Immunological evidence that a 305-kilodalton vitelline envelope polypeptide isolated from sea urchin eggs is a sperm receptor

Direct isolation of the sea urchin egg vitelline envelope with intact sperm receptors is difficult because the envelope is firmly attached to the egg plasma membrane. We now report a method for producing an inseminated egg preparation in Strongylocentrotus purpuratus (using soybean trypsin inhibitor [STI] and Ca²⁺, Mg²⁺-free seawater) that contains an elevated vitelline envelope (VE*-STI). The VE*-STI is devoid of cortical granule material, and supernumerary sperm do not detach postinsemination, suggesting that the VE*-STI contains active sperm receptors. VE*-STIs contain a 305-kD polypeptide and additional components that range from 225 to 31 kD, whereas the 305-kD polypeptide was considerably reduced in VE*s. Electrophoresis of sperm receptor hydrolase digests of VE*-STIs showed that the 305-kD polypeptide and several other envelope polypeptides are protease substrates. Univalent Fab fragments against VE*s, VE*-STIs, and 305 and 225-kD polypeptides blocked sperm binding and fertilization in an Fab concentration-dependent manner. The 305 and 225-kD polypeptides were localized in the VE*-STI using indirect immunofluorescence. Enzyme-linked immunosorbent assays showed that the 305 and 225-kD polypeptides share determinants, suggesting that the 225-kD polypeptide may be derived from the 305-kD polypeptide by the proteolysis that occurs at the cell surface during fertilization. Fab fragments against S purpuratus VE*-STI antigens neither bound to nor blocked homologous sperm binding and fertilization of Lytechinus variegatus eggs. Cross fertilizability occurred to the extent of 5% or less between L variegatus and S purpuratus, therefore, we conclude that the 305 kD-polypeptide isolated from S purpuratus is a species-specific vitelline envelope sperm receptor.     

In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.     

Characterization of hatching-associated changes in the sea urchin fertilization envelope

The embryo of the sea urchin Strongylocentrotus purpuratus hatches from the fertilization envelope (FE) via synthesis and secretion of a hatching enzyme and by ciliary activity. Although the basic characteristics of the hatching enzyme are known, little is understood about changes in the FE during hatching. We have studied the biochemical changes in FEs during hatching. Polyacrylamide gel analysis revealed an increasingly complex polypeptide spectrum of the extractable fraction of FEs isolated during development. Immunoblotting of these polypeptides (using antiserum against the soluble polypeptides extracted from FEs isolated at 30 minutes postinsemination) revealed a decrease in the soluble FE components during hatching. Immunochemical analysis of hatching medium showed a strong correlation between the soluble FE components released and the hatching interval. Immunoblotting of hatching media indicated the presence of soluble FE polypeptides of similar and lower molecular weights than those obtained for extracts of FEs. These results imply that the hatching-associated changes in the FE of S purpuratus occur via proteolysis of FE components, which are derived from the paracrystalline protein fraction, a subset of cortical granule proteins.     

Novel procedures for collection of sea urchin egg cortical granule exudate: Partial characterization and evidence for postsecretion processing

