Vitelline coat of Unio elongatulus egg: I. Isolation and biochemical characterization

In this study we isolated and purified the vitelline coat (vc) of Unio elongatulus eggs in order to investigate its protein and carbohydrate composition. SDS dissolved up to 80% of the vitelline coat protein content whereas 100 mM Ammonimum acetate (AA) at pH 11 and 1 mM lithium diiodosalicylate (LISH) dissolved only 40–50%. The ability of extremes of pH or LIS to solubilize the vitelline coats on eggs was then investigated. The results showed that pH from 7 to 11 progressively dissolved the vitelline coats without gross damage to the oocytes. SDS-PAGE of the solubilized material revealed only two components corresponding to the main components revealed by SDS-PAGE of the isolated vcs. These peptides have an apparent MW of 220 and 180 kD, are ConA positive, and seem to be connected to each other to form polycomponents. The latter feature is suggested by the electrophoretic pattern of the solubilized material under nondenaturing conditions. © 1993 Wiley-Liss, Inc.     

Differentiation of the vitelline coat and the polarized site of sperm entrance in the egg of Unio elongatulus (Mollusca, Bivalvia)

We studied the differentiation of the polarized site of sperm entrance in the egg of a freshwater bivalve, Unio elongatulus. As previously shown, in this egg model the property of sperm recognition and binding is restricted to a region characterized by a wrinkled surface which surrounds a truncated cone or crater region, at the vegetal pole of the egg. The crater is formed during oogenesis at a site opposite the oocyte attachment to the ovarian wall. It first appears as a small bleb, and later detaches, leaving in the oocyte an open narrow process filled with an orderly array of microtubules. Here the vitelline coat differentiates from the rest of the oocyte. The role of the microtubules in the formation of the region and the differential distribution of the vitelline coat components is discussed. The synthesis during development of fucosyl containing glycoproteins and their presence in an electrophoretic pattern of isolated vitelline coats were also studied using Lotus tetragonolobus (LTA) conjugated with fluorescein and peroxidase.     

Sperm binding to eggs of Ciona intestinalis. Role of Ca2+

In this paper we show that in the ascidia Ciona intestinalis extracellular Ca2+ is required for the binding of the spermatozoa to the vitelline coat (VC) glycerol-treated eggs and for fertilization to occur. Divalent cations, Mg2+ and Mn2+, cannot replace Ca2+. Once bound, the spermatozoa cannot be detached from the vitelline coat by adding of EGTA. Verapamil does not interfere with the binding of spermatozoa to the vitelline coat, whereas it blocks the Ca2+ ionophore A23187-induced sperm activation and acrosome reaction. Fertilization too was inhibited by the presence of this drug.     

Influences of cell size and aging on l-[H]norepinephrine binding sites in rat epididymal fat cells

[³H]norepinephrine binding to isolated rat fat cells was studied as a function of adipose cell age and size. Rats aged from 4 to 78 weeks were used.Scatchard analysis of norepinephrine binding revealed in old fat cells like in young ones the existence of two orders of binding sites with respectively high and low affinity for norepinephrine. The apparent association constants Ka₁ and Ka₂ associated with these binding sites did not differ consistently in the different groups of fat cells studied (Ka₁ = 1.7 to 2.2 × 10⁶ × M⁻¹; Ka₂ = 1.9 to 2.5 × 10⁴ × M⁻¹), suggesting that age and cell size do not modify the apparent affinity of norepinephrine-binding sites in rat fat cells.On the contrary, the total amount of norepinephrine bound to each of these sites was dependent upon cell age and size. In fact, maximum binding of norepinephrine to the high affinity sites was 0.9 and 9 pmol/10⁵ cells in small (diameter: 35 μ) and large (diameter: 105 μ) adipocytes, respectively, the values found for the low affinity sites being 13 and 135 pmol/10⁵ cells. When expressed per unit of fat cell area, however, the total binding capacity for these sites appeared practically constant (2.4 — 2.8 pmol × 10⁻³/mm² and 34.2 — 38.2 pmol × 10⁻³/mm² for the high and low affinity sites respectively). These data suggest that the total norepinephrine binding capacity of the fat cell is directly proportional to its surface.     

