The fatty acid composition of oophagous tadpoles (Chirixalus eiffingeri) fed conspecific or chicken egg yolk

We compared the lipid content and fatty acid composition of (1) the egg yolk of three anuran species (Chirixalus eiffingeri, Rhacophorus moltrechti and Buergeria robustus) and chicken eggs; and (2) C. eiffingeri tadpoles fed conspecific eggs or chicken egg yolk. Anuran and chicken egg yolk contained more non-polar than polar lipids but the proportions varied among species. Chicken egg yolk contained low amounts of 22:5n-3 in the polar lipid fraction, and B. robustus eggs did not contain any n-3 or n-6 non-polar lipids. The specific variation of lipid contents and fatty acid composition may relate to the maternal diet and/or breeding biology. In C. eiffingeri tadpoles that fed chicken yolk or frog egg yolk, the dominant components of polar and non-polar lipids were 16:0, 18:0, 18:1, and 18:2n-6, or 20:4n-6 fatty acids. C. eiffingeri eggs contained more n-3 fatty acids (e.g. 18:3n-3 and 20:5n-3) than chicken egg yolk, and tadpoles fed conspecific eggs contained more of these fatty acids than tadpoles fed chicken egg yolk. The compositional differences in the fatty acids between C. eiffingeri tadpoles that fed frog egg or chicken egg yolk are the reflection of the variation in the dietary sources. Our results suggest a direct incorporation of fatty acids into the body without or minimal modification, which provide an important insight into the physiological aspects of cannibalism.     

Monoclonal antibodies to the sea urchin egg vitelline layer inhibit fertilization by blocking sperm adhesion

Thirty-one mouse hybridomas were produced against the vitelline layer (VL) of the egg of the sea urchin S. purpuratus. Ascites fluids of eight of the 31 bound to the VL surface in the high ionic strength conditions of sea water. Binding was specific to the VL, since immunofluorescence showed that the antibodies elevated from the egg surface with the fertilization envelope after activation with ionophore A23187. Antibody binding was strictly species-specific, the eggs of L. pictus showing no reaction. An immunoperoxidase surface-binding assay showed a wide range in the amount of each monoclonal antibody binding to the VL surface at saturation. All eight monoclonals inhibit fertilization by inhibiting the binding of sperm to the VL. None of the eight ascites fluids reacted with egg jelly. The inhibition of fertilization correlates positively with amount of antibody binding the egg surface. In contrast to the effects of polyclonal rabbit antisera raised against whole eggs or egg cortices, these eight monoclonal antibodies to the VL do not induce the wrinkling of the egg, the cortical granule reaction, the centering of pronuclei, or any other visual indication of metabolic activation.     

Fatty acid structural requirements for activity of arachidonoyl-CoA synthetase

We have examined the fatty acid substrate specificity of arachidonoyl-CoA synthetase from human platelet membranes. A variety of positional isomers and chain-length analogs of arachidonic acid [20:4(5, 8, 11, 14)] were synthesized, and assayed for their ability to inhibit arachidonoyl-CoA formation or to serve as substrates for the synthetase. The chain-length specificity of the synthetase for delta 8,11,14 trienoic fatty acids was C19 greater than C18 = C20 much greater than C21 greater C22. Inhibition activity by positional isomers of arachidonate was 20:4(5, 8, 11, 14) approximately equal to 20:4(6, 9, 12, 15) = 20:4(7, 10, 13, 16) much greater than 20:4(4, 7, 10, 13), however, Vmax for arachidonate was greater than that for 20:4(6, 9, 12, 15). The enzyme apparently "counts" double bonds from the carboxyl terminus. As counted from the methyl terminus we found that several n-6,-9,-12 fatty acids were ineffective as inhibitors [18:3(6, 9, 12); 19:4)4, 7, 10, 13); 21:3(9, 12, 15)], whereas all methylene-interrupted tri- and tetraenoic fatty acids which contained delta 8 and delta 11 double bonds were potent inhibitors. The delta 11 double bond was best associated with optimal inhibition: 20:3(5, 11, 14) had a lower Ki than 20:3(5, 8, 14). 13-Methyl-20:3(8, 11, 14) did not inhibit the enzyme. Partially purified enzyme from calf brain, depleted of nonspecific long-chain acyl-CoA synthetase, exhibited the same fatty acid specificity as crude platelet enzyme.     

