Surface localization of the sea urchin egg receptor for sperm

The sea urchin egg receptor for sperm is thought to be involved in species-specific sperm-egg interactions at the egg surface. Recent revisions in the deduced amino acid sequence of the cloned cDNAs indicate that the protein encoded does not possess the common structural hallmarks of a membrane protein. Thus, investigation of the localization and association of the protein with the egg surface is crucial. We describe and characterize a new monoclonal antibody raised against recombinant sperm receptor protein. This antibody, in conjunction with several polyclonal antibodies, was used to study the receptor protein in eggs. Immunoprecipitation studies indicated that the antibodies recognize the high Mr (ca. 350 K) sperm receptor protein which copurified with egg plasma membrane-vitelline layer complexes. The sperm receptor protein was solubilized only by detergents and not by treatments designed to solubilize peripherally associated or lipid-anchored membrane proteins, suggesting a tight association with the membrane fraction. Confocal immunofluorescence microscopy of live eggs indicated surface staining. Finally, lysylendoproteinase C treatment of live eggs resulted in a loss of the high Mr receptor protein epitopes, and the concomitant release of a 70-kDa proteolytic fragment, which correlated with a reduced ability of the eggs to be fertilized. Taken together, these data indicate that at least some fraction of the sperm receptor protein is present on the egg surface, a requisite locale for a sperm binding protein.     

Identification and localization of a sea urchin Notch homologue: insights into vegetal plate regionalization and Notch receptor regulation

The specifications of cell types and germ-layers that arise from the vegetal plate of the sea urchin embryo are thought to be regulated by cell-cell interactions, the molecular basis of which are unknown. The Notch intercellular signaling pathway mediates the specification of numerous cell fates in both invertebrate and vertebrate development. To gain insights into mechanisms underlying the diversification of vegetal plate cell types, we have identified and made antibodies to a sea urchin homolog of Notch (LvNotch). We show that in the early blastula embryo, LvNotch is absent from the vegetal pole and concentrated in basolateral membranes of cells in the animal half of the embryo. However, in the mesenchyme blastula embryo LvNotch shifts strikingly in subcellular localization into a ring of cells which surround the central vegetal plate. This ring of LvNotch delineates a boundary between the presumptive secondary mesoderm and presumptive endoderm, and has an asymmetric bias towards the dorsal side of the vegetal plate. Experimental perturbations and quantitative analysis of LvNotch expression demonstrate that the mesenchyme blastula vegetal plate contains both animal/vegetal and dorsoventral molecular organization even before this territory invaginates to form the archenteron. Furthermore, these experiments suggest roles for the Notch pathway in secondary mesoderm and endoderm lineage segregation, and in the establishment of dorsoventral polarity in the endoderm. Finally, the specific and differential subcellular expression of LvNotch in apical and basolateral membrane domains provides compelling evidence that changes in membrane domain localization of LvNotch are an important aspect of Notch receptor function.     

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

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

The 350-kDa sea urchin egg receptor for sperm is localized in the vitelline layer

Previous studies have established by several methods that the 350-kDa egg receptor for sperm is localized on the plasma membrane-vitelline layer complex of the egg of the sea urchin Strongylocentrotus purpuratus. In addition, it has been found that molecules which are cross-reactive with anti-receptor antibody are present in the cortical granules located at the inner face of the plasma membrane. The objective of this study was to define more precisely the locale of the cell surface receptor. We have found that following fertilization, the immunoreactive receptor initially found on the egg surface moved to the fertilization envelope (FE) and then disappeared in parallel with the loss of sperm binding activity. A cross-linked, high-molecular-weight derivative of soybean trypsin inhibitor (hMW-SBTI) which was unable to pass through the elevating FE blocked the loss of both immunoreactivity and the sperm binding activity of the FE, but did not inhibit the vitelline delaminase activity that has been implicated in FE formation. Western blot analysis following SDS-PAGE of the proteins of the FE isolated in the presence of hMW-SBTI and benzamidine revealed the presence of the 350/320-kDa proteins which cross-reacted with anti-receptor antibody. Experiments in which molecules on the surface of unfertilized eggs were labeled with biotin and traced after FE formation revealed that a significant portion of the 350/320-kDa glycoproteins that were incorporated into the FE originated from the cell surface, rather than from the cortical granules. These findings provide strong evidence that in unfertilized eggs the egg receptor for sperm exists as part of the protein complex known as the vitelline layer which serves as a precursor of the FE. Evidence is presented indicating that some of the receptor in the vitelline layer is cryptic and a possible function for this cryptic form of the receptor is proposed.     