We have developed two procedures to collect total cortical granule exudate in a soluble form from eggs of the sea urchin Strongylocentrotus purpuratus. Egg suspensions were either treated with dithiothreitol to disrupt the vitelline envelope or divalent cations were removed postinsemination to prevent the normal vitelline-to-fertilization envelope transition. Rapid acidification of the insemination mixture (dithiothreitol-treated eggs) to pH 6.0 prevented precipitation of the paracrystalline protein fraction described by Bryan [1970a]. Exudate was partitioned into three fractions. The pH 8.0-insoluble fraction appeared to be identical to the paracrystalline protein fraction. The pH 8.0-soluble fraction was separated into pH 4.0-soluble and-insoluble fractions. Analysis for peroxidase and protease activities showed that peroxidase activity was localized in all three fractions whereas protease activity was restricted to the pH 4.0 insoluble fraction as reported [Carroll and Epel, 1975]. A minimum of six major proteins were detected on native polyacrylamide gels of total exudate. Under reducing and denaturing conditions, 12 polypeptides ranging from 19,000 to 165,000 in molecular weight were detected in total exudate; six polypeptides were recovered in the pH 8.0-insoluble fraction. To test the hypothesis that protease and peroxidase activities process cortical granule proteins after secretion, we inseminated eggs in solutions containing peroxidase and protease inhibitors. The paracrystalline protein fraction crystallized slowly from insemination mixtures containing both inhibitors compared to controls and there were dramatic differences in exudate electrophoretic patterns. We suggest that cortical granule protease and peroxidase activities process the exudate so that the paracrystalline protein fraction rapidly crystallizes during normal fertilization.     

Sea urchin egg-cortical granule protease has arginyl proteolytic specificity

Sea urchin eggs secrete esteroproteolytic activity at fertilization. This enzyme has been shown to be proteolytic toward embryo protein and casein, but a systematic study of its substrate specificity has not been done. In this communication we present data that demonstrates for the first time that the cortical granule protease from Strongylocentrotus purpuratus eggs cleaves arginyl residues in a protein substrate, lysozyme. We have developed a sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) assay that detects femtomole levels of trypsin and chymotrypsin protease activity [Green, 1986: Anal Biochem 152:83–88]. In the sea urchin system, we have detected protease activity from as few as 50 eggs. Correlating the RP-HPLC analysis with a spectrophotometric Nα-benzoyl-L-arginine ethyl ester assay, we have found that each egg secretes approximately 40 attomoles of trypsin-like activity. This general method should be quite useful in investigations into the natural substrate of the egg protease.     

Ionophore-induced efflux of sodium and potassium ions from sea urchin eggs

The efflux of K⁺ and Na⁺ from sea urchin eggs during Ca²⁺ ionophore A23187-induced parthenogenesis was studied in a K⁺ and Na⁺-free artificial seawater using extracellular ion-specific electrodes. We have probed this model system with monovalent cation-specific ionophores to determine if they affect K⁺ efflux in the unfertilized egg and whether any changes in ionophore sensitivity are observed during egg activation. In 500 mM choline chloride, 10 mM CaCl₂, 50 mM MgCl₂, 10 mM Tris-Cl pH 8.0, A23187 induced a rapid efflux of K⁺ and Na⁺ from the eggs after a short lag time (10–15 seconds). After the burst, the rate of K⁺ efflux remained higher than the pre-activation rate, but was lower than during the burst phase, while the rate of Na⁺ efflux became nearly zero. Monovalent cation-specific ionophores (valinomycin, gramicidin and nigericin) had no effect on K⁺ efflux from the unfertilized eggs in our model system. However, once the egg was activated by A23187, each of the above ionophores caused a prolongation of the burst phase for many minutes. These results show that the unfertilized egg plasma membrane (using our artificial conditions) is not susceptible to the monovalent cation-specific antibiotics and suggest that either the inserted cortical granule membrane or the developing fertilization envelope interacts with these ionophores to cause the change in rate-limiting step for K⁺ efflux observed egg activation.     

Gastrulation in the Sea Urchin,Strongylocentrotus purpuratus,Is Disrupted by the Small Laminin Peptides YIGSR and IKVAV