Sperm binding and fertilization envelope formation in a cell surface complex isolated from sea urchin eggs

An isolated surface complex consisting of the vitelline layer, plasma membrane, and attached secretory vesicles has been examined for its ability to bind sperm and to form the fertilization envelope. Isolated surface complexes (or intact eggs) fixed in glutaraldehyde and then washed in artificial sea water are capable of binding sperm in a species-specific manner. Sperm which bind to the isolated surface complex exhibit the acrosomal process only when they are associated with the exterior surface (vitelline layer) of the complex. Upon resuspension of the unfixed surface complex in artificial sea water, a limiting envelope is formed which, based on examination of thin sections and negatively stained surface preparations, is structurally similar to the fertilization envelope formed by the fertilized egg. These results suggest that the isolated egg surface complex retains the sperm receptor, as well as integrated functions for the secretion of components involved in assembly of the fertilization envelope.     

Polyphosphoinositide metabolism during the fertilization wave in sea urchin eggs.

A transient increase in intracellular free calcium is believed to be the signal responsible for the stimulation of the egg metabolism at fertilization and the resumption of the cell cycle. We have studied how the polyphosphoinositides (PPI) turn over at fertilization in sea urchin eggs, in order to determine the relationship between the metabolism of these lipids and the calcium signal. We compare the patterns of PPI turnover that occur during the first minute following fertilization in eggs in which PPI are labelled to steady state with [3H]inositol or [3H]arachidonate with that in which PPI are labelled for a shorter period with [3H]inositol. When eggs are labelled to apparent isotopic equilibrium with either [3H]inositol or [3H]arachidonate, no early increase in [3H]PtdInsP2 occurs while PtdIns decreases slightly. On the contrary, when not labelled to isotopic equilibrium, all [3H]PPI increase during the first 15 seconds following fertilization. We find that, within seconds, fertilization triggers a 600-fold increase in the turnover of PPI, producing an amount of InsP3 apparently sufficient to trigger calcium release. We suggest that phosphoinositidase C and PtdInsP kinase, responsible respectively for the hydrolysis and synthesis of PtdInsP2, are both stimulated to a comparable degree in the first 30 seconds following fertilization and that net changes in the amount of PtdInsP2 at fertilization are very sensitive to the relative levels of activation of the two enzymes. Activating the eggs with the calcium ionophore A23187 showed that both these enzymes are sensitive to calcium, suggesting that calcium-dependent InsP3 production might play a role in the initiation and/or the propagation of the fertilization calcium wave.(ABSTRACT TRUNCATED AT 250 WORDS)     

The vitelline envelope to fertilization envelope conversion in eggs of Xenopus laevis

Fertilization of the Xenopus laevis egg causes the conversion of the vitelline envelope to the fertilization envelope, a change reflected in the loss of sperm penetrability of the egg and the appearance of an electron-dense layer on the outer aspect of the fertilization envelope. As seen by one-dimensional gel electrophoresis, two components with molecular weights of 69,000 and 64,000 in the vitelline envelope were converted to 66,000 and 61,000 in the fertilization envelope. By two-dimensional gel electrophoresis, the components in the 69,000 and 64,000 molecular weight regions of the vitelline envelope were seen to shift to more basic isoelectric points upon conversion to the fertilization envelope. Peptide mapping by limited proteolysis suggested that the 69,000 and 64,000 molecular weight components shared the same polypeptide chains but the smaller glycoprotein lacked a carbohydrate side chain found on the larger species. Similar sites on each glycoprotein were affected when the vitelline envelope was converted to the fertilization envelope. No N-terminal amino acids could be identified on the envelope components, indicating that these glycoproteins have blocked N-termini. Ionophore A23187-activation of jellied eggs (but not dejellied eggs) caused the molecular weight changes in the absence of sperm. Thus, factors from the jelly and the cortical granules but not from sperm apparently are involved in the processing of the 69,000 and 64,000 molecular weight components.     

