Sulfhydryl groups are involved in the activation of sea urchin eggs

Unfertilized sea urchin eggs exposed to the sulfhydryl reagents Ag⁺ or N-ethylmaleimide either elevated fertilizationlike membranes, formed surface protrusions, developed a clear cortical layer devoid of organelles, or cytolysed. The relative fraction of each modification varied from batch to batch and was also dose and time dependent. With Ag⁺ and higher doses of N-EMI (10^?3 M), the most common effect was the elevation of a membrane indicating cortical exocytosis, while at lower doses of N-EMI protrusions were predominant. Glutathione (GSH) protected eggs against these reagents also in a dose-dependent manner. Eggs exposed to equimolar amounts of N-EMI and GSH, which otherwise formed membranes, produced protrusions, while increasing GSH tenfold afforded complete protection. We suggest there are two targets for the sulfhydryl reagents–the first, SH groups on proteins that regulate the release of Ca²⁺ from the intracellular sequestering mechanism which subsequently triggers cortical exocytosis; the second, SH groups on the egg surface that may regulate cortical organization.     

How calcium may cause exocytosis in sea urchin eggs

The process of secretory granule-plasma membrane fusion can be studied in sea urchin eggs. Micromolar calcium concentrations are all that is required to bring about exocytosisin vitro. I discuss recent experiments with sea urchin eggs that concentrate on the biophysical aspects of granule-membrane fusion. The backbone of biological membranes is the lipid bilayer. Sea urchin egg membrane lipids have negatively charged head groups that give rise to an electrical potential at the bilayer-water interface. We have found that this surface potential can affect the calcium required for exocytosis. Effects on the surface potential may also explain why drugs like trifluoperazine and tetracaine inhibit exocytosis: they absorb to the bilayer and reduce the surface potential. The membrane lipids may also be crucial to the formation of the exocytotic pore through which the secretory granule contents are released. We have measured calcium-induced production of the lipid, diacylglycerol. This lipid can induce a phase transition that will promote fusion of apposed lipid bilayers. The process of exocytosis involves the secretory granule core as well as the lipids of the membrane. The osmotic properties of the granule contents lead to swelling of the granule during exocytosis. Swelling promotes the dispersal of the contents as they are extruded through the exocytotic pore. The movements of water and ions during exocytosis may also stabilize the transient fusion intermediate and consolidate the exocytotic pore as fusion occurs.     

Cross fertilization between sea urchin eggs and oyster spermatozoa

Interphylum crossing was examined between sea urchin eggs (Temnopleurus hardwicki) and oyster sperm (Crassostrea gigas). The eggs could receive the spermatozoa with or without cortical change. The fertilized eggs that elevated the fertilization envelope began their embryogenesis. Electron microscopy revealed that oyster spermatozoa underwent acrosome reaction on the sea urchin vitelline coat, and their acrosomal membrane fused with the egg plasma membrane after the appearance of an intricate membranous structure in the boundary between the acrosomal process and the egg cytoplasm. Oyster spermatozoa penetrated sometimes into sea urchin eggs without stimulating cortical granule discharge and consequently without fertilization envelope formation. The organelles derived from oyster spermatozoa seemed to be functionally inactive in the eggs whose cortex remained unchanged.     

Heat labile activating substance for ornithine decarboxylase activity in sea urchin eggs

In sea urchin eggs, the activity of ornithine decarboxylase (ODC) [ E C 4.1.1.17] is detectable only in the particulate fraction yielded by centrifuging egg homogenates at 10,000g for 30 minutes. ODC activity in the particulate fraction isolated from fertilized eggs is higher than that from unferti-lized eggs. ODC activity in the particulate fraction isolated from either unfertilized or fertilized eggs is enhanced by adding the supernatant fraction obtained by centrifugation at 105,000g for two hours. Heating this supernatant at 70°C for 15 minutes results In complete loss of the stimulating capacity for ODC activity. Sea urchin eggs seem to contain heat labile activating substance(s) for ODC activity. The substance does not pass through the ultrafiltration membrane Diafro UM–10. Only eggs and unhatched embryos, in which mitosis occurs frequently, contain the activating substance. In the presence of the activating substance, Ca²⁺enhanced ODC activity.     