Effect on fertilization and localized binding of lectins in the ascidian Phallusia mammillata

The effect of concanavalin A (conA), fucose-binding protein (FBP), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA) on fertilization of the ascidian Phallusia mammillata was investigated. ConA, FBP, and RCA had no influence on fertilization and did not bind to the chorion or sperm, as determined with FITC-conjugated conA and by electron microscopy with gold-labelled FBP. WGA (100 μg/ml) prevented fertilization of eggs by sperms in concentrations which gave 100% fertilization in controls (2 × 10⁷ sperm/ml). N-Acetyl-glucosamine (50 mM) abolished the effect of WGA, whereas an excess (100 mM) of this competitive sugar alone did not affect fertilization. FITC-conjugated and gold-labelled WGA revealed binding sites on the chorion, but not on follicle cells nor sperms. Electron microscopy showed that WGA gold-markers are bound to the fibrillar network forming the outer layer of the chorion and indicate that WGA inhibits fertilization by interfering with sperm binding to the chorion. Binding of WGA to the chorion may either mask sperm binding receptors or cause chorion resistance to sperm enzymes.     

Polyspermic fertilization of sea urchin eggs treated with protease inhibitors: Localization of sperm receptor sites at the egg surface

The normal elevation of the fertilization membrane and the establishment of the block to polyspermy are retarded in Arbacia punctulata eggs by specific protease inhibitors, soybean trypsin inhibitor (SBTI), leupeptin, and antipain. Ultrastructural observations show that the vitelline layer remains attached to the plasma membrane of fertilized SBTI treated eggs at numerous sites (cortical projections). Quantitive morphometric analysis indicates that the vitelline layer elevates from about 65% of the surface of SBTI treated eggs during the first 3 min post insemination. However, the vulnerability of SBTI treated eggs to refertilization (polyspermy) only declined during the subsequent gradual detachment of the vitelline layer from the cortical projections over the next 15 min. Antipain and leupeptin (10^?5 to 10^?3M) also promoted polyspermy in Arbacia eggs by a process of refertilization extending for a 10- to 15-min period after the initial monospermic insemination. Normal cleavage and development was obtained when eggs were placed in leupeptin and antipain (10^?3M) after the fertilization membrane had elevated. The data indicate that the normal secretory function (or functions) of the cortical granule protease in establishing the block to polyspermy is retarded by these protease inhibitors, and that the vitelline layer is transformed into a mechanical barrier to prevent penetration by supernumerary sperm during its detachment from the plasma membrane of the egg. Furthermore, the vitelline layer in unfertilized eggs appears to be a mosaic structure, with sperm receptor sites localized in regions of the egg's surface, which give rise to cortical projections in the presence of SBTI.     

A thermodynamic study of sperm-egg interaction.

We have studied the binding of spermatozoa to the receptor sites on the vitelline coat (VC) of glycerol-treated eggs (ghost eggs) of the Ascidian, Ciona intestinalis (Protochordate). Glycerol treatment cytolyses the egg without affecting the ability of the VC to bind spermatozoa in a species-specific manner; however, in this system binding is not followed by the acrosome reaction. The ghost eggs are metabolically inert. As a base line for our analysis, we have studied the concentration-dependent heat evolved and oxygen consumption of spermatozoa when diluted in sea water. The process has been analyzed on the basis of equations derived by Liquori and Tripiciano to describe cell growth. Upon binding to the ghost eggs, the spermatozoa produce an explosive heat evolution (excess heat) which is not accompanied by oxygen consumption. The excess heat produced plotted against sperm concentration (at constant egg concentrations) gives an asymmetric bell-shaped curve. This is interpreted as being due to the competitive effect of sperm agglutination at a high sperm concentration. It is concluded that only spermatozoa that attach singly (monomeric spermatozoa) to the egg undergo metabolic activation.     