Release of arachidonic acid from vascular endothelial cells: fatty acyl specificity is observed with receptor-mediated agonists and with the calcium ionophore A23187 but not with melittin

Vascular endothelial cells respond to a variety of physiological and pharmacological stimuli by releasing free arachidonic acid from membrane phospholipids, thus initiating synthesis of prostacyclin. Previous work in our laboratory has demonstrated that the thrombin-stimulated deacylation is specific for arachidonate and structurally similar polyunsaturated fatty acids that contain a delta-5 double bond. We now report that histamine, bradykinin, and the calcium ionophore A23187 exhibit the same fatty acid specificity as does thrombin. Experiments with both human umbilical vein and calf pulmonary artery endothelial cells indicate that these agonists stimulate the release of previously incorporated [14C]arachidonate but not 8,11,14-[14C]eicosatrienoate or [14C]docosatetraenoate. By contrast, melittin stimulates the release of 8,11,14-eicosatrienoate, docosatetraenoate, and oleate as well as arachidonate. These results suggest that histamine, bradykinin, and A23187 activate a common calcium-dependent phospholipase A2. Melittin appears either to alter the substrate specificity of the receptor-linked phospholipase A2 activity or to activate additional enzymes as well.     

Substrate specificity of the agonist-stimulated release of polyunsaturated fatty acids from vascular endothelial cells

Stimulation of vascular endothelial cells with agonists such as histamine and thrombin results in release of arachidonic acid from membrane lipids and subsequent eicosanoid synthesis. As shown previously, the agonist-stimulated deacylation is specific for arachidonate, eicosapentaenoate, and 5,8,11-eicosatrienoate. This study has utilized radiolabeled fatty acids differing in chain length and position of double bonds to further elucidate the fatty acyl specificity of agonist-stimulated deacylation. Replicate wells of confluent human umbilical vein endothelial cells were incubated with 14C-labeled fatty acids and then challenged with histamine, thrombin, or the calcium ionophore A23187. Comparison of the results obtained with isomeric eicosatetraenoic fatty acids with initial double bonds at carbons 4, 5, or 6 indicated that the deacylation induced by all three agonists exhibited marked specificity for the cis-5 double bond. Lack of stringent chain length specificity was indicated by agonist-stimulated release of 5,8,11,14- tetraenoic fatty acids with 18, 19, 20, and 21 carbons. Release of 5,8,14-[14C]eicosatrienoate was two-to threefold that of 5,11,14-[14C]eicosatrienoate, thus indicating that the cis-8 double bond may also contribute to the stringent recognition by the agonist-sensitive phospholipase. The present study has also demonstrated that histamine, thrombin, and A23187 do not stimulate release of docosahexaenoate from endothelial cells.     

Tyrosine kinase-dependent activation of phospholipase Cgamma is required for calcium transient in Xenopus egg fertilization