Protein synthetic activity in swimming sea urchin spermatozoa

Summary

Free swimming Paracentrotus lividus spermatozoa show a significant rate of protein synthesis which remains nearly linear over a period of 90 min. This protein synthetic activity is scarcely affected by emetine but completely suppressed by chloramphenicol, suggesting its possible mitochondrial origin.     

Nuclear histones of unfertilized sea urchin eggs

Histones have been isolated from the nuclei of unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. The electrophoresis of these histones exhibits a pattern different from that of the sperm or embryo of the same species.     

Fertilization induced release of glycogen from bound state in sea urchin eggs

Glycogen in sea urchin eggs is found in both the precipitate and the supernatant fractions obtained by adding perchloric acid to the egg homogenate. Glycogen in the acid-insoluble fraction is apparently protein-bound (bound glycogen) while the acid-extractable form (free glycogen) seems to bind with less protein. The greatest amount of bound glycogen is found in the particulate fraction obtained by centrifugation of the egg homogenate at 10,000g for 30 minutes. The supernatant fraction obtained by centrifugation at 105,000g for two hours contained the largest amount of free glycogen of all the fractions obtained. The bound glycogen decreases and the free glycogen increases markedly following fertilization, while the total level of glycogen does not change. The glycogen release from the bound state occurs in vitro and the rate of release is higher in fertilized eggs than in unfertilized eggs. Polyamines (putrecine, spermidine, and spermine) cause an increase in the rate of glycogen release in the egg homogenate. cAMP, AMP, and ADP exert no effect on glycogen release in vitro, whereas ATP slightly enhances the rate of glycogen release. Na⁺ and K⁺ hardly accelerate the rate of glycogen release, and divalent cations, such as Ca²⁺ and Mg²⁺, cause an increase in the rate of glycogen release.     

Replication origins are already licensed in G1 arrested unfertilized sea urchin eggs

Fertilization relieves the oocyte from a cell cycle arrest, inducing progression towards mitotic cycles. While the signalling pathways involved in oocyte to embryo transition have been widely investigated, how they specifically trigger DNA replication is still unclear. We used sea urchin eggs whose oocytes are arrested in G1 to investigate in vivo the molecular mechanisms regulating initiation of replication after fertilization. Unexpectedly, we found that CDC6, Cdt1 and MCM3, components of the pre-replication complexes (pre-RC) which license origins for replication, were already loaded on female chromatin before fertilization. This is the first demonstration of a cell cycle arrest in metazoan in which chromatin is already licensed for replication. In contrast pre-RC assemble on chromatin post-fertilization as in other organisms. These differences in the timing of pre-RC assembly are accompanied by differences in Cdk2 requirement for DNA replication initiation between female and male chromatin post-fertilization. Finally, we demonstrated that a concomitant inhibition of MAP kinase and ATM/ATR pathways releases the block to DNA synthesis. Our findings provide new insight into the mechanisms contributing to the release of G1 arrest and the control of S-phase entry at fertilization.     

Detection of phospholipase Cγ in sea urchin eggs

Phosphorylation on tyrosine and turnover of polyphosphoinositide metabolism are rapidly stimulated after fertilization. However, the interconnection between these pathways remains to be determined. In the present paper it is demonstrated that eggs of two different sea urchin species contain tyrosine phosphorylated proteins with calcium-sensitive phospholipase C activity. We have investigated whether phospholipase Cγ (PLCγ), characteristic of tyrosine kinase receptors, could be responsible for this activity. Western blot and immunocytochemistry performed with antibodies directed against PLCγ revealed the presence of this protein in cortical regions. It was also observed that PLCγ displayed calcium-sensitive activity. The present results suggest that PLCγ may be part of the cascade of events leading to the calcium signal responsible for egg activation at fertilization.     

