Distributional Change of Membrane-associated Calcium in Sea Urchin Eggs during Fertilization

The source and sink for the intracellular calcium released during fertilization were examined in sea urchin eggs, Hemicentrotus pulcherrimus , with chlortetracycline as a fluorescent chelate probe. In order to distinguish the differential distribution of membrane-associated calcium in various compartments in cytoplasm, eggs were stratified by centrifugation before or after fertilization. Only the layer containing mainly mitochondria exhibited the chlortetracycline-fluorescence in unfertilized eggs. After fertilization, a new fluorescent band emerged in the membrane-rich clear layer of stratified eggs. Chlortetracycline-fluorescence in the clear layer was gradually redistributed surrounding the prophase nucleus and then incorporated into the mitotic apparatus. From these observations, we postulate that the major source(s) of released free calcium ions at fertilization is in the mitochondira layer and membranes in the clear layer are newly activated as the calcium sequestering system after fertilization.     

Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos

The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism.     

Polarization of the Surface Membrane and Cortical Layer of Sea Urchin Blastomeres, and its Inhibition by Cytochalasin B

Sea-urchin blastomeres have two domains of the plasma membrane which can be distinguished immunocytochemically. An egg-surface antibody (anti-ES), which binds to the membrane of the entire surface region of eggs before cleavage, binds to the membrane of the outer surface region of blastomeres after cleavage, but not to that of the cleavage furrow region or interblastomeric surface region.
The anti-ES binding sites on the egg membrane were chased after cleavage by labeling the egg plasma membrane with FITC conjugated monovalent anti-ES (FITC-Fab anti-ES) before the first cleavage, and then allowing the eggs to cleave. The surface fluorescence increased in intensity in the cleavage furrow region with progress of furrowing, but after completion of the furrowing, the fluorescence became uniform and finally decreased in the interblastomeric surface region.
The distributions of pigment granules and NBD-phallacidin stainable microfilaments in the cortex after completion of furrowing were polarized in the same way as the anti-ES binding area. As cytochalasin B completely inhibited the polarization in both the surface and cortical layer but colchicine did not, polarization of the anti-ES binding area was concluded to be due to the post-cleavage polarized distribution of submembranous microfilaments in the cortical layer.     

Electric Field-induced Fusion of Sea Urchin Eggs

An electrical method is described which permits the fusion of denuded eggs of the sea urchin species Paracentrotus lividus . In a nearly non-conductive medium, containing 1.2 M glucose at pH 6.0–8.4, eggs or fertilized stages are brought into close membrane contact by dielectrophoresis arising from the application of a highly inhomogeneous alternating electric field. During this process the eggs aligne parallel to the field forming egg-chains. In well pigmented eggs pigmentcapping is observed in the areas of cell contact. After completion of the alignment, the application of an additional single high field pulse of μs duration induces fusion of two or more eggs. The mechanism underlying the fusion process is the reversible electric breakdown of membranes in the zones of cell-to-cell contact. Fusion proceeds within 1–10 min at 10–20°C. Fused eggs have intact nuclei, can be fertilized, but undergo abortive cleavage.     

Movements of Echinochrome Granules during the Early Development of Sea Urchin Eggs

ECHINOCHROME pigment granules in unfertilized eggs of the sea urchin Arbacia punctulata undergo randomly-directed saltatory movements^1,2. After fertilization, nearly all these granules migrate to the egg cortex and become embedded. Subsequent pigment granule movements may represent mass cortical changes rather than independent granule movements^2,3. At the fourth cleavage, a quartet of micromeres containing little or no pigment forms at the vegetal pole. By the two or four-cell stage, pigment granules have begun to move out of this region, leaving a “clear area” on each blastomere (Fig. 1 and refs. 4, 5). To investigate possible mechanisms for these movements and their relation to cortical events     

Effect of Cytochalasin B on Eggs of Atlantic Salmon and Rainbow Trout

Cytochalasin B action has been investigated in polyploidisation and gynogenesis in Atlantic salmon and rainbow trout. Solutions of 10, 20, 30 and 50 mg/1 were used to treat eggs from 35 h° to the 2-, 4- and 8-cell stages for rainbow trout, from 35 h° to the 2- and 4-cell stages, and 50 h° to the 8-cell stage for salmon. Survival rate was low in all treated groups. Low frequencies of polyploid and gynogenetic individuals were observed in embryos and alevins by means of two methods: DNA analysis by flow cytometry and chromosome counting. All polyploids observed were triploids without evidence of mosaicism. A few spontaneous diploid gynogenetic rainbow trout were registrated, as well as spontaneous triploid salmon, in controls.     