In a previous study (K.-I. Sato et al., 1999, Dev. Biol. 209, 308-320), we presented evidence that a Src-related protein-tyrosine kinase (PTK), named Xyk, may act upstream of the calcium release in fertilization of the Xenopus egg. In the present study, we examined whether PTK activation of phospholipase Cgamma (PLCgamma) plays a role in the fertilization-induced calcium signaling. Immunoprecipitation studies show that Xenopus egg PLCgamma is tyrosine phosphorylated and activated within a few minutes after fertilization but not after A23187-induced egg activation. Consistently, we observed a fertilization-induced association of PLCgamma with Xyk activity that was not seen in A23187-activated eggs. A Src-specific PTK inhibitor, PP1, blocked effectively the fertilization-induced association of PLCgamma with Xyk activity and up-regulation of PLCgamma, when microinjected into the egg. In addition, a PLC inhibitor, U-73122, inhibited sperm-induced inositol 1,4,5-trisphosphate production and the calcium transient and subsequent calcium-dependent events such as cortical contraction, elevation of fertilization envelope, and tyrosine dephosphorylation of p42 MAP kinase, all of which were also inhibited by PP1. On the other hand, A23187 could cause the calcium response and calcium-dependent events in eggs injected with PP1 or U-73122. These results support the idea that Xenopus egg fertilization requires Src-family PTK-dependent PLCgamma activity that acts upstream of the calcium-dependent signaling pathway.     

Changing localizations of site-specific sperm surface antigens during sea urchin fertilization

Indirect immunofluorescence studies show that monoclonal antibody (mAb) J18/2 binds site-specifically to surface antigens localized over the acrosome and tail regions of mature Strongylocentrotus purpuratus spermatozoa. Within 5 min after induction of the acrosome reaction by exposure to egg jelly or ionophore A23187, these surface antigens become detectable over the lateral region of the head so that the entire surface of the spermatozoon is labeled. Polyspermically fertilized S. purpuratus eggs fixed at varying times after insemination and exposed to mAb J18/2 reveal that these surface antigens are quickly incorporated into the egg plasma membrane and begin to disperse as early as 1.5 min after insemination. At subsequent times, they undergo further dispersal so that by 45 min they are distributed over the entire surface of the egg. These results suggest that the sperm surface components recognized by mAb J18/2 gain the ability to disperse laterally during the acrosome reaction and proceed to do so in the egg plasma membrane after fertilization.     

Phospholipid metabolism of stimulated lymphocytes. Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [3H]arachidonate and [14C]oleate or [3H]arachidonate and [14C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3-7-fold) and phosphatidylethanolamine (2-3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

Elongation of C20 polyunsaturated fatty acids by human skin fibroblasts

Human skin fibroblasts actively elongate a portion of incorporated C20 polyunsaturated fatty acids to their respective C22 derivatives. As much as 40% of incorporated [14C]eicosapentaenoate is elongated within 8 h and 85% by 48 h. Elongation of [14C]arachidonate is initially less than half that of [14C]eicosapentaenoate and plateaus at 20-30% of incorporated 14C-labeled fatty acid. The elongation of 5,8,11-[14C]eicosatrienoate is intermediate between that of 20:4(n-6) and 20:5(n-3). Docosatetraenoate is not an effective inhibitor of the elongation of arachidonate, thus suggesting that the observed plateau is not due to product inhibition. When concentrations of exogenous fatty acids are increased, these cells elongate substantial quantities of C20 polyunsaturated fatty acids; elongation of eicosapentaenoate is consistently more extensive than that of arachidonate. Eicosapentaenoate is also an effective inhibitor of the elongation of [14C]arachidonate. Increases in exogenous arachidonate up to 10 microM result in an increase in elongation of [14C]arachidonate both in absolute quantities and as a percentage of that incorporated; the arachidonate thus acts as a positive modulator of its own elongation. Increased eicosapentaenoate also enhances the elongation of [14C]eicosapentaenoate, but only at lower concentrations (0.02-0.15 microM). The factors which regulate the elongation of C20 polyunsaturated fatty acids in human skin fibroblasts serve to permit extensive elongation of eicosapentaenoate while retaining incorporated arachidonate primarily in its C20 form.     