Cytological effects of urethan on cleavage induction in parthenogenetically activated sea urchin eggs

The effect of urethan on artificial induction of cleavage in eggs of the sea urchin, Hemicentrotus pulcherrimus, was studied. When the eggs were exposed for 20 minutes to seawater containing urethan (final concentration, 0.08 M) after butyric acid-activation and then treated with hypertonic seawater, the cleavage rate was enchanced by about 1.5 times as compared with the nontreated eggs. In the eggs exposed to urethan–seawater for over 70 minutes many clear spots appeared throughout the cytoplasm. Simultaneously, the pigment granules, which had been embedded within the cortex, migrated to the inner cytoplasm and encircled a monastral centrosphere and clear spots. The clear spots were composed of microtubules much like cytasters, and in the central region of them centrioles were not yet found. The eggs in which the pigment granules disappeared from the cortex may be more susceptible to cleavage induction by succeeding hypertonic treatment.     

Activation of maternal centrosomes in unfertilized sea urchin eggs.

Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 microM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material.     

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.     

Zymogen activation and characterization of a major gelatin-cleavage activity localized to the sea urchin extraembryonic matrix

The hyaline layer (HL) is an apically located extracellular matrix (ECM) which surrounds the sea urchin embryo from the time of fertilization until metamorphosis occurs. While gelatin-cleavage activities were absent from freshly prepared hyaline layers, a dynamic pattern of activities developed in layers incubated at 15 or 37 degrees C in Millipore-filtered sea water (MFSW). Cleavage activities at 90, 55, 41, and 32 kDa were evident following incubation at either temperature. The activation pathway leading to the appearance of these species was examined to determine the minimum salt conditions required for processing and to establish precursor-product relationships. In both qualitative and quantitative assays, the purified 55 kDa gelatinase activity was inhibited by 1,10-phenanthroline (a zinc-specific chelator) and ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA). Calcium reconstituted the activity of the EGTA-inhibited enzyme with an apparent dissociation constant (calcium) of 1.2 mM. Developmental substrate gel analysis was performed using various stage embryos. The 55 and 32 kDa species comigrated with gelatin-cleavage activities present in sea urchin embryos. Collectively, the results reported here document a zymogen activation pathway which generates a 55 kDa, gelatin-cleaving activity within the extraembryonic HL. This species displayed characteristics of the matrix metalloproteinase class of ECM modifying enzymes.     

Multiple molecular forms of acid nitrophenyl phosphatase in sea urchin eggs and embryos

Acid nitrophenyl phosphatases from sea urchin eggs and embryos were investigated by gel filtration. Four different forms were found in Hemicentrotus pulcherrimus, and three forms in Anthocidaris crassispina and Pseudocentrotus depressus. The first and second forms (designated AcP-1 and AcP-2) had the highest activity in the range of pH 5.6–6.0. The third (designated AcP-3) had an apparent optimum pH between pH 4.3 and 4.8, and the fourth (designated AcP-4) showed the maximum activity at pH 3.0. AcP-1 was much more thermolabile than AcP-2 and AcP-3 at 56°C. NaF inhibited AcP-2, AcP-3, and AcP-4 but not AcP-1. AcP-1, AcP-2, and AcP-3 were observed in the three species, whereas AcP-4 was not detected in A. crassispina and P. depressus. AcP-1, AcP-2, and AcP-3 were separted by gel filtration. AcP-1 and AcP-2 of A. crassispina and H. pulcherrimus were studied in developing embryos. The behavior of these forms in gel filtration changed during development, from unfertilized eggs to the pluteus stage.     

