Quantitation of a src-like tyrosine protein kinase during fertilization of the sea urchin egg

Fertilization of the sea urchin egg is known to involve an increase in overall protein tyrosine kinase activity which precede the first cell division. In order to determine the types of tyrosine kinases that are involved in fertilization, we have used immunological and other criteria to identify a c-src related protein kinase in eggs of the sea urchin L. variegatus. Using an immune complex assay, we have measured the level of this c-src related protein kinase during fertilization and early embryonic development. Fertilization results in a decrease in the c-src kinase detectable by this technique suggesting that c-src does not contribute to the fertilization induced increase in protein tyrosine kinase activity.     

Sea urchin fertilization stimulates CaM kinase-II (multifunctional [type II] Ca2+/CaM kinase) activity and association with p34cdc2

Upon fertilization, the sea urchin egg synthesizes proteins which impart a Ca²⁺ dependence to M-phase onset. A potential target of this Ca²⁺ dependence may be CaM kinase-II (the multifunctional [type II] Ca²⁺/calmodulin [CaM]-dependent protein kinase) which is necessary for nuclear envelope breakdown in fertilized sea urchin eggs. This study was intended to determine whether sea urchin CaMK-II is activated after fertilization and whether it interacts with other known M-phase regulators, such as p34^cdc2. We report that total CaMK-II activity, measured by solution assays, increases after fertilization, peaking just prior to cleavage. Interestingly, total CaMK-II activity continues to fluctuate, peaking again prior to second and third cleavage. Gel assays also reveal enhanced levels of the 56 and 62 kDa potential CaMK-II phosphoproteins after fertilization. Finally, CaMK-II activity and only the 62 kDa phosphoprotein physically associate with p34^cdc2, but again only after fertilization. These changes in CaMK-II activity and p34^cdc2-association after fertilization may ensure that Ca²⁺ signals are targeted to the M-phase machinery at the appropriate developmental times.     

Role of phospholipase Cgamma at fertilization and during mitosis in sea urchin eggs and embryos

It is well known that stimulation of egg metabolism after fertilization is due to a rise in intracellular free calcium concentration. In sea urchin eggs, this first calcium signal is followed by other calcium transients that allow progression through mitotic control points of the cell cycle of the early embryo. How sperm induces these calcium transients is still far from being understood. In sea urchin eggs, both InsP3 and ryanodine receptors contribute to generate the fertilization calcium transient, while the InsP3 receptor generates the subsequent mitotic calcium transients. The identity of the mechanisms that generate InsP3 after fertilization remains an enigma. In order to determine whether PLCgamma might be the origin of the peaks of InsP3 production that punctuate the first mitotic cell cycles of the fertilized sea urchin egg, we have amplified by RT-PCR several fragments of sea urchin PLCgamma containing the two SH2 domains. The sequence shares similarities with SH2 domains of PLCgamma from mammals. One fragment was subcloned into a bacterial expression plasmid and a GST-fusion protein was produced and purified. Antibodies raised to the GST fusion protein demonstrate the presence of PLCgamma protein in eggs. Microinjection of the fragment into embryos interferes with mitosis. A related construct made from bovine PLCgamma also delayed or prevented entry into mitosis and blocked or prolonged metaphase. The bovine construct also blocked the calcium transient at fertilization, in contrast to a tandem SH2 control construct which did not inhibit either fertilization or mitosis. Our data indicate that PLCgamma plays a key role during fertilization and early development.     

Studies of the mechanism of the electrical polyspermy block using voltage clamp during cross-species fertilization

Prevention of polyspermic fertilization in sea urchins (Jaffe, 1976, Nature (Lond.). 261:68-71) and the worm Urechis (Gould-Somero, Jaffe, and Holland, 1979, J. Cell Biol. 82:426-440) involves an electrically mediated fast block. The fertilizing sperm causes a positive shift in the egg's membrane potential; this fertilization potential prevents additional sperm entries. Since in Urechis the egg membrane potential required to prevent fertilization is more positive than in the sea urchin, we tested whether in a cross-species fertilization the blocking voltage is determined by the species of the egg or by the species of the sperm. With some sea urchin (Strongylocentrotus purpuratus) females, greater than or equal to 90% of the eggs were fertilized by Urechis sperm; a fertilization potential occurred, the fertilization envelope elevated, and sometimes decondensing Urechis sperm nuclei were found in the egg cytoplasm. After insemination of sea urchin eggs with Urechis sperm during voltage clamp at +50 mV, fertilization (fertilization envelope elevation) occurred in only nine of twenty trials, whereas, at +20 mV, fertilization occurred in ten of ten trials. With the same concentration of sea urchin sperm, fertilization of sea urchin eggs occurred, in only two of ten trials at +20 mV. These results indicate that the blocking voltage for fertilization in these crosses is determined by the sperm species, consistent with the hypothesis that the fertilization potential may block the translocation within the egg membrane of a positively charged component of the sperm.     

