ehrlich procedure. (e) The sialic acid was not cleaved by N-acetylneuraminic acid aldolase. (f) Infrared spectrum studies of the sialic acid indicated the presence of methyl and O-acetyl ester groups.

Isolation and Properties of a New Type of Sialopolysaccharide-Protein Complex from the Jelly Coat of Sea Urchin Eggs

Abstract A heteropolysaccharide-protein complex containing 70% sialic acid was isolated from the jelly coat of the eggs of the sea urchin, Pseudocentrotus depressus. The yield of this sialopolysaccharide-protein complex was 0.32% of the whole jelly powder. Purified by centrifugation and chromatography on Bio-Gel, the complex was found to be homogeneous by the criteria of electrophoresis and ultracentrifugation. Its sedimentation coefficient, s020 ,w, was 5.1, and the material carried a strong negative charge. The carbohydrate composition of the complex, expressed in percentages of the dry weight, was sialic acid, 70.0; fucose, 9.2; hexose, 3.7; and hexosamine, 2.4. Analyses for glycolic acid strongly indicated that all of the sialic acid occurred in glycolyl form. The molar ratio of sulfate to fucose was 1.42:1. The combined weights of monosaccharide, sulfate, and amino acid residues accounted for 96.0% of the complex weight. Thin layer chromatography of the sialopolysaccharide-protein complex after hydrolysis in mild acid showed five spots stained by Ehrlich's reagent. The major spot migrated as N-glycolylneuraminic acid. No indication of N-acetylneuraminic acid appeared. It is proposed that there are several new, undescribed neuraminic acids in the jelly coat, since the sialic acid yielded only 45% of the color in the reaction with thiobarbituric acid as compared with the direct Ehrlich assay and, moreover, no release of sialic acid from the complex by neuraminidase treatment was obtained.