Helper Function of Follicle Cells in Sperm-Egg Interactions of the Ascidian, Ciona intestinalis

Studies were made on the involvement in sperm-egg interactions of follicle cells of Ciona intestinalis , which are tall, vacuolated cells attached to the outer surface of the egg vitelline coat. The basal surface of the follicle cells is polygonal. The borders between cells could easily be observed by the binding of fluorescent SBA (soy bean agglutinin), a lectin recognizing N-acetylgalactosamine (GaINAc) residues. At fertilization many spermatozoa aggregate along these polygonal borders of cells on the vitelline coat, through which they entered the perivitelline space. The removal of follicle cells was sometimes associated with loss of SBA-binding sites, and in such cases the sperm did not show a hexagonal pattern of aggregation, but became dispersed all over the vitelline coat. Removal of the follicle sometimes delayed fertilization. Examination of sections of gametes stained with DAPI, a fluorescent dye staining DNA, showed that removal of the follicle reduced the number of spermatozoa bound to the vitelline coat and, more especially, the number of spermatozoa penetrating through the vitelline coat. The blockage of GalNAc residues on the vitelline coat with SBA did not appreciably affect the time course of fertilization or the number of sperm associated with eggs. These findings are discussed in relation to the role of follicle cells in facilitating sperm aggregation on the vitelline coat and their penetration through it.     

Synthesis and function of the fibrous layers covering the eggs of Siphlonurus lacustris (Ephemeroptera, Siphlonuridae)

Ultrastructural analysis (transmission and electron scanning microscopy) of the eggs of the mayfly Siphlonurus lacustris (Eaton) showed that they are wrapped in a thick coat composed of a network of tightly entwined filaments. Groups of twisted filaments form slightly uplifted buttons that are scattered on the coat surface. After experimentally induced egg deposition, egg–water interaction promotes marked cohesion of the eggs and their firm adhesion to the substrate. Egg masses include numerous gametes; the covering of those located close to the substrate greatly extends to anchor the whole mass. Eggs removed from the coat reveal a slightly punctuated smooth chorion and tagenoform micropyles (three to five). The coat increases egg size by about 20%. The lack of female reproductive accessory glands in Ephemeroptera transfers the synthesis of the adhesive coats to the follicle cells, which are typically competent for insect egg shell deposition (vitelline envelope and chorionic layers). This covering results from electron‐dense granules that give rise to filaments progressively organized to form superimposed layers variously orientated around the egg. In addition to egg adhesion to the substrate, a trophic function and protection from shear stress are postulated for this covering.     

Galactosyltransferase activities in subcellular fractions of two YC8 lymphoma cell variants: does a relationship exist between antigenic determinants and acceptor sites for galactose?

Experiments have been undertaken to correlate physiological changes, observed in two YC8 cells variants (P and L) and some of their immunological and enzymatic properties. These cell lines show different responses towards antilymphocyte and anti-Moloney sera. Subcellular fractionations have been made. The A fractions (d: 1.14/1.16) have the highest ouabain-inhibited Mg²⁺-stimulated (Na⁺-K⁺)-dependent ATPase and galactosyltransferase activities. Some properties of the latter enzyme have been studied: whereas optima pH and requirements for Mn²⁺ ions have been found to be the same for both cell line enzymes, on the contrary, different kinetic parameters have been shown with respect to sugar donor (UDP-galactose) on endogeneous or exogeneous (ovomucoid) acceptors. Apparent Km for UDP-galactose is 1.7 × 10⁻⁶ M (P-cells) and 3.3 × 10⁻⁶ M (L-cells), on endogeneous acceptors, and P-cell V max < L-cell V max; on ovomucoid it is 0.61 × 10⁻⁶ M, for both cell lines. These results suggest the presence on L-cells of more endogeneous acceptor sites, the higher affinity of P-cells for UDP-galactose being balanced by less endogeneous acceptor sites for galactose. When ovomucoid is added, galactose transfer on endogeneous acceptor sites of both cells is negligible. Apparent Km for ovomucoid is 8.6 × 10⁻⁵ M (P-cells) and 4.3 × 10⁻⁵ M (L-cells). These data support the above-mentioned hypothesis: L-cell enzymes would be more rapidly saturated than P-cell enzymes because of the higher number of endogeneous sites on L-cells.This supposed acquired character of L-cells as well as their immunological behaviour could explain the modified properties of L-cells as compared to P-cells.     