Laminin is present on the apical and basolateral sides of epithelial cells of very early sea urchin blastulae. We investigated whether small laminin-peptides, known to have cell binding activities, alter the development of sea urchin embryos. The peptide YIGSR-NH₂ (850 μM) and the peptide PA22-2 (5 μM), which contains the peptide sequence IKVAV (Tashiro et al., J. Biol. Chem. 264, 16174, 1989), typically blocked archenteron formation when added to the sea water soon after fertilization. At lower doses, the YIGSR peptide allowed invagination of the archenteron but blocked archenteron extension and differentiation and evagination of the feeding arms. The effect of YIGSR and PA22-2 peptides declined when added to progressively older stages until no effect was seen when added at the mesenchyme blastula stage (24 hours after fertilization). Control peptides GRGDS, YIGSE, and SHA22, a dodeca-peptide with a scrambled IKVAV sequence, had no effect on development. The YIGSK peptide containing a conserved amino acid modification had only a small effect on gastrulation. The results suggest that YIGSR and IKVAV peptides specifically disrupt cell/extracellular matrix interactions required for normal development of the archenteron and feeding arms. Our recent finding that YTGIR is at the cell binding site of the B1 chain of S. purpuratus laminin supports this conclusion. Evidently, laminin or other laminin-like molecules are among the many extracellular matrix components needed for the invagination and extension of the archenteron during the gastrulation movements of these embryos.     

Statistical and Frequency-Amplitude Characteristics of Ultraweak Emissions of the Loach Eggs and Embryos under the Normal Conditions and during Their Optic Interactions. 1. Characteristics of Ultraweak Emission in Normal Development and the Optic Role of Egg Envelopes

Ultraweak emissions of groups comprising several dozens of unfertilized and fertilized loach eggs, embryos, larvae, and their egg envelopes were measured on a photomultiplier tube. The envelopes absorbed the light from external sources but readily gave it back in the absence of embryos. We carried out statistical and frequency-amplitude analyses of ultraweak emissions and studied the autocorrelation structure of their frequency spectra. The frequencies of signals with different intensity underwent regular changes during development. Cascades of short-term (1 ms) flashes timed (during cleavage) to furrowing were a characteristic element of ultraweak emission. The Fourier spectra of developing embryos had pronounced frequency-amplitude peaks and higher, than in unfertilized eggs and inanimate samples, mutual correlation during successive time intervals. Stage-specific translational symmetry of the frequency spectra of ultraweak emissions was demonstrated, which suggests the presence in groups of embryos of a coordinated system of harmonic oscillators. The latter underwent regular changes during development. The measurement of ultraweak emissions represents a unique non-invasive method of analysis of these oscillators.     

Sea urchin ovoperoxidase: oocyte-specific member of a heme-dependent peroxidase superfamily that functions in the block to polyspermy

Ovoperoxidase is one of several oocyte-specific proteins that are stored within sea urchin cortical granules, released during the cortical reaction, and incorporated into the newly formed fertilization envelope. Ovoperoxidase plays a particularly important role in this process, crosslinking the envelope into a hardened matrix that is insensitive to biochemical and mechanical challenges and thus providing a permanent block to polyspermy. Here we present the primary structures of two ovoperoxidases as predicted from cDNAs cloned from the sea urchins Strongylocentrotus purpuratus (AF035380) and Lytechinus variegatus (AF035381). We also present a proposed scheme for the post-translational processing of ovoperoxidase based upon comparisons between the cDNA and protein structures and taking into account previously published reports. The sea urchin ovoperoxidase sequences conform to a profile shared by members of a heme-dependent animal peroxidase family, including the mammalian myelo-, lacto-, eosinophil, and thyroid peroxidases. Using in situ RNA hybridizations, we showed that the mRNA of S. purpuratus ovoperoxidase (4 kb) is present exclusively in oocytes, and is turned over rapidly following germinal vesicle breakdown. Taking into account our immunoblot and N-terminal sequencing data along with reports from similar peroxidases, we propose that ovoperoxidases are synthesized in a pre-pro form and proteolytically processed to result in the 70 and 50 kDa forms that are found in the fertilization envelope. The sequence and structural data presented here will facilitate our continuing studies of the biogenesis of cortical granules and the fertilization envelope. Additionally, since ovoperoxidase activities have been reported in a wide range of animals, these cDNAs will be useful in uncovering similar peroxidases used in the fertilization reactions of other metazoan eggs.     