Lipoxins are major lipoxygenase products of rainbow trout macrophages

Rainbow trout macrophages synthesize lipoxins as major lipoxygenase products entirely from endogenous fatty acids. High-performance liquid chromatographic analysis of the supernatants from macrophages challenged with calcium ionophore A23187 revealed a range of lipoxygenase products including mono-hydroxy fatty acids, leukotrienes B4 and B5 and four major peaks with retention times and UV spectra characteristic of lipoxins (lambda max 302 nm). Cochromatography with authentic standards, UV spectroscopy and radiolabeling with [14C]arachidonate and eicosapentaenoate allowed tentative identification of the two largest peaks as lipoxin A4 and A5.     

Total lipid and fatty acid compositions of <Emphasis Type="Italic">Lertha sheppardi</Emphasis> (Neuroptera: Nemopteridae) during its main life stages

Total lipid and the fatty acid compositions of phospholipid and triacylglycerol fractions, prepared from eggs, 3^rd instars of larvae, pupae, male and female adults of Lertha sheppardi, were analyzed by gas chromatography and gas chromatography-mass spectrometry. The effect of diet (adults’ nutrition) on fatty acid composition of L. sheppardi adults was also investigated. Total lipid of L. sheppardi considerably increased in adults compared with immature stages. There was a significant decrease in total lipid level in larval stage in contrast with egg stage. Qualitative analysis revealed the presence of 14 fatty acids during all stages. The major components were C16 and C18 saturated and unsaturated components which are ubiquitous to most animal species. In addition to these components, one odd-chain (C17:0) and prostaglandin precursor fatty acids were found. The fatty acid profiles of phospholipids and triacylglycerols were substantially different. In phospholipid fraction, monounsaturated fatty acids were the major proportion of fatty acids in both sex of adults and pupae, whereas polyunsaturated fatty acids were the most dominant fatty acids in eggs and 3^rd instars. Results of triacylglycerol fraction revealed that fatty acid composition of eggs had higher level of C16:1, C18:0 and C18:3n-3 content than that of 3^rd instars and pupae, which suggests accumulation of energetic and structural reserve materials during embryonic development. At more advanced developmental stages, mainly in adult females, the amount of C16:1 increased once again, which may be related to the need for accumulation of sufficient energy and of carbon reservoir in the developing new vitellum. Percentages of C18:1 were significantly high in adult stages compared to other stages. These findings indicate that the accumulation and consumption of fatty acids fluctuate through different development stages. Diet did not effect the fatty acid composition of L. sheppardi adults.     

Gamete Interactions in Xenopus laevis: Identification of Sperm Binding Glycoproteins in the Egg Vitelline Envelope

A quantitative assay was developed to study the interaction of Xenopus laevis sperm and eggs. Using this assay it was found that sperm bound in approximately equal numbers to the surface of both hemispheres of the unfertilized egg, but not to the surface of the fertilized egg. To understand the molecular basis of sperm binding to the egg vitelline envelope (VE), a competition assay was used and it was found that solubilized total VE proteins inhibited sperm-egg binding in a concentration-dependent manner. Individual VE proteins were then isolated and tested for their ability to inhibit sperm binding. Of the seven proteins in the VE, two related glycoproteins, gp69 and gp64, inhibited sperm-egg binding. Polyclonal antibody was prepared that specifically recognized gp69 and gp64. This gp69/64 specific antibody bound to the VE surface and blocked sperm binding, as well as fertilization. Moreover, agarose beads coated with gp69/64 showed high sperm binding activity, while beads coated with other VE proteins bound few sperm. Treatment of unfertilized eggs with crude collagenase resulted in proteolytic modification of only the gp69/64 components of the VE, and this modification abolished sperm-egg binding. Small glycopeptides generated by Pronase digestion of gp69/64 also inhibited sperm-egg binding and this inhibition was abolished by treatment of the glycopeptides with periodate. Based on these observations, we conclude that the gp69/64 glycoproteins in the egg vitelline envelope mediate sperm-egg binding, an initial step in Xenopus fertilization, and that the oligosaccharide chains of these glycoproteins may play a critical role in this process.     