Na is essential for activation of the inseminated sea urchin egg

The presence of at least 2.5 mM Na during the first several min after insemination is required for the activation of sea urchin eggs. Of those chemical species examined that exist entirely as cations, and which did not activate the unfertilized egg, only Li substitutes for Na. Ammonium and other amines, with pKa values between 9 and 10.8, in the complete absence of Na (a) can induce nuclear activation of unfertilized eggs, and (b) permit the development of the early fertilized egg through the stage that normally requires Na.     

Changes in the topography of the sea urchin egg after fertilization

Rats of various postnatal ages were utilized to study developmental changes in the distribution of Na, K and H2O between the various compartments of the lateral ventricular plexus (LVP). During the 3 weeks after birth, as the LVP grows from 0.5 to 0.8 mg, there is a significant increase in plexus K which is accompanied by a progressive decrease in Na and H2O. Also, during this postnatal period the decrease in [3h]inulin space in the plexus is proportional to the decrease in the Na space. Between 3 weeks and adulthood, the [3h]inulin and Na spaces are both augmented to a similar extent; moreover, during this same period of development there is a trebling of the residual [51cr]erythrocyte volume. Despite the substantial changes in the volume of the extracellular fluid and of the residual blood in the plexus with age, the calculated concentrations (mEquiv./kg H2O) of choroid cell Na (30-35) and K (145-155) are similar for all ages investigated. The derived data for cellular ionic concentration, together with the analysis of the ionic concentration gradients (cerebrospinal fluid/plasma H2O), suggest that the transport mechanism which translocates Na and K across the choroidal membrane is operative as early as 3-4 days postnatal. The important role of the choroid plexus in central nervous system homeostasis is discussed in relation to the developing brain.     

Muscle-type tropomyosin of sea urchin egg increases the actin-binding of nonmuscle-type tropomyosin

Tropomyosin isoforms in eggs of several species of sea urchins are classified into two types, muscle-type and nonmuscle-type, based on their antigenicities. Their actin-binding abilities were investigated using muscle-type isoform (32K) and nonmuscle-type isoform (30K), which were purified by the method previously reported and separated by isoelectric focusing from eggs of sea urchin, Strongylocentrotus intermedius. Co-sedimentation assays revealed that 32K could stoichiometrically bind to actin filaments independently of the 30K, but 30K alone bound very poorly. The actin-binding of 30K was, however, considerably increased in the presence of 32K, and the molar ratio of the bound 30K and 32K was approximately 1:1. The increase in the actin-binding of 30K is probably caused by the interaction of 30K with 32K in a head-to-tail manner, as indicated by the higher specific viscosity of the mixture than that of 32K alone.     

Calcium can disrupt the SNARE protein complex on sea urchin egg secretory vesicles without irreversibly blocking fusion

The homotypic fusion of sea urchin egg cortical vesicles (CV) is a system in which to correlate the biochemistry and physiology of membrane fusion. Homologues of vesicle-associated membrane protein (VAMP), syntaxin, and SNAP-25 were identified in CV membranes. A VAMP and syntaxin immunoreactive band at a higher apparent molecular mass (approximately 70 kDa) was detected; extraction and analysis confirmed that the band contained VAMP, SNAP-25, and syntaxin. This complex was also identified by immunoprecipitation and by sucrose gradient analysis. VAMP in the complex was insensitive to proteolysis by tetanus toxin. All criteria identify the SNARE complex as that described in other secretory systems. Complexes exist pre-formed on individual CV membranes and form between contacting CV. Most notably, CV SNARE complexes are disrupted in response to [Ca2+]free that trigger maximal fusion. N-Ethylmaleimide, which blocks fusion at or before the Ca2+-triggering step, blocks complex disruption by Ca2+. However, disruption is not blocked by lysophosphatidylcholine, which transiently arrests a late stage of fusion. Since removal of lysophosphatidylcholine from Ca2+-treated CV is known to allow fusion, complex disruption occurs independently from the membrane fusion step. As Ca2+ disrupts rather than stabilizes the complex, the presumably coiled-coil SNARE interactions are not needed at the time of fusion. These findings rule out models of fusion in which SNARE complex formation goes to completion ("zippers-up") after Ca2+ binding removes a "fusion-clamp."     