Motility and centrosomal organization during sea urchin and mouse fertilization

Motility and the behavior and inheritance of centrosomes are investigated during mouse and sea urchin fertilization. Sperm incorporation in sea urchins requires microfilament activity in both sperm and eggs as tested with Latrunculin A, a novel inhibitor of microfilament assembly. In contrast the mouse spermhead is incorporated in the presence of microfilament inhibitors indicating an absence of microfilament activity at this stage. Pronuclear apposition is arrested by microfilament inhibitors in fertilized mouse oocytes. The migrations of the sperm and egg nuclei during sea urchin fertilization are dependent on microtubules organized into a radial monastral array, the sperm aster. Microtubule activity is also required during pronuclear apposition in the mouse egg, but they are organized by numerous egg cytoplasmic sites. By the use of an autoimmune antibody to centrosomal material, centrosomes are detected in sea urchin sperm but not in unfertilized eggs. The sea urchin centrosome expands and duplicates during first interphase and condenses to form the mitotic poles during division. Remarkably mouse sperm do not appear to have the centrosomal antigen and instead centrosomes are found in the unfertilized oocyte. These results indicate that both microfilaments and microtubules are required for the successful completion of fertilization in both sea urchins and mice, but at different stages. Furthermore they demonstrate that centrosomes are contributed by the sperm during sea urchin fertilization, but they might be maternally inherited in mammals.     

The oxidative burst at fertilization is dependent upon activation of the dual oxidase Udx1

The sea urchin egg is a quiescent cell...until fertilization, when the egg is activated. The classic respiratory burst at fertilization is the result of prodigious hydrogen peroxide production, but the mechanism for this synthesis is not known. Here we quantitate the kinetics of hydrogen peroxide synthesis at a single-cell level using an imaging photon detector, showing that 60 nM hydrogen peroxide accumulates within the perivitelline space of each zygote. We find that the NADPH oxidation activity is enriched at the cell surface and is sensitive to a pharmacological inhibitor of NADPH oxidase enzymes. Finally, we show that a sea urchin dual oxidase homolog, Udx1, is responsible for generating the hydrogen peroxide necessary for the physical block to polyspermy. Phylogenetic analysis of the enzymatic modules in Udx1 suggests a potentially conserved role for the dual oxidase family in hydrogen peroxide production and regulation during fertilization.     

Cloning and characterization of a phospholipase C-beta isoform from the sea urchin Lytechinus pictus

Calcium is a ubiquitous intracellular signaling molecule controlling a wide array of cellular processes including fertilization and egg activation. The mechanism for triggering intracellular Ca(2+) release in sea urchin eggs during fertilization is the generation of inositol-1,4,5-trisphosphate by phospholipase C (PLC) hydrolysis of phosphatidylinositol-4,5-bisphosphate. Of the five PLC isoforms identified in mammals (beta, gamma, delta, epsilon and zeta), only PLCgamma and PLCdelta have been detected in echinoderms. Here, we provide direct evidence of the presence of a PLCbeta isoform, named suPLCbeta, within sea urchin eggs. The coding sequence was cloned from eggs of Lytechinus pictus and determined to have the greatest degree of homology and identity with the mammalian PLCbeta4. The presence of suPLCbeta within the egg was verified using a specifically generated antibody. The majority of the enzyme is localized in the non-soluble fraction, presumably the plasma membrane of the unfertilized egg. This distribution remains unchanged 1 min postfertilization. Unlike PLCbeta4, suPLCbeta is activated by G protein betagamma subunits, and this activity is Ca(2+)-dependent. In contrast to all known PLCbeta enzymes, suPLCbeta is not activated by Galphaq-GTPgammaS subunit suggesting other protein regulators may be present in sea urchin eggs.     

The appearance of beta-1,3-glucanase in hatching embryos of the sea urchin, Echinometra vanbrunti.