Distal cis-acting elements restrict expression of the CyIIIb actin gene in the aboral ectoderm of the sea urchin embryo

The distal region of the S. purpuratus actin CyIIIb gene, between −400 and −1400 nucleotides, contains at least three distinct cis-acting elements (C1R, C1L and E1) which are necessary for correct expression of fusion reporter genes in transgenic sea urchin embryos. The contribution of these elements in the temporal and spatial regulation of the gene was analyzed by single and double site-directed mutagenesis in fusion constructs which carry the bacterial chloramphenicol acetyl transferase (CAT) gene as a reporter. Following microinjection of the transgenes in sea urchin embryos, the activity of the mutants was compared to the wild type in time and space by measuring CAT activity at the blastula and pluteus embryonic stages and by in situ hybridization to the CAT mRNA at pluteus stage. Our results indicate that E1 involved in the temporal regulation of CyIIIb and that all three elements are necessary and sufficient to confer aboral (dorsal) ectoderm specificity to the proximal promoter. This is achieved by suppressing the promoter's activity in all other tissues by the cooperative interaction of the cis-acting elements. The C1R element, binding site of the nuclear receptors SpCOUP-TF and SpSHR2, is by itself sufficient to restrict expression in the ectoderm, whereas the aboral ectoderm restricted expression requires in addition the presence of both C1L adn E1. It is therefore evident, that the actin CyIIIb gene is exclusively expressed in the aboral ectoderm by a combinatorial repression in all other cell lineages of the developing embryo.     

RhoGTPase signalling at epithelial tight junctions: Bridging the GAP between polarity and cancer

The establishment and maintenance of epithelial polarity must be correctly controlled for normal development and homeostasis. Tight junctions (TJ) in vertebrates define apical and basolateral membrane domains in polarized epithelia via bi-directional, complex signalling pathways between TJ themselves and the cytoskeleton they are associated with. RhoGTPases are central to these processes and evidence suggests that their regulation is coordinated by interactions between GEFs and GAPs with junctional, cytoplasmic adapter proteins. In this InFocus review we determine that the expression, localization or stability of a variety of these adaptor proteins is altered in various cancers, potentially representing an important mechanistic link between loss of polarity and cancer. We focus here, on two well characterized RhoGTPases Cdc42 and RhoA who's GEFs and GAPs are predominantly localized to TJ via cytoplasmic adaptor proteins.     

RhoH modulates pre-TCR and TCR signalling by regulating LCK

Pre-T-cell receptor (pre-TCR) and TCR signals govern the development of T-lymphocytes. RhoH, a hematopoietic-specific and GTPase-deficient member of the RhoGTPase family, is required in the development of T-lymphocytes. Here we found that RhoH binds and modulates LCK, the non-receptor tyrosine kinase crucial in initiating pre-TCR and TCR signallings. In both pre-TCR and TCR signalling transduction, LCK is phosphorylated by CSK to maintain the inactive state of LCK at rest. Upon being activated, CSK phosphorylation is removed and LCK autophosphorylation leads to LCK activation and further phosphorylates ZAP70 to initiate further downstream signalling. At rest, LCK may be recruited to the plasma membrane by RhoH, which also binds CSK, resulting in LCK inactivation. Additionally, the presence of RhoH enhances the inactivation phosphorylation of LCK by CSK. RhoH was found to bind preferentially inactive LCK, indicating that, upon ligand-mediated TCR activation, LCK is dephosphorylated resulting in LCK autoactivation and its release from RhoH. Thus RhoH is a critical part of the microenvironment for maintaining the inactive state of LCK. Furthermore, we found that the reduction of RhoH levels results in LCK autoactivation and constitutive activation of the TCR pathway. Our findings indicate that RhoH is a key adapter protein that maintains LCK in the inactive state, contributing to the regulation of both pre-TCR and TCR signalling during T-cell development. The data also supports a model for ligand-independent signal transduction by pre-TCR.