Induction of Fertilization Membrane Formation and Cyanide-insensitive Respiration in Sea Urchin Eggs by the Treatment with Dimethylsulfoxide Followed by an Incubation in an Ice Bath

In unfertilized eggs of the sea urchin Hemicentrotus pulcherrimus , fertilization membrane formation was induced by an incubation with dimethylsulfoxide (DMSO) for several min at 20°c followed by another incubation in an ice bath. The number of eggs with fertilization membrane, thus obtained, increased in relation to the concentration of DMSO between 1 and 3% (v/v) and was higher than 75% at concentrations above 3%. Fertilization membrane formation by this treatment occurred in Ca²⁺ free- or Ca²⁺, Mg²⁺ free- artificial sea water containing EGTA (50 mM) and was inhibited by verapamil. In the presence of DMSO, the membrane formation was also induced by 2, 4-dinitrophenol or cyanide in considerable number of eggs at 20°c. Eggs remained fertilizable, even when they were kept with DMSO for 1 hr at 20°c. DMSO slightly enhanced respiratory rate in unfertilized eggs and substantially reduced it in fertilized eggs. DMSO-treated eggs exhibited cyanide-insensitive respiratory burst following chilling in an ice bath or by adding DNP or cyanide, in a similar manner to the burst induced by sperm.     

The Interactions of Sea Urchin Gametes During Fertilization

The interactions between sea urchin spermatozoa and ova duringfertilization usually exhibit a high degree of species specificity.Under natural conditions and reasonable gamete concentrations,most interspecific inseminations fail to yield zygotes. Macromoleculeson the external surfaces of the apposing gametes must surelybe responsible for successful gamete recognition, adhesion andfusion. Species specific recognition between surface componentsof sperm and egg could occur during at least three events comprisingthe fertilization process. The first event is the interactionof the sperm plasma membrane with the egg jelly coat. This inducesthe sperm acrosome reaction resulting in the exocytosis of the"bindin" -containing acrosome granule and also the extrusionof the acrosome process from the anterior tip of the sperm.The second event is the adhesion of the bindin-coated acrosomeprocess to glycoprotein "bindin receptors" on the external surfaceof the egg vitelline layer. The third event is the penetrationof the vitelline layer and the fusion of sperm and egg plasmamembranes. With the isolations of the component of egg jellywhich induces the acrosome reaction, sperm bindin from the acrosomevesicle and the egg surface bindin receptor from the vitellinelayer, there is hope of discovering the molecular basis of thismost interesting intercellular interaction which results inthe activation of embryonic development.     

Inhibitory Effect of Calmodulin Antagonists and Calcium Antagonists on Fertilization-Induced Cyanide-Insensitive Respiration in Sea Urchin Eggs

During initial several minutes after fertilization, sea urchin eggs exhibited high rate of respiration which was only slightly inhibited by cyanide. This cyanide-insensitive respiration was inhibited by calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and chlorpromazine, which were added within 1 min after insemination. The inhibitory effect of W-7 on cyanide-insensitive respiration was higher than that of W-5. Cyanide-sensitive respiration of fertilized eggs observed after this initial period was not inhibited by these compounds. Ca²⁺ influx in eggs just after fertilization was inhibited by calcium antagonists but was rather enhanced by calmodulin antagonists. Fertilization-induced stimulation of cyanide-insensitive respiration probably results from calmodulin-dependent reactions which are activated by Ca²⁺ influx.     

Extreme Toxicity of Ethanol During Activation of Sea Urchin Eggs

We have examined the effects of ethanol on early fertilization events and later development in the sea urchin Strongylocentrotus purpuratus . Eggs can still be fertilized in ethanol concentrations as high as 480 mM (2.0%); egg cytolysis was rapidly observed postinsemination in 50% of the cells at 220 mM ethanol. Yet, sperm motility was essentially normal in 250 mM ethanol; 940 mM ethanol was required to affect a 50% reduction. To determine the effect of ethanol on K⁺-efflux from eggs induced by fertilization, we used parthenogenetic activation induced by the Ca²⁺-ionophore A23187. Surprisingly, ethanol at only 0.2 mM caused an abnormal K⁺-efflux, but only when added between 1 and 3 min after induction of activation. The K⁺-efflux rates of unfertilized eggs were not influenced by up to 730 mM ethanol. Finally, normal embryonic development through the mesenchyme blastula stage was observed in egg suspensions which were treated for 30 min with ethanol concentrations as high as 240 mM, but washed with normal seawater prior to insemination. Normal plutei were obtained from cultures which were continuously cultured in 24 mM ethanol from 15 min postinsemination. We conclude that an extreme ethanol sensitivity of embryogenesis is apparent only during the cortical reaction.     