Two characteristics of fertilizing sea urchin eggs are the elevation of a fertilization membrane and the excretion of a β-glucanase. Of 13 species tested, one species, Echinometra vanbrunti, lacks both characteristics. No β-glucanase exists in the eggs and cleavage stages. However, β-glucanase appears at hatching and is secreted to the sea water during and after the hatching period. The enzyme may function in the hatching process. The hypothesis is presented that the β-glucanase excreted by eggs of other sea urchin species may function in the elevation of the fertilization membrane.     

Inhibition of sea urchin fertilization by jaspisin, a specific inhibitor of matrix metalloendoproteinase

Jaspisin, originally isolated from a marine sponge as an inhibitor of the hatching of the sea urchin (Hemicentrotus pulcherrimus) embryo, causes inhibition of sea urchin fertilization. Electron microscopic examination revealed that the acrosome reaction was induced in jaspisin-treated sperm when they were incubated with an intact egg. The acrosome-reacted sperm bound to the vitelline layer by the acrosomal material surrounding the acrosomal process. However, fusion of the acrosomal process and the egg plasma membrane failed to take place. Membrane potential changes were monitored using eggs preloaded with a membrane potential-sensitive fluorochrome, di-8-ANEPPS. Depolarization of the membrane potential, normally observed in the fertilized egg was not observed in the egg inseminated in the presence of jaspisin, indicating the absence of electrical continuity between the jaspisin-treated egg and sperm. Jaspisin inhibited the activities of matrix metallo-endoproteinase members but not of other types of proteinases. These results provide strong, albeit indirect, evidence that a matrix metallo-endoproteinase(s) is involved in the process of gamete fusion during sea urchin fertilization.     

Synaptotagmin I is involved in the regulation of cortical granule exocytosis in the sea urchin

Cortical granules are stimulus-dependent secretory vesicles found in the egg cortex of most vertebrates and many invertebrates. Upon fertilization, an increase in intracellular calcium levels triggers cortical granules to exocytose enzymes and structural proteins that permanently modify the extracellular surface of the egg to prevent polyspermy. Synaptotagmin is postulated to be a calcium sensor important for stimulus-dependent secretion and to test this hypothesis for cortical granule exocytosis, we identified the ortholog in two sea urchin species that is present selectively on cortical granules. Characterization by RT-PCR, in-situ RNA hybridization, Western blot and immunolocalization shows that synaptotagmin I is expressed in a manner consistent with it having a role during cortical granule secretion. We specifically tested synaptotagmin function during cortical granule exocytosis using a microinjected antibody raised against the entire cytoplasmic domain of sea urchin synaptotagmin I. The results show that synaptotagmin I is essential for normal cortical granule dynamics at fertilization in the sea urchin egg. Identification of this same protein in other developmental stages also shown here will be important for interpreting stimulus-dependent secretory events for signaling throughout embryogenesis.     

Propranolol induces polyspermy during sea urchin fertilization

Propranolol, a β-adrenergic receptor blocker, is found to induce polyspermy in sea urchin eggs. Unfertilized sea urchin eggs treated for 10 min with 50 μM of propranolol, and then inseminated, become polyspermic and show a fertilization envelope which is barely visible to the light microscope. Examination of treated eggs by transmission and scanning electron microscopy shows that the drug does not alter the cortex of the unfertilized egg. However, after insemination an incomplete cortical reaction occurs. This might well account for both polyspermy and the defective elevation of the fertilization envelope. Since the effects of the drug are reversed by simultaneous treatment with adrenalin, perhaps propranolol interferes with the monoaminergic system that has been proposed to be active. The involvement of the monoaminergic system in the fertilization process is present in the sea urchin egg. © 1996 Wiley-Liss, Inc.     