Plasma Membrane Glycoproteins of Ciona intestinalis Spermatozoa that Interact with the Egg1

In the ascidian Ciona intestinalis the species-specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)-binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS-PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with ³H-Con A. Affinity chromatography on Con A-agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A-retained fraction was determined with binding and fertilization assays.     

Pre-fusion events of sperm-oocyte interaction in the marine shrimp,Sicyonia ingentis

The non-motile sperm of Sicyonia ingentis, mixed with eggs by a spawning female, undergo a primary binding to the vitelline envelope (VE) of the oocyte. Once bound to the VE, sperm undergo exocytosis of the acrosomal vesicle, penetrate the VE, and secondarily bind to a surface coat that is closely associated with the oolemma. Unreacted sperm preincubated with solubilized VE components exhibit diminished binding to VEs in a concentration dependent manner. The ligand responsible for this binding is a carbohydrate moiety in the VE. The ligand preferentially binds to the anterior tip of unreacted sperm, as demonstrated with anti-VE polyclonal antibodies. Acrosome intact sperm will not bind to surface coats; however, acrosome reacted sperm do bind to surface coats via an externalized acrosomal granule.     

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.     

Immunoelectron Microscopic Demonstration of Cortical Granule Lectins in Coelomic, Unfertilized and Fertilized Eggs of Xenopus laevis

Immunoelectron microscopic studies demonstrated cortical granule lectins (CGLs) in coelomic, unfertilized and fertilized eggs of Xenopus laevis . An antiserum raised against purified cortical granule lectin 1 specifically reacted with the CGLs in immunoblotting and agar diffusion tests. When ultrathin sections were treated with the antiserum and protein A-gold solution, gold particles, indicating antigenic sites, were seen over cortical granules of coelomic and unfertilized eggs, and over the perivitelline space, the vitelline coat and the condensed region of the fertilization layer of fertilized eggs. The pre-fertilization layer immediately adjacent to the outer margin of the vitelline coat in unfertilized eggs was free from gold particles. These observations suggest that released CGLs permeate through the vitelline coat of fertilized eggs and interact with the pre-fertilization layer mainly at the outer margin of the vitelline coat, resulting in formation of the fertilization layer which acts as a block to polyspermy.     

Effects of protease inhibitors on sperm-related events in sea urchin fertilization

Evidence for sperm-borne proteolytic enzymes exposed during the acrosome reaction in sea urchin sperm has been accumulating. To investigate the possible role(s) such enzymes have in fertilization, we studied the effects of several protease inhibitors on sperm-related events. Soybean trypsin inhibitor, Nα-p-tosyl-l-lysine, chloromethyl ketone, phenylmethylsulfonyl fluoride, and chymostatin neither reduced the number of acrosome reactions nor interfered with gamete binding. p-Nitrophenyl-p′-guanidinobenzoate caused sperm to fuse into irregular clumps, rendering them unable to fertilize eggs. However, l-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), an inhibitor of chymotrypsin, prevented the acrosome reaction in Strongylocentrotus purpuratus, S. droebachiensis, and Lytechinus pictus. The effects of TPCK on sperm in subsequent steps of fertilization were also investigated. First, gamete binding assays were performed on fixed eggs. This precluded any effects TPCK might have had on egg-derived secretions (e.g., proteases). Binding of prereacted sperm occurred with both fixed and living eggs. However, fertilization of living eggs in the presence of TPCK was greatly reduced, even though sperm had been prereacted with egg jelly. Vitelline coats were then removed from eggs by trypsin treatment. Eggs in TPCK fertilized and developed normally after the above treatment. These observations are consistent with the hypothesis of a sperm protease participating in the acrosome reaction and the penetration of the egg vitelline coat in the sea urchin.     