Acetylcholinesterase in sea urchin spermatozoa

Flagella contain the bulk of spermatozoan acetylcholinesterase. Brief sonication of sea urchin sperm suspended in Tris-buffered (pH 8.0), Ca, Mg-free artificial sea water (F-ASW) containing 10 mM ethylene diaminetetracetic acid, (EDTA) doubled the specific activity over that of the intact spermatozoa. Lipids were removed from the solubilized supernatant of the tail membrane fraction by ether extraction. Hydrolysis of acetylthiocholine in the presence of dithiobisnitrobenzoic acid (DTNB) was monitored spectrophotometrically at 412 nm by the Ellman procedure. The enzyme was purified by affinity chromatography on a Sepharose cyanogen bromide gel to which the cholinesterase inhibitor trimethyl (para-aminophenyl) ammonium chloride was coupled. The enzyme was eluted from the column with a discontinous NaCl gradient (0.1–0.5 M). The active fraction recovered at 0.35 M NaCl contained 0.007% of the initial total sperm cell protein with a 500-fold increase in specific activity. Twenty-four hr centrifugation on a 5–20% sucrose density gradient at 50,000g in a Beckman L5-75 centrifuge yielded peaks at 14.7 S and 9.1 S. In the presence of 1% Triton X-100, three peaks appeared: 23.3 S, 13.7 S, and 9.1 S. These sedimentation coefficients resemble those of the electroplax acetylcholinesterase (AChE) forms A₈ and A₄. Eserine completely inhibited the activity of the purified enzyme, which exhibits a substrate optimum at 4 mM acetylcholine. The activity is depressed by 75% at 10 mM ACh and by 90% at 25 mM. The Km was 2.1 × 10^?4 M. In the sperm cell the enzyme that terminates the action of intracellularly synthesized ACh may be involved in controlling ionophoric channels that regulate transmembrane transport of calcium.     

Characterization of a metalloproteinase: A late stage specific gelatinase activity in the sea urchin embryo

We have partially purified and characterized an 87 kDa gelatinase activity expressed in later stage sea urchin embryos. Cleavage activity was specific for gelatin and no cleavage of sea urchin peristome type I collagen, bovine serum albumin or casein was detected. Magnesium and Zn²⁺ inhibited the gelatinase and Ca²⁺ protected against inhibition. Ethylenediamine tetracetic acid, ethylenebisoxyethylenenitriol tetraacetic acid and 1,10-phenanthroline were inhibitory, suggesting that the gelatinase is a Ca²⁺- and Zn²⁺-dependent metalloproteinase. No inhibition was detected with serine or cysteine protease inhibitors and the vertebrate matrix metalloproteinase (MMP) inhibitor, Batimastat, was also ineffective. The vertebrate MMP activator p-aminophenylmercuric acetate was without effect. These results allow us to identify both similarities and differences between echinoderm and vertebrate gelatinases. J. Cell. Biochem. 66: 337–345, 1997. © 1997 Wiley-Liss, Inc.     

Effect of maitotoxin on sea urchin egg fertilization and on Ca2+ permeabilities of eggs and intracellular stores.

Maitotoxin (MTX), a potent marine toxin involved in ciguatera poisoning, inhibited sea urchin egg fertilization in a dose-dependent manner with an IC50 of 7.5 x 10(-3) MU (mouse-unit)/ml. It did not affect male gametes fertilizing capabilities but provoked exocytosis in female gametes. It induced a K+ loss simultaneously with a Na+ entry into unfertilized eggs and increased the Ca2+ influx at higher concentrations. On isolated cortex preparations, high concentrations of MTX reduced the rate of ATP-dependent Ca2+ accumulation into reticulum compartments and caused a leakage of Ca2+ from a preparation pre-loaded with 45Ca2+. Verapamil (10(-4) M) similarly blocked the increase of egg permeability to Ca2+ and the effect on Ca2+ sequestering into intracellular compartment, induced by MTX. Ion transport perturbations which evolved relatively slowly are probably not the direct cause of fertilization inhibition which could be related to a modification of the plasma membrane of the female gametes by this hydrophilic toxin.     

Ionic and permeability requirements for exocytosis in vitro in sea urchin eggs

Summary We study exocytosis in the planar isolated cortex of the egg of the sea urchinLytechinus pictus. Solutins bathing the exocytotic apparatus need not contain appreciable amounts of ions: fusion follows addition of submicromolar calcium to solutions containing only nonelectrolyte. We examine the effects of altering the granule membrane permeability to small molecules with ionophores and digitonin. Introducing holes in the secretory granule membrane to the extent of allowing free passage of small molecules does not cause seretion in vitro. We add the amphipathic compound digitonin at 12 to 15 M concentrations and demonstrate that the granule membrane can become permeable to lucifer yellow, yet that granules remain intact. Granules still undergo exocytosis after digitonin treatment at such concentrations upon subsequent addition of calcium. Higher concentrations of digitonin lead to granule content swelling and vesicle bursting. We conclude that cortical granule hydration during exocytosis is not mediated by small ionic channels.