SCN— Blocks Hardening of the Fertilization Envelope of Sea Urchin Eggs and Adhesion of Blastomeres

Sea urchin eggs kept in artificial sea water (ASW) containing 0.01–0.3 M NaSCN in place of NaCI from within 2 min after insemination formed thin, enlarged fertilization envelopes, which were broken on mild agitation of egg suspensions more easily than those formed in Ca²⁺-free ASW. The blastomeres of almost all embryos derived from eggs treated with 0.2M SCN^— for 1 hr dissociated spontaneously, and did not reassociate with other blastomeres appreciably. Thus SCN^— probably denaturated some compound(s) participating in blastomere binding and hardening of the fertilization envelope. Abnormal arrangements of blastomeres, probably due to incomplete blastomere dissociation, were observed in embryos derived from eggs treated with 0.1 M SCN^— for 1 hr. Treatment of fertilized or unfertilized eggs with 0.05–0.1 M SCN^— for a short period caused concentration-dependent block of morphogenic processes such as formation of the archenteron and pluteus arms in the post-hatching period. The effects of SCN^— on morphogenesis were not inhibited by furosemide or 4,4'-diisothiocyano 2,2'-disulfonic stilbene. Presumably, the denaturation of several compounds in the egg surface by SCN^— causes abnormal morphogenesis of embryos. The inhibitory effects of SCN^— on hardening of the fertilization envelope, blastomere binding and morphogenesis were greater in the absence of Ca²⁺.     

Accumulation of WGA Receptors in the Cleavage Furrow during Cytokinesis of Sea Urchin Eggs

Sea urchin eggs stained with fluorescein-conjugated wheat germ agglutinin (F-WGA) before or after fixation showed a marked accumulation of fluorescence at the cleavage furrow in the first and the second cell divisions. WGA receptors (WGA-binding membrane glycoproteins) were redistributed to the equatorial region through several steps in compressed eggs. Accumulated WGA receptors showed a distribution similar to that of contractile-ring microfilaments throughout most of the steps. Therefore, the former is probably associated with the latter directly or indirectly. Labeling with F-WGA provides a simple method to detect contractile-ring microfilaments in living eggs. Treatment of eggs with colcemid shortly before cytokinesis dispersed the ring-like accumulation of WGA receptors together with contractile-ring microfilaments. This result suggests that microtubule structures, probably asters, are involved in the redistribution of WGA receptors. Cytochalasin B prevented furrowing when it was applied shortly before cytokinesis. While contractile-ring microfilaments showed a spotty distribution in the expected furrow region, WGA receptors were normally redistributed. Furthermore, a higher concentration of the drug allowed the appearance of accumulated WGA receptors in compressed eggs although the development into a ring-like configuration was inhibited. These observations suggest the possibility that the redistribution of WGA receptors is involved in the formation of contractile ring.     

Aster Formation in Sea Urchin Eggs Induced by the injection of Enzyme-Treated Sperm Centrioles

To determine the responsible components of isolated sperm centrioles for the aster induction in sea urchin eggs, the sperm centriolar fraction was treated with various enzymes and was injected into the unfertilized eggs, then the aster formation in first division was observed after fertilization.
Treatment with 1 μg/ml or higher concentration of trypsin inhibited the centriolar activity for aster induction, whereas the treatment with 50 μg/ml of DNase 1, 80 μg/ml of RNase A, 40 μg/ml of RNase T1, or 0.1 μg/ml of trypsin had no inhibitory effect to induce asters. Injection of 0.5 μg/ml of RNase A or 1 mUg/ml of RNase T1 into the egg caused the detention of mitosis at the streak stage. To examine the temperature effect for aster induction, the centriolar fraction was pre-treated with boiling temperature, and it was found that the fraction became incapable to induce any aster.
Results obtained suggest that the effective components of the sperm centriolar fraction to induce asters in the fertilized sea urchin eggs are the proteins but not the nucleic acids. The aster inducing activity is destroyed by heat treatment.     