Phospholipid metabolism of stimulated lymphocytes: Preferential incorporation of polyunsaturated fatty acids into plasma membrane phospholipid upon stimulation with concanavalin A

Rabbit thymocytes were isolated and incubated for various lengths of time with concanavalin A. The cultures were pulsed for the last 12.5 min of incubation with equimolar mixtures of radioactively labelled fatty acids, either [³H]arachidonate and [¹⁴C]oleate or [³H]arachidonate and [¹⁴C]palmitate, and the uptake of each fatty acid into phospholipid of plasma membrane was determined. Upon binding of the mitogen, the fatty acids were incorporated at an increased rate with a new steady state being reached between 12.5 and 42.5 min after stimulation. Initially after 12.5 min, when the two fatty acids were added together, no preferential incorporation of the polyunsaturated fatty acid arachidonate was seen compared to the saturated or monounsaturated ones, palmitate or oleate. However shortly thereafter arachidonate, when compared to palmitate or oleate, started to be preferentially incorporated into plasma membrane phospholipid so that by 4 h after activation, only arachidonate was incorporated at an increased rate: the uptake of palmitate and oleate had reverted to that of unstimulated cells. In contrast, when palmitate or oleate were added alone, after 4 h of activation incorporation was increased similar to that of arachidonate, suggesting that all long chain fatty acids compete for the same activated enzyme(s). A detailed analysis of incorporation into phospholipid species showed that all fatty acids were taken up with the highest rate into phosphatidylcholine. After activation, fatty acid incorporation was increased by approx. 50% for phosphatidylcholine: the highest stimulation rates were observed with phosphatidylinositol (3–7-fold) and phosphatidylethanolamine (2–3-fold). The data suggest that shortly after stimulation with mitogens, the membrane phospholipids start to change by replacing saturated and monounsaturated fatty acids by polyunsaturated ones, thus creating a new membrane.     

Kinetics of actin assembly attending fertilization or artificial activation of sea urchin eggs

Changes in the state of actin assembly triggered by fertilization or by artificial activation of sea urchin eggs were quantified using the DNase I inhibition assay. Insemination of Lytechinus pictus or Strongylocentrotus purpuratus eggs induces a cyclic variation in the level of G-actin as follows: between 0 and 30 s after insemination, the G-actin content decreases. This is followed by an increase in the amount of monomeric actin between 30 and 60 s, and then from 60 s to 5 min postinsemination there is a progressive decrease in the egg's level of G-actin. This latter decrease is more pronounced in S. purpuratus eggs than in L. pictus eggs. Using sperm mimetics that trigger an increase in intracellular calcium concentration (A23187 in sodium-free seawater), a cytoplasmic alkalinization (NH4Cl), a plasma membrane depolarization (seawater enriched with potassium ions), or all three of these phenomena (A23187 in normal seawater), each phase depicted at fertilization correlates with the following metabolic events accompanying egg awakening: phase 1, of uncertain origin (possibly related to plasma membrane depolarization); phase 2, elevation of intracellular calcium concentration; phase 3, alkalinization of the intracellular milieu but only if the transient intracellular calcium rise has taken place.     

The alphaBbetaC integrin is expressed on the surface of the sea urchin egg and removed at fertilization