Signal transduction pathways that contribute to CDK1/cyclin B activation during the first mitotic division in sea urchin embryos

In sea urchins, fertilization triggers a rapid rise in protein synthesis necessary for activation of CDK1/cyclin B, the universal cell cycle regulator. It has been shown that FRAP/mTOR is required for eIF4E release from the translational repressor 4E-BP, a process that occurs upstream of de novo cyclin B synthesis. Here, we investigate whether PI 3-kinase acts independently or upstream from FRAP/mTOR in the signal transduction pathway that links fertilization to the activation of the CDK1/cyclin B complex in sea urchin egg. We found that wortmannin, a potent inhibitor of PI 3-kinase, partially inhibited the global increase in protein synthesis triggered by fertilization. Furthermore, wortmannin treatment induced partial inhibition of cyclin B translation triggered by fertilization, in correlation with an intermediate effect of the drug on 4E-BP degradation and on the dissociation of the 4E-BP/eIF4E complex induced by fertilization. Our results presented here suggest that PI 3-kinase activity is required for completion of mitotic divisions of the sea urchin embryo. Incubation of eggs with wortmannin or microinjection of wortmannin or LY 294002 affects drastically mitotic divisions induced by fertilization. In addition, we found that wortmannin treatment inhibits dephosphorylation of the tyrosine inhibitory site of CDK1. Taken together, these data suggest that PI 3-kinase acts upstream of at least two independent targets that function in the CDK1/cyclin B activation triggered by fertilization of sea urchin oocytes. We discuss the significance of these results concerning the cascade of reactions that impinge upon the activation of the CDK1/cyclin B complex that follows sea urchin oocyte fertilization.     

Chromosome changes during the in vitro ageing of MRC-5 human fibroblasts.

We have measured the release of fertilization acid from sea urchin eggs in specific cation-free media by activating the eggs with the calcium ionophore A23187. The fertilization acid is normal in calcium-free sea water and several other substituted media, indicating that specific cations present in sea water are not required for release of the acid. The fertilization acid may result from release of cortical granule contents, but we suggest the possibility that other cellular processes are involved.     

Function of a sea urchin egg Src family kinase in initiating Ca2+ release at fertilization

Egg activation at fertilization requires the release of Ca(2+) from the egg's endoplasmic reticulum, and recent evidence has indicated that a Src family kinase (SFK) may function in initiating this signaling pathway in echinoderm eggs. Here, we identify and characterize a SFK from the sea urchin Strongylocentrotus purpuratus, SpSFK1. SpSFK1 RNA is present in eggs, and an antibody made against a SpSFK1 peptide recognizes an approximately 58-kDa egg membrane-associated protein in eggs of S. purpuratus as well as another sea urchin Lytechinus variegatus. Injection of both species of sea urchin eggs with dominant-interfering Src homology 2 domains of SpSFK1 delays and reduces the release of Ca(2+) at fertilization. Injection of an antibody against SpSFK1 into S. purpuratus eggs also causes a small increase in the delay between sperm-egg fusion and Ca(2+) release. In contrast, when injected into eggs of L. variegatus, this same antibody has a dramatic stimulatory effect: it causes PLCgamma-dependent Ca(2+) release like that occurring at fertilization. Correspondingly, in lysates of L. variegatus eggs, but not S. purpuratus eggs, the antibody stimulates SFK activity. Injection of L. variegatus eggs with another antibody that recognizes the L. variegatus egg SFK also causes PLCgamma-dependent Ca(2+) release like that at fertilization. These results indicate that activation of a Src family kinase present in sea urchin eggs is necessary to cause Ca(2+) release at fertilization and is capable of stimulating Ca(2+) release in the unfertilized egg via PLCgamma, as at fertilization.     

THE MEMBRANE CAPACITANCE OF THE SEA URCHIN EGG

1. The surface of the unfertilized sea urchin egg is folded and the folds are reversibly eliminated by exposing the egg to hypotonic sea water. If the plasma membrane is outside the layer of cortical granules, unfolding may explain why the membrane capacitance per unit area decreases (and does not increase) when a sea urchin egg is put into hypotonic sea water. 2. The degree of surface folding markedly increases after fertilization, which provides an explanation for the increase in membrane capacitance per unit area observed after fertilization. 3. The percentage reduction in membrane folding in fertilized eggs after immersion in hypotonic sea water is probably sufficient to explain the decrease in membrane capacitance per unit area observed in these conditions.     

Fertilization in echinoderms

For more than 150years, echinoderm eggs have served as overly favored experimental model systems in which to study fertilization. Sea urchin and starfish belong to the same phylum and thus share many similarities in their fertilization patterns. However, several subtle but fundamental differences do exist in the fertilization of sea urchin and starfish, reflecting their phylogenetic bifurcation approximately 500 million years ago. In this article we review some of the seminal and recent findings that feature similarities and differences in sea urchin and starfish at fertilization.     