Participation of sperm proteasome in fertilization of the phlebobranch ascidian Ciona intestinalis

Sperm proteasomes are thought to be involved in sperm binding to and in sperm penetration through the vitelline coat of the eggs of the stolidobranch ascidian Halocynthia roretzi. However, it is not known whether they are involved in the fertilization of eggs of other ascidians. Therefore, we investigated whether sperm proteasomes are also involved in the fertilization of the eggs of the primitive phlebobranch ascidian Ciona intestinalis. Fertilization of the eggs of C. intestinalis was potently inhibited by the proteasome inhibitors MG115 and MG132 but not by the cysteine protease inhibitor E-64-d. On the other hand, neither fertilization of the vitelline coat-free eggs nor sperm binding to the vitelline coat was inhibited by the two proteasome inhibitors at a concentration sufficient to inhibit fertilization of intact eggs. These results indicate that the proteasome plays an essential role in sperm penetration through the vitelline coat rather than in sperm binding to the coat or in sperm-egg membrane fusion. The proteasome activity, which was detected in the sperm extract using Suc-Leu-Leu-Val-Tyr-MCA as a substrate, was strongly inhibited by both MG115 and MG132, and was weakly inhibited by chymostatin, whereas neither leupeptin nor E-64-d inhibited the activity. The molecular mass of the enzyme was estimated to be 600-kDa by Superose 12 gel filtration, and the activity in sperm extract was immunoprecipitated with an anti-proteasome antibody. These results indicate that the proteasome present in sperm of C. intestinalis is involved in fertilization, especially in the process of sperm penetration through the vitelline coat, probably functioning as a lysin. Mol. Reprod. Dev. 50:493–498, 1998. © 1998 Wiley-Liss, Inc.     

Infection of Blissus antillus (Hemiptera: Lygaeidae) Eggs by the Entomopathogenic Fungi Metarhizium anisopliae and Beauveria bassiana

This study determined the pathogenicity and virulence of Beauveria bassiana and Metarhizium anisopliae to eggs of the chinch bug Blissus antillus (Hemiptera: Lygaeidae). Eggs were inoculated under laboratory conditions by immersion in concentrations of 1 × 10⁴ and 5 × 10⁶ conidia/ml. Inoculated eggs were kept under controlled conditions. Evaluations were carried out daily for 20 days. M. anisopliae isolates were highly virulent to eggs, even at 1 × 10⁴ conidia/ml. All B. bassiana isolates tested were considered to be of low virulence or avirulent. The most virulent isolate tested was ESALQ 818 (M. anisopliae), which caused 96.7% infection, when eggs were immersed in suspensions of 1 × 10⁴ conidia/ml. Conidial production on infected eggs was observed to be highest for M. anisopliae isolate CG144, with a mean value of 11.6 × 10⁵ conidia/ml/egg. Infection of Blissus eggs oviposited on plant stems was greater when M. anisopliae isolate CG144 was formulated in mineral oil (63.5% mortality) than when formulated in Tween 80 (27.1% mortality).     