Insights into the Molecular Mechanisms Involved in Sea Urchin Fertilization Envelope Assembly

Sea urchin fertilization envelope assembly provides an ideal model system for investigating the production and modification of an extracellular matrix. The contents of secretory vesicles and the egg glycocalyx mix to initiate assembly. Limited proteolysis and covalent crosslinking by a transglutaminase act as early events to modify the nascent envelope. A subset of secreted proteins binds to this matrix through ionic interactions that require divalent cations. For example, one secreted protein, proteoliaisin, is responsible for attaching ovoperoxidase to the envelope. Ovoperoxidase hardens the envelope by using hydrogen peroxide, produced by the egg during the respiratory burst, to form dityrosine crosslinks between a subset of fertilization envelope proteins. Numerous spatial and temporal regulatory mechanisms exist to ensure that proper assembly occurs in an environment isolated from the normal cytosolic regulatory machinery.     

Phorbol Myristate Acetate Induces the Phosphorylation of Plasma Membrane-Associated Proteins in Sea Urchin Eggs

Immediately after fertilization sea urchin eggs undergo an increase in cytoplasmic pH from 6.8 to 7.2. This pH change occurs by activation of a Na⁺/H⁺ antiporter, and is a necessary signal for later steps in metabolic activation of development. Activators of protein kinase C such as phorbol myristate acetate (PMA) and diacylglycerol produce a similar pH increase in eggs. Phosphorylation of the antiporter or a regulatory protein may be a step in activating Na⁺/H⁺ exchange. Here we show that treatment of sea urchin eggs ( S. purpuratus ) with PMA results in increased phosphorylation of over a dozen proteins. Of these, three proteins of Mr=240, 92 and 80 kD are located in the egg cortex; under-representation of these bands in isolated cortical granules suggests that they are plasma membrane-associated. Phosphorylation of the 92 kD band is concentration-dependent over a range of 10 to 1000 nM PMA and occurs over a time-course of 1 to 3 min. Phosphoamino acid analysis indicates that phosphorylation is on serine residues. Phosphorylation appeares to be mediated by protein kinase C since the inactive PMA analogue, 4α-phorbol 12, 13-didecanoate, does not induce phosphorylation nor does experimental alkalinization of the egg cytoplasm.     

A Century of Sea Urchin Development

SYNOPSIS. In the last quarter of the nineteenth century severalinvestigators including Richard and Oskar Hertwig, Theodor Boveri,Hans Driesch, Curt Herbst, T. H. Morgan and others turned theirattention to sea urchin eggs and early embryos. This favorablecombination of outstanding investigators and the sea urchinembryo as an experimental organism contributed to a fundamentalunderstanding of the cell, fertilization and heredity. The advantagesof the sea urchin continued to be recognized as experimentalembryologists used these embryos to develop the concepts ofgradients, regulative development and inductive interactions.Then, as developmental biology arose from chemical embryology,the sea urchin embryo once again emerged as an ideal experimentalanimal, pivotal in the understanding of the molecular and developmentalbiology of eukaryotic organisms.     

Activation of Sea Urchin Eggs by Halothane and Its Inhibition by Dantrolene

Eggs of the sea urchin, Hemicentrotus pulcherrimus , were stimulated by halothane, known to induce Ca²⁺ release from sarcosome, to cause fertilization membrane formation in normal and Ca²⁺ free artificial sea water. In the absence of external Ca²⁺, halothane-induced formation of fertilization membrane was inhibited by dantrolene, an inhibitor of Ca²⁺ release from sarcosome, but was not blocked by nifedipine, a Ca²⁺ antagonist specific to Ca²⁺ channels in plasma membrane. Ca²⁺ release from sedimentable fraction isolated from eggs was induced by halothane and was inhibited by dantrolene, but was not blocked by nifedipine. In normal artificial sea water, halothane-caused egg activation was not inhibited either by dantrolene or by nifedipine, but was blocked in the presence of both compounds. ⁴⁵Ca²⁺ influx was substantially stimulated by halothane in eggs exposed to ⁴⁵CaCl₂. Halothane-induced ⁴⁵Ca²⁺ influx into eggs was inhibited by nifedipine but was not blocked by dantrolene. When Ca²⁺ release from intracellular organellae is blocked, Ca²⁺ transport through Ca²⁺ channels in plasma membrane probably acts as a "fail-safe" system to induce an increase in cytosolic Ca²⁺ level, resulting in egg activation.