Cyclin E/Cdk2 is required for sperm maturation, but not DNA replication, in early sea urchin embryos

The cell cycle is driven by the activity of cyclin/cdk complexes. In somatic cells, cyclin E/cdk2 oscillates throughout the cell cycle and has been shown to promote S-phase entry and initiation of DNA replication. In contrast, cyclin E/cdk2 activity remains constant throughout the early embryonic development of the sea urchin and localizes to the sperm nucleus following fertilization. We now show that cyclin E localization to the sperm nucleus following fertilization is not unique to the sea urchin, but also occurs in the surf clam, and inhibition of cyclin E/cdk2 activity by roscovitine inhibits the morphological changes indicative of male pronuclear maturation in sea urchin zygotes. Finally, we show that inhibition of cyclin E/cdk2 activity does not block DNA replication in the early cleavage cycles of the sea urchin. We conclude that cyclin E/cdk2 activity is required for male pronuclear maturation, but not for initiation of DNA replication in early sea urchin development.     

Inhibition of sea urchin fertilization by fatty acid ethanolamides and cannabinoids

The influence of saturated and unsaturated fatty acid ethanolamides as well as Δ⁹-tetrahydrocannabinol (Δ⁹-THC), WIN 55,212-2 and cannabinoid CB₁ receptor antagonist SR 141716 on sea urchin fertilization was studied. The ethanolamides of arachidonic, oleic and linoleic acids but not saturated fatty acid (C₁₄–C₂₀) derivatives inhibited fertilization when pre-incubated with sperm cells. Δ⁹-THC and WIN 55,212-2 also inhibited fertilization, Δ⁹-THC being ten times as potent as WIN 55,212-2. Selective cannabinoid CB₁ receptor antagonist SR 141716 also blocked fertilization and did not antagonize the action of Δ⁹-THC. The obtained results indicate that different unsaturated fatty acid ethanolamides may control sea urchin fertilization, and that sea urchin sperm cell cannabinoid receptor may differ from the known cannabinoid receptor subtypes.     

Evidence that hatching enzyme of the sea urchin Strongylocentrotus purpuratus is a chymotrypsin-like protease

The sea urchin blastula secretes a hatching enzyme (HE) that dissolves the fertilization envelope. HE was collected from the supernatant seawater of cultures of hatched Strongylocentrotus purpuratus blastulae, and concentrated 20 times by ultrafiltration. The proteolytic activity of HE using casein as substrate was inhibited by the chymotrypsin inhibitors, chymostatin and N-tosyl-L-phenylalanine chloromethyl ketone. The activity was not inhibited by inhibitors (antipain, elastatinal, pepstatin, phosphoramidon, soybean trypsin inhibitor, and N alpha-p-tosyl-L-lysine chloromethyl ketone) of other types of proteases. HE did not hydrolyze the synthetic trypsin substrate, alpha-N-benzoyl-L-arginine ethyl ester, but did hydrolyze the synthetic substrate of chymotrypsin, N-benzoyl-L-tyrosine ethyl ester (BTEE). The BTEEase activity of HE was completely inhibited by the chymotrypsin inhibitors chymostatin and 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC). Chymostatin inhibited the natural hatching of sea urchin blastulae. Application of HE to freshly fertilized sea urchin eggs, 2 h after insemination, caused premature dispersal of the hardened fertilization envelope. Chymostatin and NCDC inhibited HE-induced lysis of the fertilization envelope, while inhibitors of other types of proteases were ineffective. These data suggest that sea urchin HE is a chymotrypsin-like protease we call "chymotrypsin."     

False fertilization in sea urchin eggs induced by diabolin, a 120K kelp protein

A 120K lectin-like protein was isolated from the kelp Laminaria diabolica (Oni-kombu), with a unique activity to induce false fertilization specifically in the eggs of the sea urchin Hemicentrotus pulcherrimus. The protein designated as "diabolin" rendered the unfertilized egg forms and elevated the fertilization envelope without insemination at 18 nM half-maximally. Those eggs with elevated fertilization envelopes, however, could not enter into normal cleavage or further development, and hence the proliferation of the sea urchin was hindered. Diabolin, thus, by its unique defense mechanism protects the kelp from the predator sea urchin. It was partially sequenced and found to have the highest homology with phytoene dehydrogenase from the plant virus Erwinia uredovora. A question was left to be solved as to how the kelp on the southeast coast of Hokkaido Island could develop the defense mechanism against the sea urchin on Honshu Island separated by Tsugaru Straits.