Integrins are expressed on the surface of some vertebrate eggs where they are thought to have a role in fertilization. The objective of this study is to determine if integrins are expressed on sea urchin eggs. The alphaB and betaC subunits were cloned using the homology polymerase chain reaction. Monoclonal and polyclonal antibodies were developed against bacterially expressed fragments of the extracellular domains of the betaC subunit and the alphaB subunit. As well, a monoclonal antibody was developed against a synthesized peptide corresponding to part of the cytoplasmic domain of betaC. Analysis of biotinylated egg cortex extracts immunoprecipitated with either anti-betaC or anti-alphaB yields bands of 130 and 225 kDa. Immunoblots confirm that betaC is part of the complex immunoprecipitated with anti-alphaB. Confocal immunofluorescence and immunogold electron microscopy show that betaC is present on the surface of the unfertilized egg at the tips of microvilli and in cortical granules. During the cortical reaction, immunoreactivity with antibodies to the extracellular domains of betaC and alphaB disappears from the egg surface, and microvillar casts on the fertilization envelope become immunoreactive. With antibodies to the cytoplasmic domain of betaC, immunoreactivity is lost from the surface of the egg, but the fertilization envelope does not immediately become immunoreactive. In immunoblots of egg cortex there are immunoreactive bands of the predicted sizes for alphaB and betaC. However, in fertilization envelopes, a second band that is slightly lower in molecular weight is also present. Eggs fertilized in the presence of soybean trypsin inhibitor have elongated microvilli that remain bound to the elevating fertilization envelope and immunoreactive to anti-betaC antibodies. Eggs fertilized in the presence of an ovoperoxidase inhibitor, 3-amino-1,2,4-triazole, have a patchy distribution of betaC immunoreactivity in fertilization envelopes. Together, these data suggest that alphaBbetaC integrins are expressed on the surface of unfertilized eggs and, during the cortical reaction, the extracellular domains are cleaved by proteases and cross-linked into the fertilization envelope by ovoperoxidase. The alphaBbetaC integrin receptors may have several potential functions prior to their removal at fertilization, including attachment of the vitelline envelope to the egg surface and anchoring the cortical cytoskeleton.     

Probable participation of phospholipase A2 reaction in the process of fertilization-induced activation of sea urchin eggs

In sea urchin eggs activated by sperm, A23187 or melittin, BPB (4-bromophenacyl bromide, a phospholipase A₂ inhibitor) blocked fertilization envelope formation and transient CN⁻-insensitive respiration in a concentration-dependent manner. BPB had virtually no effect on the increase in [Ca²⁺]_i, (cytosolic Ca²⁺ level), the activity of phosphorylase a and the rate of protein synthesis, as well as acid production and augmentation of CN⁻-sensitive respiration. BPB also inhibited fertilization envelope formation and augmentation of CN⁻-insensitive respiration induced by melittin. Melittin, known to be an activator of phospholipase A₂, induced the envelope formation, acid production, augmentation of CN⁻-insensitive and sensitive respiration, but did not cause any increase in [Ca²⁺]_i, the phosphorylase a activity and the rate of protein synthesis. An activation of phospholipase A₂ induced by Ca²⁺ or melittin seems to result in cortical vesicle discharge and production of fatty acids, which are to be utilized in CN⁻-insensitive lipid peroxidase reactions. Activation of other examined cell functions in eggs activated by sperm or A23187, probably results from Ca²⁺-triggered sequential reactions other than Ca²⁺-caused activation of phospholipase A₂.     

Fertilization envelope formation induced by melittin in sea urchin eggs does not depend on Ca2+ signalling

In sea urchin eggs, 10 μg/mL melittin was found to induce fertilization envelope formation without any increase in [Ca²⁺]_i (the intracellular free Ca²⁺ level). On the other hand, 10 μmol/L Br-A23187 and 100 μg/mL SDS induced fertilization envelope formation associated with [Ca²⁺]_i increase. If EGTA was injected into eggs to make an intracellular concentration of 2 mmol/L, [Ca²⁺]_i became quite low and was not altered by melittin, or by Br-A23187 and SDS. In eggs containing EGTA, fertilization envelope formation was induced by melittin even in Ca²⁺-free artificial sea water, but not by Br-A23187 or SDS. Thus [Ca²⁺]_i is essential for induction of a fertilization envelope in sea urchin eggs by Br-A23187 or SDS but not by melittin. Melittin probably activates some Ca²⁺-independent reaction downstream of Ca²⁺-dependent reactions in a sequential reaction system that finally results in fertilization envelope formation.