Identification of a new sea urchin vitelline envelope sperm binding glycoprotein

The sea urchin egg vitelline envelope (VE) is composed of eight major glycopolypeptides that are heavily mannosylated and contain fucose and N-acetylglucosamine moieties based on lectin staining. In the present study, the macromolecular composition of the VE and the potential role of a purified VE glycoprotein in initial gamete binding was investigated. The VE components were solubilized from the surface of intact, dejellied eggs with dithiothreitol in divalent cation-free seawater, and analyzed using native, reduced electrophoresis and immunoblotting. Three major VE glycoproteins, VE-A, VE-B and VE-C, and one minor component, VE-D, were identified with antisera against whole VE preparations and against glutaraldehyde-fixed, unfertilized eggs. The electrophoretically purified glycoproteins resolved into a common subunit doublet and one unique subunit each of decreasing size on blots of sodium dodecylsulfate polyacrylamide gels. Lectin affinity chromatography was used for analysis and purification of reduced VE components; a glycoprotein eluted from Con A columns with methyl-mannoside comigrated with VE-B when analyzed by immunoblotting. Whole VE preparations and VE-B obtained from Con A columns were found to inhibit fertilization when preincubated with sperm, thus directly establishing a role for VE-B in gamete binding.     

The nucleus negatively controls the synthesis of mitochondrial proteins in the sea urchin egg

Enucleation of Paracentrotus lividus eggs, followed by parthenogenetic activation induces a sharp increase in the synthesis of mitochondrial proteins as shown by electrofluorography after in vivo labeling with radioactive amino acids. These results further substantiate the hypothesis that the cell nucleus negatively controls mitochondrial replication in the sea urchin egg.     

Identification and characterization of a constitutive HSP75 in sea urchin embryos

STUDY OBJECTIVE: To evaluate the influence of occupational exposure to carcinogens in explaining the association between socioeconomic status and lung cancer. DESIGN: A prospective cohort study. Data on diet, other lifestyle factors, sociodemographic characteristics and job history were collected by means of a self administered questionnaire. Follow up for incident cancer was established by record linkage with a national pathology register and with regional cancer registries. SETTING: Population originating from 204 municipalities in The Netherlands. PARTICIPANTS: These comprised 58 279 men aged 55-69 years in September 1986. After 4.3 years of follow up there were 470 microscopically confirmed incident lung cancer cases with complete data on dietary habits and job history. MEASUREMENTS AND MAIN RESULTS: Estimation of occupational exposure to asbestos, paint dust, polycyclic aromatic hydrocarbons, and welding fumes was carried out by two experts, using information on job history from the baseline questionnaire. Socioeconomic status was measured by means of highest attained level of education and two indicators based on occupation. In the initial multivariate analyses of socioeconomic status and lung cancer, adjustment was made for age, smoking habits, intake of vitamin C, beta-carotene and retinol, and history of chronic obstructive pulmonary disease or asthma. Additional adjustment for occupational exposure to the four carcinogens mentioned above did not change the inverse association between the level of education and lung cancer risk (initial model: RR highest/lowest level of education = 0.53; 95% CI 0.34, 0.82; additional model: RR highest/lowest level of education = 0.53; 95% CI 0.34, 0.84). Nor was the association between the two occupation based indicators of socioeconomic status and lung cancer risk influenced by occupational exposure to carcinogens. The effect of occupational exposure on the association between the level of education and lung cancer risk did not differ between ex-smokers and current smokers. CONCLUSIONS: Occupational exposure to asbestos, paint dust, polycyclic aromatic hydrocarbons, and welding fumes could not explain the inverse association between socioeconomic status and lung cancer risk. More research which explicitly addresses possible explanations for the association between socioeconomic status and lung cancer risk is needed.     

Relative changes in F-actin during the first cell cycle: evidence for two distinct pools of F-actin in the sea urchin egg

The cortical actin cytoskeleton undergoes dramatic rearrangements during fertilization of sea urchin eggs. To characterize these changes further, we quantified the relative changes in filamentous actin (F-actin) during fertilization and the first cell cycle in both intact eggs and in isolated cortices by quantitative fluorescence microscopy. The level of F-actin in the intact egg decreased after fertilization and continued to decrease throughout the first cell cycle. By 60 min after fertilization, the level of F-actin had decreased to 50% of the unfertilized sea urchin egg. By cytokinesis, the level of F-actin had decreased to 30% of the unfertilized egg. After completion of cell division, individual blastomeres had 10% of the F-actin in the unfertilized egg. In contrast, there was an increase in cortical F-actin to 370% of the level in the unfertilized egg after fertilization. This increase corresponded to the formation of microvilli. There was little change in the level of cortical F-actin during the first cell cycle. We draw parallels to other systems that increase the amount of F-actin in the Triton-insoluble cytoskeleton by recruiting actin from a Triton-soluble pool of F-actin.     