Leukotriene B4 binding to human neutrophils

[³H] Leukotriene B₄ (LTB₄) binds concentration dependency to intact human polymorophonuclear leukocytes (PMN's). The binding is saturable, reaches equilibrium in 10 min at 4°C, and is readily reversible. Mathematical modeling analysis reveals biphasic binding of [³H] LTB₄ indicating two discrete populations of binding sites. The high affinity binding sites have a dissociation constant of 0.46 × 10⁻⁹M and B_max of 1.96 × 10⁴ sites per neutrophil; the low affinity binding sites have a dissociation constant of 541 × 10⁻⁹M and a B_max of 45.6 × 10⁴ sites per neutrophil. Competitive binding experiments with structural analogues of LTB₄ demonstrate that the interaction between LTB₄ and the binding site is stereospecific, and correlates with the relative biological activity of the analogs. At 25°C[³H] LTB₄ is rapidly dissociated from the binding site and metabolized to 20-OH and 20-COOH-LTB₄. Purification of neutrophils in the presence of 5-lipoxygenase inhibitors significantly increases specific [³H] LTB₄ binding, suggesting that LTB₄ is biosynthesized during the purification procedure. These data suggest that stereospecific binding and metabolism of LTB₄ in neutrophils are tightly coupled processes.     

Influence of buffer ions and divalent cations on coated vesicle disassembly and reassembly

Disruption of the coat of coated vesicles is accompanied by the release of clathrin and other proteins in soluble form. The ability of solubilized coated vesicle proteins to reassemble into empty coats is influenced by Mg²⁺, Tris ion concentration, pH, and ionic strength. The proteins solubilized by 2 M urea spontaneously reassemble into empty coats following dialysis into isolation buffer (0.1 M MES–1 mM EGTA–1 mM MgCl₂–0.02% NaN₃, pH 6.8). Such reassembled coats have sedimentation properties similar to untreated coated vesicles. Clathrin is the predominant protein of reassembled coats; most of the other proteins present in native coated vesicles are absent. We have found that Mg²⁺ is important in the coat assembly reaction. At pH 8 in 0.01 M or 0.1 M Tris, coats dissociate; however, 10 mM MgCl₂ prevents dissociation. If the coats are first dissociated at pH 8 and then the MgCl₂ raised to 10 mM, reassembly occurs. These results suggest that Mg²⁺ stabilizes the coat lattice and promotes reassembly. This hypothesis is supported by our observations that increasing Mg²⁺ (10 μM–10 mM) increases reassembly whereas chelation of Mg²⁺ by (EGTA) inhibits reassembly. Coats reassembled in low-Tris (0.01 M, pH 8) supernatants containing 10 mM MgCl₂ do not sediment, but upon dialysis into isolation buffer (pH 6.8), these coats become sedimentable. Nonsedimentable coats are noted also either when partially purified clathrin (peak I from Sepharose CL4B columns) is dialyzed into low-ionic-strength buffer or when peaks I and II are dialyzed into isolation buffer. Such nonsedimentable coats may represent intermediates in the assembly reaction which have normal morphology but lack some of the physical properties of native coats. We present a model suggesting that tightly intertwined antiparallel clathrin dimers form the edges of the coat lattice.     

Designing of MIP based QCM sensor having thymine recognition sites based on biomimicking DNA approach

Quartz crystal microbalance (QCM) sensors coated with molecular imprinted polymers (MIP) have been developed for the determination of thymine. In this method, methacryloylamidoadenine (MA-Ade) have used as a new monomer and thymine template for inspiration of DNA nucleobases interaction. The thymine can be simultaneously hydrogen binding to MA-Ade and fit into the shape-selective cavities. Thus, the interaction between nucleobases has an effect on the binding ability of the QCM sensors. The binding affinity of the thymine imprinted sensors has investigated by using the Langmuir isotherm. The thymine imprinted QCM electrodes have shown homogeneous binding sites for thymine (K_a: 1.0 × 10⁵ M⁻¹) while heterogeneous binding sites for uracil. On the other hand, recognition selectivity of the QCM sensor based on thymine imprinted polymer toward to uracil, ssDNA and ssRNA has been reported in this work.