Identification of ArgBP1, an Arg protein tyrosine kinase binding protein that is the human homologue of a CNS-specific Xenopus gene

Cardiac fibroblasts constitute greater than 90% of the non-myocyte cells in the heart. Previously, it was established that cardiac fibroblasts are predisposed to transformation into a phenotype with muscle-specific features and that transforming growth factor-beta 1 (TGF-beta 1) is a specific inducer of this event. In this study the hypothesis that TGF-beta 1-induced phenotypic modulation of cardiac fibroblasts is associated with their altered proliferative capacity is tested. Therefore the effects of TGF-beta 1 on DNA synthesis in cardiac fibroblasts under normal conditions of cell culture and in response to a potent mitogen, basic fibroblasts growth factor (bFGF) were determined. The results showed that TGF-beta 1 at 15 ng/ml (a concentration that induces fibroblast "transformation") had a regulatory effect on proliferative capacity of cardiac fibroblasts which varied as the function of cell density in culture. In subconfluent and confluent cultures, pre-treatment of cardiac fibroblasts with TGF-beta 1 for 24 h resulted in a dramatic shift in the bFGF-induced stimulation of DNA synthesis. TGF-beta 1-induced inhibition of DNA synthesis in cardiac fibroblasts coincided with their phenotypic modulation as evidenced by the expression of sarcomeric actin mRNA and morphological changes. Cross-linking studies with [125I]-labeled TGF-beta 1 showed the presence of conventional types I, II and III TGF-beta 1 receptor complexes on cardiac fibroblasts and their binding to TGF-beta 1 under the experimental conditions. In summary, these data indicate that the proliferative capacity of cardiac fibroblasts is controlled by TGF-beta 1. They further suggest that the TGF-beta 1-induced phenotypic modulation of cardiac fibroblasts may be extended to include their altered proliferative capacity.     

Purification of EGIP-D-binding protein from the embryos of the sea urchin Anthocidaris crassispina

We report three cases of male breast myofibroblastoma. This uncommon benign tumor arises from breast mesenchyma and is more frequently seen in adult men. Mammographic findings consist of a well-delimited, round to oval dense mass, variable in size but usually 1-4 cm in diameter. No microcalcifications were observed. Ultrasonography confirms the solid nature of the lesion, showing a well-circumscribed, homogeneous, hypoechoic mass, compressible with pressure. Although FNA cytology may support the diagnosis, surgical biopsy should be performed. Tumorectomy is the treatment of choice. To our knowledge, no more than 40 cases of breast myofibroblastoma have been reported. This is the first report in the literature which emphasizes the mammographic and ultrasonographic features of this tumor.     

Membrane fusion is induced by a distinct peptide sequence of the sea urchin fertilization protein bindin

Fertilization in the sea urchin is mediated by the membrane-associated acrosomal protein bindin, which plays a key role in the adhesion and fusion between sperm and egg. We have investigated the structure/function relationship of an 18-amino acid peptide fragment "B18," which represents the minimal membrane binding motif of the protein and resembles a putative fusion peptide. The peptide was found to mimic the behavior of its parent protein bindin with respect to (a) its high affinity for lipid bilayers, (b) the ability to aggregate and fuse vesicles, (c) the binding of Zn2+ by a histidine-rich motif, (d) the tendency to self-assemble, and (e), as indicated earlier, the adhesion to cell surface polysaccharides. Fluorescence and light scattering assays were used here to monitor peptide-induced lipid mixing, leakage, and aggregation of large unilamellar sphingomyelin/cholesterol vesicles. For these activities, B18 requires the presence of Zn2+ ions, with which it forms oligomeric complexes and assumes a partially alpha-helical conformation, as observed by circular dichroism. We conclude that aggregation and fusion involves a "trans-complex" between peptides on apposing vesicles that are connected by Zn2+ bridges.